当前期刊: BMC Microbiology Go to current issue    加入关注   
显示样式:        排序: 导出
  • Sampling methods affect Nematode-Trapping Fungi biodiversity patterns across an elevational gradient
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-16
    Wei Deng; Jia-Liang Wang; Matthew B. Scott; Yi-Hao Fang; Shuo-Ran Liu; Xiao-Yan Yang; Wen Xiao

    Understanding the patterns of species richness across elevational gradients is a key concept for contemporary research in ecology and evolution, and critical to understanding large-scale trends in biodiversity, global change and conservation. However, patterns of elevational species richness between taxonomic groups, regions and latitudes are inconsistent, so that various, sometimes conflicting hypotheses exist. Several scholars have pointed out that research on elevational distribution patterns is often biased by the sampling design employed. To test this hypothesis, we analyzed species richness of Nematode-Trapping Fungi (NTF) across an elevation gradient at two mountainous sites in western Yunnan Province, P.R. China. We tested for potential differences in the results when using different sampling designs. A total of 3 genera, 17 species, 222 strains of NTF were isolated and identified from Gaoligongshan and Cangshan. Species accumulation curves for both sites and sampling modes had acceptable leveling, demonstrating sufficient sampling effort. At Gaoligongshan, the elevation distribution patterns of NTF were different under two sampling patterns. When reducing the analyzed altitude range in Gaoligongshan, the elevation distribution pattern of the NTF changed. A similar elevation distribution pattern was observed in Cangshan when testing the same altitude range. In general, when treating the same dataset using different sampling designs, the resulting distribution patterns of species richness and occurrence frequencies were clearly different. Moreover, after removal of the samples located within lower-altitude zones affected by anthropogenic interferences, the distribution pattern of NTF in the two sites tended to become uniform. The sampling design, and in particular the elevation interval between plots, has a significant effect on the assessment of species distribution in mountainous regions. Other factors such as human activities and the multi-dimensionality of biodiversity also contribute to result biases. It is recommended that sampling design is given careful consideration in future studies on the elevational gradients of species richness, using stratified approaches according to the most relevant factors.

  • Genome-resolved metagenomics analysis provides insights into the ecological role of Thaumarchaeota in the Amazon River and its plume
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-15
    Otávio H. B. Pinto; Thais F. Silva; Carla S. Vizzotto; Renata H. Santana; Fabyano A. C. Lopes; Bruno S. Silva; Fabiano L. Thompson; Ricardo H. Kruger

    Thaumarchaeota are abundant in the Amazon River, where they are the only ammonia-oxidizing archaea. Despite the importance of Thaumarchaeota, little is known about their physiology, mainly because few isolates are available for study. Therefore, information about Thaumarchaeota was obtained primarily from genomic studies. The aim of this study was to investigate the ecological roles of Thaumarchaeota in the Amazon River and the Amazon River plume. The archaeal community of the shallow in Amazon River and its plume is dominated by Thaumarchaeota lineages from group 1.1a, which are mainly affiliated to Candidatus Nitrosotenuis uzonensis, members of order Nitrosopumilales, Candidatus Nitrosoarchaeum, and Candidatus Nitrosopelagicus sp. While Thaumarchaeota sequences have decreased their relative abundance in the plume, Candidatus Nitrosopelagicus has increased. One genome was recovered from metagenomic data of the Amazon River (ThauR71 [1.05 Mpb]), and two from metagenomic data of the Amazon River plume (ThauP25 [0.94 Mpb] and ThauP41 [1.26 Mpb]). Phylogenetic analysis placed all three Amazon genome bins in Thaumarchaeota Group 1.1a. The annotation revealed that most genes are assigned to the COG subcategory coenzyme transport and metabolism. All three genomes contain genes involved in the hydroxypropionate/hydroxybutyrate cycle, glycolysis, tricarboxylic acid cycle, oxidative phosphorylation. However, ammonia-monooxygenase genes were detected only in ThauP41 and ThauR71. Glycoside hydrolases and auxiliary activities genes were detected only in ThauP25. Our data indicate that Amazon River is a source of Thaumarchaeota, where these organisms are important for primary production, vitamin production, and nitrification.

  • Prevalence and antimicrobial susceptibility of enterotoxigenic extra-intestinal Bacteroides fragilis among 13-year collection of isolates in Kuwait
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-15
    Wafaa Jamal; Fatima Bibi Khodakhast; Ameerah AlAzmi; Jόzsef Sόki; Ghayda AlHashem; Vincent O. Rotimi

    Some strains of Bacteroides fragilis species are associated with diarrhea as a result of enterotoxin production (bft or fragilysin). Fragilysin is activated by C11 protease (fpn) and together with C10 protease (bfp) play a significant role in its invasiveness. The objectives of this study were to investigate the proportion of clinical isolates from extra-intestinal sources that are toxin producers and characterize the genes mediating toxin production. Clinical isolates submitted to our reference laboratory over the last 13 years were screened for toxin production using PCR technique. All stool isolates were excluded. The isolates were tested for their susceptibility to 8 antimicrobial agents by E test. Carbapenem resistance gene cfiA was detected by PCR. A total of 421 B. fragilis isolates were viable. Out of these, bft was detected in 210 (49.9%) isolates. Of the 210 bft-positive isolates, 171 (81.4%), 33 (15.7%) and 6 (2.8%) harbored bft-1, bft-2, and bft-3 genes, respectively. Twenty (9.5%) of the bft-positive strains originated from bloodstream infections. Twenty-five, 20 and 9 strains harbored bfp-1, bfp-2 and bfp-3 gene, respectively. Two, 3, 4 bfp isotypes were detected simultaneously in some of strains. The resistance rates against amoxicillin-clavulanic acid was 32%, clindamycin 62%, cefoxitin 26%, imipenem 11%, meropenem 17%, metronidazole 4%, piperacillin 61% and tigecycline 14%. A chromosomally located cfiA gene that encode metallo-β-lactamase was identified in only 34 isolates (16.2%). The prevalence of enterotoxin-producing B. fragilis was high among the extra-intestinal isolates. Metronidazole was the most active agent against all isolates. There was no statistically significance difference between resistance rates among bft-positive and bft-negative isolates except for clindamycin.

  • Comparisons of microbiological characteristics and antibiotic resistance of Klebsiella pneumoniae isolates from urban rodents, shrews, and healthy people
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-14
    Xue-shan Zhong; Yong-zhi Li; Jing Ge; Gang Xiao; Yun Mo; Yu-qi Wen; Jing-ping Liu; Yi-quan Xiong; Min Qiu; Shu-ting Huo; Ming-ji Cheng; Qing Chen

    The comparisons of molecular characterization and antibiotic resistance of Klebsiella pneumoniae (KP) isolates from humans and other animal hosts are not well studied. Our goal was to compare the molecular epidemiology of KP strains that were isolated from urban rodents, shrews, and healthy people. K. pneumoniae (KP) isolates were isolated from fecal samples of rodents, shrews and healthy adults in 2015 in southern China. In total, 465 fecal samples were collected, of which 85 from rodents, 105 from shrews, and 275 from healthy adults. Antimicrobial susceptibility and production of extended-spectrum β-lactamases (ESBL) of the isolates were tested. PCR-based methods were used to detect specific genes, including ESBL genes (blaTEM, blaSHV, and blaCTX-M) in ESBL-producing isolates, capsular serotypes (K1, K2, K5, K20, K54, and K57) in hypervirulent KPs (hvKPs), and virulence genes (magA, wcaG, rmpA, uge, kfu, and aerobactin) in hvKP isolates. Multilocus sequence type (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to exclude the homology of these isolates. The carriage rate of KP in urban rodents and shrews (78.42%) was higher than that in healthy adults (66.18%) (χ2 = 8.206, P = 0.004). The prevalence rates of ESBL-producing isolates among rodents, shrews, and humans were 7.94, 12.79, and 17.03%, respectively. The positive rates of CTX-M, TEM and SHV types in ESBL-producing isolates were 29.79, 27.66, and 17.02%, respectively. Serotype K1, K5, K20, and K57 were detected in both small mammals and humans. PFGE typing revealed thirty-six clusters. PFGE cluster A was clustered by samples of shrews and healthy adult, with a similarity of 88.4%. MLST typing revealed thirty-eight types. ST23 and ST35 were detected in samples of shrews and healthy adults. ST37 was detected in samples of 2 rodents and a healthy adult. Overlapping serotypes of hvKP were observed in both the animals and humans. The same PFGE or MLST types were also found in isolates derived humans, rodents and shrews. Therefore, urban rodents and shrews might play a certain role in the transmission of drug-resistant and hypervirulent KP.

  • Biodegradation of endocrine disruptor Bisphenol A by Pseudomonas putida strain YC-AE1 isolated from polluted soil, Guangdong, China
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-13
    Adel Eltoukhy; Yang Jia; Ruth Nahurira; M. A. Abo-Kadoum; Ibatsam Khokhar; Junhuan Wang; Yanchun Yan

    Bisphenol A is an important organic chemical as an intermediate, final and inert ingredient in manufacturing of many important products like polycarbonate plastics, epoxy resins, flame retardants, food–drink packaging coating, and other. BPA is an endocrine disruptor compound that mimics the function of estrogen causing damage to reproductive organs. Bacterial degradation has been consider as a cost effective and eco-friendly method for BPA degradation compared with physical and chemical methods. This study aimed to isolate and identify bacterial strain capable to degrade and tolerate high concentrations of this pollutant, studying the factors affecting the degradation process and study the degradation mechanism of this strain. YC-AE1 is a Gram negative bacterial strain isolated from soil and identified as Pseudomonas putida by 16S rRNA gene sequence and BIOLOG identification system. This strain found to have a high capacity to degrade the endocrine disruptor Bisphenol A (BPA). Response surface methodology using central composite design was used to statistically optimize the environmental factors during BPA degradation and the results obtained by significant model were 7.2, 30 °C and 2.5% for optimum initial pH, temperature and inoculum size, respectively. Prolonged incubation period with low NaCl concentration improve the biodegradation of BPA. Analysis of variance (ANOVA) showed high coefficient of determination, R2 and Adj-R2 which were 0.9979 and 0.9935, respectively. Substrate analysis found that, strain YC-AE1 could degrade a wide variety of bisphenol A-related pollutants such as bisphenol B, bisphenol F, bisphenol S, Dibutyl phthalate, Diethylhexyl phthalate and Diethyl phthalate in varying proportion. Pseudomonas putida YC-AE1 showed high ability to degrade a wide range of BPA concentrations (0.5–1000 mg l− 1) with completely degradation for 500 mg l− 1 within 72 h. Metabolic intermediates detected in this study by HPLC-MS were identified as 4,4-dihydroxy-alpha-methylstilbene, p-hydroxybenzaldeyde, p-hydroxyacetophenone, 4-hydroxyphenylacetate, 4-hydroxyphenacyl alcohol, 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl) propanoate. This study reports Pseudomonas putida YC-AE1 as BPA biodegrader with high performance in degradation and tolerance to high BPA concentration. It exhibited strong degradation capacity and prominent adaptability towards a wide range of environmental conditions. Moreover, it degrades BPA in a short time via two different degradation pathways.

  • First identification and genotyping of Enterocytozoon bieneusi in humans in Myanmar
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-13
    Yujuan Shen; Baiyan Gong; Xiaohua Liu; Yanchen Wu; Fengkun Yang; Jie Xu; Xiaofan Zhang; Jianping Cao; Aiqin Liu

    Intestinal pathogen infections are widespread among impoverished populations. Enterocytozoon bieneusi is the most common pathogen of intestinal microsporidian species in humans worldwide. However, no epidemiological information is available on E. bieneusi infection in humans in Myanmar. The present study comprised the first identification and genotyping of E. bieneusi in humans conducted in Myanmar. A total of 172 fecal specimens were collected from the Wa people (one each) in four villages of Pangsang Township of the Matman District of Shan State, Myanmar, and each participant completed a questionnaire. E. bieneusi was identified and genotyped using polymerase chain reaction (PCR) and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. The average prevalence of E. bieneusi was 8.72% (15/172), ranging from 3.85 to 13.89% by village. E. bieneusi infection was not related to any of the risk factors studied. Six genotypes were identified, comprising two known genotypes Peru6 (n = 10) and D (n = 1) and four novel genotypes (MMR23, MMR25, MMR86, and MMR87) (one each), and two people infected with genotype Peru6 were from the same family. A phylogenetic analysis based on a neighbor-joining tree of the ITS sequences of E. bieneusi indicated that all the six genotypes were clustered into group 1. This is the first identification and genotyping of E. bieneusi in humans in Myanmar. The observations that the two people infected with genotype Peru6 were from the same family, and that all six genotypes obtained in the present study fell into zoonotic group 1, showed the potential for anthropogenic and zoonotic transmissions. The present data argue for the importance of epidemiological control and prevention from medical sectors.

  • Correction to: Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-10
    Andrew Jenkins; Cecilie Raasok; Benedikte N. Pedersen; Kristine Jensen; Åshild Andreassen; Arnulf Soleng; Kristin Skarsfjord Edgar; Heidi Heggen Lindstedt; Vivian Kjelland; Snorre Stuen; Dag Hvidsten; Bjørn-Erik Kristiansen

    After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.

  • Larva of greater wax moth Galleria mellonella is a suitable alternative host for the fish pathogen Francisella noatunensis subsp. orientalis
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-09
    Winarti Achmad Sarmin Djainal; Khalid Shahin; Matthijs Metselaar; Alexandra Adams; Andrew P. Desbois

    Francisella noatunensis subsp. orientalis (Fno) is the etiological agent of francisellosis in cultured warm water fish, such as tilapia. Antibiotics are administered to treat the disease but a better understanding of Fno infection biology will inform improved treatment and prevention measures. However, studies with native hosts are costly and considerable benefits would derive from access to a practical alternative host. Here, larvae of Galleria mellonella were assessed for suitability to study Fno virulence. Larvae were killed by Fno in a dose-dependent manner but the insects could be rescued from lethal doses of bacteria by antibiotic therapy. Infection progression was assessed by histopathology (haematoxylin and eosin staining, Gram Twort and immunohistochemistry) and enumeration of bacteria recovered from the larval haemolymph on selective agar. Fno was phagocytosed and could survive intracellularly, which is consistent with observations in fish. Virulence of five Fno isolates showed strong agreement between G. mellonella and red Nile tilapia hosts. This study shows that an alternative host, G. mellonella, can be applied to understand Fno infections, which will assist efforts to identify solutions to piscine francisellosis thus securing the livelihoods of tilapia farmers worldwide and ensuring the production of this important food source.

  • Evaluation of resazurin-based assay for rapid detection of polymyxin-resistant gram-negative bacteria
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-08
    Huaiyu Jia; Renchi Fang; Jie Lin; Xuebin Tian; Yajie Zhao; Lijiang Chen; Jianming Cao; Tieli Zhou

    Colistin resistance is considered a serious problem due to a lack of alternative antibiotics. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test is a resazurin reduction-based technique that relies on the visual detection of bacterial growth in the presence of a defined concentration of colistin. The aim of this study was to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test in the detection of colistin susceptibility in common clinical Gram-negative bacteria. A total of 253 clinical isolates from a teaching hospital, including Acinetobacter baumanii (n = 58, 8 colistin-resistant), Pseudomonas aeruginosa (n = 61, 11 colistin-resistant), Klebsiella pneumoniae (n = 70, 20 colistin-resistant) and Escherichia coli (n = 64, 14 colistin-resistant) were tested in this study. The sensitivity and specificity of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test compared to Broth microdilution method was 100 and 99%, respectively. Our results suggest that Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test could be used as an accurate detection method for colistin resistance.

  • HpaXpm, a novel harpin of Xanthomonas phaseoli pv. manihotis, acts as an elicitor with high thermal stability, reduces disease, and promotes plant growth
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-06
    Yue Liu; Xiaoyun Zhou; Wenbo Liu; Jiamin Huang; Qinghuan Liu; Jianzhang Sun; Xinfeng Cai; Weiguo Miao

    Harpins are proteins secreted by the type III secretion system of Gram-negative bacteria during pathogen–plant interactions that can act as elicitors, stimulating defense and plant growth in many types of non-host plants. Harpin-treated plants have higher resistance, quality and yields and, therefore, harpin proteins may potentially have many valuable agricultural applications. Harpins are characterized by high thermal stability at 100 °C. However, it is unknown whether harpins are still active at temperatures above 100 °C or whether different temperatures affect the activity of the harpin protein in different ways. The mechanism responsible for the heat stability of harpins is also unknown. We identified a novel harpin, HpaXpm, from the cassava blight bacteria Xanthomonas phaseoli pv. manihotis HNHK. The predicted secondary structure and 3-D structure indicated that the HpaXpm protein has two β-strand domains and two major α-helical domains located at the N- and C-terminal regions, respectively. A phylogenetic tree generated using the maximum likelihood method grouped HpaXpm in clade I of the Hpa1 group along with harpins produced by other Xanthomonas spp. (i.e., HpaG-Xag, HpaG-Xcm, Hpa1-Xac, and Hpa1Xm). Phenotypic assays showed that HpaXpm induced the hypersensitive response (HR), defense responses, and growth promotion in non-host plants more effectively than Hp1Xoo (X. oryzae pv. oryzae). Quantitative real-time PCR analysis indicated that HpaXpm proteins subjected to heat treatments at 100 °C, 150 °C, or 200 °C were still able to stimulate the expression of function-related genes (i.e., the HR marker genes Hin1 and Hsr203J, the defense-related gene NPR1, and the plant growth enhancement-related gene NtEXP6); however, the ability of heat-treated HpaXpm to induce HR was different at different temperatures. These findings add a new member to the harpin family. HpaXpm is heat-stable up to 200 °C and is able to stimulate powerful beneficial biological functions that could potentially be more valuable for agricultural applications than those stimulated by Hpa1Xoo. We hypothesize that the extreme heat resistance of HpaXpm is because the structure of harpin is very stable and, therefore, the HpaXpm structure is less affected by temperature.

  • In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-06
    Vuong Van Hung Le; Catrina Olivera; Julian Spagnuolo; Ieuan G. Davies; Jasna Rakonjac

    Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, sodium deoxycholate (DOC) in growth inhibition and killing of enterobacteria. Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a β-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of nitric oxide-detoxifying enzyme Hmp results in a three-fold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gram-negative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

  • Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-07
    Diansy Zincke; Michael H. Norris; Berzhan Kurmanov; Ted L. Hadfield; Jason K. Blackburn

    The exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains. In this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant−) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA. Screening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

  • Temporal analysis of mRNA expression profiles in Orientia infected C3HeB/FeJ mouse
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-06
    Chien-Chung Chao; Ruoting Yang; Zhiwen Zhang; Tatyana Belinskaya; Chye-Teik Chan; Stacy-Ann Miller; Rasha Hammamieh; Marti Jett; Wei-Mei Ching

    Scrub typhus causes up to 35% mortality if left untreated. One billion people living in the endemic regions are at risk. In spite of its heavy disease burden in some of the most populated areas in the world, there is no vaccine available. Although the disease can be effectively treated by proper antibiotics, timely and accurate diagnosis remains a challenge. Orientia tsutsugamushi infects a variety of mammalian cells in vitro and replicates in the cytoplasm of the infected cells. Microarray analysis has been used extensively to study host-pathogen interactions in in vitro models to understand pathogenesis. However there is a lack of in vivo studies. In this study, C3HeB/FeJ (C3H) mice were infected by O. tsutsugamushi via the intraperitoneal route and monitored gene expression at 10 different time points post infection. We observed two distinct types of expression profiles in the genes that we analyzed. There are two valleys (4–18 h and 2–4 days) with low number of differentially expressed genes (DEG) with three peaks with high number of DEG at 2 h, 1-day and 7-day post infection. Further analysis revealed that pathways like complement and coagulation cascade, and blood clotting cascade pathways showed significant global changes throughout entire time course. Real time quantitative Polymerase Chain Reaction (RT-qPCR) confirmed the change of expression for genes involved in complement and coagulation cascade. These results suggested dynamic regulation of the complement and coagulation cascades throughout most of the time post infection while some other specific pathways, such as fatty acid metabolism and tryptophan metabolism, are turned on or off at certain times post infection. The findings highlight the complex interconnection among all different biological pathways. It is conceivable that specific pathways such as cell growth control and cell development in the host are affected by Orientia in the initial phase of infection for Orientia to grow intracellularly. Once Orientia is replicating successfully inside the host as infection progresses, the infection could activate pathways involved in cellular immune responses to defend for host cell survival and try to eliminate the pathogen.

  • An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-02
    Shi-qi An; Miguel A. Valvano; Yan-hua Yu; Jeremy S. Webb; Guillermo Lopez Campos

    Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

  • Method comparison for the direct enumeration of bacterial species using a chemostat model of the human colon
    BMC Microbiol. (IF 3.287) Pub Date : 2020-01-02
    Ines B. Moura; Charmaine Normington; Duncan Ewin; Emma Clark; Mark H. Wilcox; Anthony M. Buckley; Caroline H. Chilton

    Clostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. In this study, antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. Three in vitro models were exposed to clindamycin to induce simulated CDI. All models were treated with vancomycin, and two received an FMT. Populations of total bacteria, Bacteroides spp., Lactobacillus spp., Enterococcus spp., Bifidobacterium spp., C. difficile, and Enterobacteriaceae were monitored using both methods. Total clostridia were monitored by selective culture. Using qPCR analysis, we additionally monitored populations of Prevotella spp., Clostridium coccoides group, and Clostridium leptum group. Both methods showed an exacerbation of disruption of the colonic microbiota following vancomycin (and earlier clindamycin) exposure, and a quicker recovery (within 4 days) of the bacterial populations in the models that received the FMT. C. difficile proliferation, consistent with CDI, was also observed by both qPCR and culture. Pearson correlation coefficient showed an association between results varying from 98% for Bacteroides spp., to 62% for Enterobacteriaceae. Generally, a good correlation was observed between qPCR and bacterial culture. Overall, the molecular assays offer results in real-time, important for treatment efficacy, and allow the monitoring of additional microbiota groups. However, individual quantification of some genera (e.g. clostridia) might not be possible without selective culture.

  • Application of LpxC enzyme inhibitor to inhibit some fast-growing bacteria in human gut bacterial culturomics
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-30
    Fengyi Hou; Yuxiao Chang; Zongyu Huang; Ni Han; Lei Bin; Huimin Deng; Zhengchao Li; Zhiyuan Pan; Lei Ding; Hong Gao; Ruifu Yang; Fachao Zhi; Yujing Bi

    Culturomics can ascertain traces of microorganisms to be cultivated using different strategies and identified by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry or 16S rDNA sequencing. However, to cater to all requirements of microorganisms and isolate as many species as possible, multiple culture conditions must be used, imposing a heavy workload. In addition, the fast-growing bacteria (e.g., Escherichia) surpass the slow-growing bacteria in culture by occupying space and using up nutrients. Besides, some bacteria (e.g., Pseudomonas) suppress others by secreting antibacterial metabolites, making it difficult to isolate bacteria with lower competence. Applying inhibitors to restrain fast-growing bacteria is one method to cultivate more bacterial species from human feces. We applied CHIR-090, an LpxC enzyme inhibitor that has antibacterial activity against most Gram-negative bacteria, to culturomics of human fresh feces. The antibacterial activity of CHIR-090 was first assessed on five Gram-negative species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, and Bacteroides vulgatus), all of which are commonly isolated from the human gut. Then, we assessed suitable concentrations of the inhibitor. Finally, CHIR-090 was applied in blood culture bottles for bacterial cultivation. In total, 102 species from five samples were identified. Of these, we found one new species, two species not reported previously in the human gut, and 11 species not previously isolated from humans. CHIR-090 can suppress E. coli, P. aeruginosa, K. pneumoniae, Pro. vulgaris, but not B. vulgatus. Compared with the non-inhibitor group, CHIR-090 increased bacteria isolation by 23.50%, including four species not reported in humans and one new species. Application of LpxC enzyme inhibitor in culturomics increased the number of species isolated from the human gut.

  • Primary progressive multiple sclerosis in a Russian cohort: relationship with gut bacterial diversity
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-30
    Madina Kozhieva; Natalia Naumova; Tatiana Alikina; Alexey Boyko; Valentin Vlassov; Marsel R. Kabilov

    Gut microbiota has been increasingly acknowledged to shape significantly human health, contributing to various autoimmune diseases, both intestinal and non-intestinal, including multiple sclerosis (MS). Gut microbiota studies in patients with relapsing remitting MS strongly suggested its possible role in immunoregulation; however, the profile and potential of gut microbiota involvement in patients with primary progressive MS (PPMS) patients has received much less attention due to the rarity of this disease form. We compared the composition and structure of faecal bacterial assemblage using Illumina MiSeq sequencing of V3-V4 hypervariable region of 16S rRNA genes amplicons in patients with primary progressive MS and in the healthy controls. Over all samples 12 bacterial phyla were identified, containing 21 classes, 25 orders, 54 families, 174 genera and 1256 operational taxonomic units (OTUs). The Firmicutes phylum was found to be ultimately dominating both in OTUs richness (68% of the total bacterial OTU number) and in abundance (71% of the total number of sequence reads), followed by Bacteroidetes (12 and 16%, resp.) and Actinobacteria (7 and 6%, resp.). Summarily in all samples the number of dominant OTUs, i.e. OTUs with ≥1% relative abundance, was 13, representing much less taxonomic richness (three phyla, three classes, four orders, six families and twelve genera) as compared to the total list of identified OTUs and accounting for 30% of the sequence reads number in the healthy cohort and for 23% in the PPMS cohort. Human faecal bacterial diversity profiles were found to differ between PPMS and healthy cohorts at different taxonomic levels in minor or rare taxa. Marked PPMS-associated increase was found in the relative abundance of two dominant OTUs (Gemmiger sp. and an unclassified Ruminococcaceae). The MS-related differences were also found at the level of minor and rare OTUs (101 OTUs). These changes in OTUs’ abundance translated into increased bacterial assemblage diversity in patients. The findings are important for constructing a more detailed global picture of the primary progressive MS-associated gut microbiota, contributing to better understanding of the disease pathogenesis.

  • An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-30
    Elisabeth A. Slade; Robin M. S. Thorn; Amber Young; Darren M. Reynolds

    The majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time. The collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide− 1 was achieved by 30 h of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2–1.6 log reduction in biofilm density at 72 h compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 h at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation. The collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies.

  • Gut bacteria of weevils developing on plant roots under extreme desert conditions
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-30
    Fengqun Meng; Nitsan Bar-Shmuel; Reut Shavit; Adi Behar; Michal Segoli

    Many phytophagous insects, whose diet is generally nitrogen-poor, rely on gut bacteria to compensate for nutritional deficits. Accordingly, we hypothesized that insects in desert environments may evolve associations with gut bacteria to adapt to the extremely low nutrient availability. For this, we conducted a systematic survey of bacterial communities in the guts of weevils developing inside mud chambers affixed to plant roots in the Negev Desert of Israel, based on 16S rRNA gene amplicon sequencing. Our analyses revealed that gut bacterial communities in weevil larvae were similar across a wide geographical range, but differed significantly from those of the mud chambers and of the surrounding soils. Nevertheless, a high proportion of bacteria (including all of the core bacteria) found in the weevils were also detected in the mud chambers and soils at low relative abundances. The genus Citrobacter (of the Enterobacteriaceae family) was the predominant group in the guts of all individual weevils. The relative abundance of Citrobacter significantly decreased at the pupal and adult stages, while bacterial diversity increased. A mini literature survey revealed that members of the genus Citrobacter are associated with nitrogen fixation, recycling of uric acid nitrogen, and cellulose degradation in different insects. The results suggest that although weevils could potentially acquire their gut bacteria from the soil, weevil host internal factors, rather than external environmental factors, were more important in shaping their gut bacterial communities, and suggest a major role for Citrobacter in weevil nutrition in this challenging environment. This study highlights the potential involvement of gut bacteria in the adaptation of insects to nutritional deficiencies under extreme desert conditions.

  • Long-term impact of fecal transplantation in healthy volunteers
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-30
    Oleg V. Goloshchapov; Evgenii I. Olekhnovich; Sergey V. Sidorenko; Ivan S. Moiseev; Maxim A. Kucher; Dmitry E. Fedorov; Alexander V. Pavlenko; Alexander I. Manolov; Vladimir V. Gostev; Vladimir A. Veselovsky; Ksenia M. Klimina; Elena S. Kostryukova; Evgeny A. Bakin; Alexander N. Shvetcov; Elvira D. Gumbatova; Ruslana V. Klementeva; Alexander A. Shcherbakov; Margarita V. Gorchakova; Juan José Egozcue; Vera Pawlowsky-Glahn; Maria A. Suvorova; Alexey B. Chukhlovin; Vadim M. Govorun; Elena N. Ilina; Boris V. Afanasyev

    Fecal microbiota transplantation (FMT) has been recently approved by FDA for the treatment of refractory recurrent clostridial colitis (rCDI). Success of FTM in treatment of rCDI led to a number of studies investigating the effectiveness of its application in the other gastrointestinal diseases. However, in the majority of studies the effects of FMT were evaluated on the patients with initially altered microbiota. The aim of our study was to estimate effects of FMT on the gut microbiota composition in healthy volunteers and to monitor its long-term outcomes. We have performed a combined analysis of three healthy volunteers before and after capsule FMT by evaluating their general condition, adverse clinical effects, changes of basic laboratory parameters, and several immune markers. Intestinal microbiota samples were evaluated by 16S rRNA gene and shotgun sequencing. The data analysis demonstrated profound shift towards the donor microbiota taxonomic composition in all volunteers. Following FMT, all the volunteers exhibited gut colonization with donor gut bacteria and persistence of this effect for almost ∼1 year of observation. Transient changes of immune parameters were consistent with suppression of T-cell cytotoxicity. FMT was well tolerated with mild gastrointestinal adverse events, however, one volunteer developed a systemic inflammatory response syndrome. The FMT leads to significant long-term changes of the gut microbiota in healthy volunteers with the shift towards donor microbiota composition and represents a relatively safe procedure to the recipients without long-term adverse events.

  • Pan-genome diversification and recombination in Cronobacter sakazakii, an opportunistic pathogen in neonates, and insights to its xerotolerant lifestyle
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-27
    Isaiah Paolo A. Lee; Cheryl P. Andam

    Cronobacter sakazakii is an emerging opportunistic bacterial pathogen known to cause neonatal and pediatric infections, including meningitis, necrotizing enterocolitis, and bacteremia. Multiple disease outbreaks of C. sakazakii have been documented in the past few decades, yet little is known of its genomic diversity, adaptation, and evolution. Here, we analyzed the pan-genome characteristics and phylogenetic relationships of 237 genomes of C. sakazakii and 48 genomes of related Cronobacter species isolated from diverse sources. The C. sakazakii pan-genome contains 17,158 orthologous gene clusters, and approximately 19.5% of these constitute the core genome. Phylogenetic analyses reveal the presence of at least ten deep branching monophyletic lineages indicative of ancestral diversification. We detected enrichment of functions involved in proton transport and rotational mechanism in accessory genes exclusively found in human-derived strains. In environment-exclusive accessory genes, we detected enrichment for those involved in tryptophan biosynthesis and indole metabolism. However, we did not find significantly enriched gene functions for those genes exclusively found in food strains. The most frequently detected virulence genes are those that encode proteins associated with chemotaxis, enterobactin synthesis, ferrienterobactin transporter, type VI secretion system, galactose metabolism, and mannose metabolism. The genes fos which encodes resistance against fosfomycin, a broad-spectrum cell wall synthesis inhibitor, and mdf(A) which encodes a multidrug efflux transporter were found in nearly all genomes. We found that a total of 2991 genes in the pan-genome have had a history of recombination. Many of the most frequently recombined genes are associated with nutrient acquisition, metabolism and toxin production. Overall, our results indicate that the presence of a large accessory gene pool, ability to switch between ecological niches, a diverse suite of antibiotic resistance, virulence and niche-specific genes, and frequent recombination partly explain the remarkable adaptability of C. sakazakii within and outside the human host. These findings provide critical insights that can help define the development of effective disease surveillance and control strategies for Cronobacter-related diseases.

  • Composite genome sequence of Bacillus clausii, a probiotic commercially available as Enterogermina®, and insights into its probiotic properties
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-30
    Indu Khatri; Gaurav Sharma; Srikrishna Subramanian

    Some of the spore-forming strains of Bacillus probiotics are marketed commercially as they survive harsh gastrointestinal conditions and bestow health benefits to the host. We report the composite genome of Bacillus clausii ENTPro from a commercially available probiotic Enterogermina® and compare it with the genomes of other Bacillus probiotics. We find that the members of B. clausii species harbor high heterogeneity at the species as well as genus level. The genes conferring resistance to chloramphenicol, streptomycin, rifampicin, and tetracycline in the B. clausii ENTPro strain could be identified. The genes coding for the bacteriocin gallidermin, which prevents biofilm formation in the pathogens Staphylococcus aureus and S. epidermidis, were also identified. KEGG Pathway analysis suggested that the folate biosynthesis pathway, which depicts one of the important roles of probiotics in the host, is conserved completely in B. subtilis and minimally in B. clausii and other probiotics. We identified various antibiotic resistance, bacteriocins, stress-related, and adhesion-related domains, and industrially-relevant pathways, in the genomes of these probiotic bacteria that are likely to help them survive in the harsh gastrointestinal tract, facilitating adhesion to host epithelial cells, persistence during antibiotic treatment and combating bacterial infections.

  • Phenotypic and molecular characterization of antimicrobial resistance in Trueperella pyogenes strains isolated from bovine mastitis and metritis
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-27
    Mobin Rezanejad; Sepideh Karimi; Hassan Momtaz

    Trueperella pyogenes is one of the most clinically imperative bacteria responsible for severe cases of mastitis and metritis, particularly in postpartum dairy cows. The bacterium has emergence of antibiotic resistance and virulence characters. The existing research was done to apprise the phenotypic and genotypic evaluation of antibiotic resistance and characterization of virulence factors in the T. pyogenes bacteria of bovine mastitis and metritis in postpartum cows. Two-hundred and twenty-six bovine mastitic milk and 172 uterine swabs were collected and transferred to laboratory. Samples were cultured and T. pyogenes isolates were subjected to disk diffusion and DNA extraction. Distribution of virulence and antibiotic resistance genes was studied by PCR. Thirty-two out of 226 (14.15%) mastitic milk and forty-one out of 172 (23.83%) uterine swab samples were positive for T. pyogenes. Isolates of mastitic milk harbored the highest prevalence of resistance toward gentamicin (100%), penicillin (100%), ampicillin (90.62%), amoxicillin (87.50%) and trimethoprim-sulfamethoxazole (87.50%), while those of metritis harbored the highest prevalence of resistance toward ampicillin (100%), amoxicillin (100%), gentamicin (97.56%), penicillin (97.56%) and cefalexin (97.56%). AacC, aadA1, aadA2 and tetW were the most generally perceived antibiotic resistance genes. All bacteria harbored plo (100%) and fimA (100%) virulence factors. NanP, nanH, fimC and fimE were also the most generally perceived virulence factors. All bacteria harbored plo and fimA virulence factors which showed that they can use as specific genetic markers with their important roles in pathogenicity of T. pyogenes bacteria. Phenotypic pattern of antibiotic resistance was confirmed by genotypic characterization of antibiotic resistance genes.

  • Variation of growth characteristics of pneumococcus with environmental conditions
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-26
    Adrienn Tóthpál; Katherine Desobry; Shreyas S. Joshi; Anne L. Wyllie; Daniel M. Weinberger

    Pneumococcus is exposed to a variety of temperature and oxygen levels in the upper respiratory tract and as it invades the lung, tissues, and blood. We sought to determine the effect of environmental variability on growth in vitro and to assess variability between strains. We evaluated the effect of temperature and oxygen on the growth of 256 isolates representing 53 serotypes, recovered from healthy carriers and disease patients. Strains were grown at a range of temperatures, anaerobically or in ambient air with catalase, and were monitored by reading the optical density. Regression models evaluated variation in the characteristics of the growth curves. Most isolates grew to the maximal density at low temperatures (~33C) and under aerobic conditions. There was considerable variability between strains, and some of this variability was linked to serotype. However, capsule-switch experiments suggest that the production of different capsules might not be sufficient to explain this variation, suggesting there could be interactions between the capsule and genetic background. Pneumococcal strains vary in how they respond to environmental variations, some of this variation can be explained by the capsule type being produced, but capsule production itself is not sufficient to explain the variability. This variability could help to explain why different lineages of pneumococcus are more common in carriage or disease.

  • Diet and irradiation effects on the bacterial community composition and structure in the gut of domesticated teneral and mature Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae)
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Deane N. Woruba; Jennifer L. Morrow; Olivia L. Reynolds; Toni A. Chapman; Damian P. Collins; Markus Riegler

    Mass-rearing, domestication and gamma irradiation of tephritid fruit flies used in sterile insect technique (SIT) programmes can negatively impact fly quality and performance. Symbiotic bacteria supplied as probiotics to mass-reared fruit flies may help to overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known. We have used next-generation sequencing to characterise the bacterial community composition and structure within Queensland fruit fly, Bactrocera tryoni (Froggatt), by generating 16S rRNA gene amplicon libraries derived from the guts of 58 individual teneral and mature, female and male, sterile and fertile adult flies reared on artificial larval diets in a laboratory or mass-rearing environment, and fed either a full adult diet (i.e. sugar and yeast hydrolysate) or a sugar only adult diet. Overall, the amplicon sequence read volume in tenerals was low and smaller than in mature adult flies. Operational taxonomic units (OTUs), belonging to the families Enterobacteriaceae (8 OTUs) and Acetobacteraceae (1 OTU) were most prevalent. Enterobacteriaceae dominated laboratory-reared tenerals from a colony fed a carrot-based larval diet, while Acetobacteraceae dominated mass-reared tenerals from a production facility colony fed a lucerne chaff based larval diet. As adult flies matured, Enterobacteriaceae became dominant irrespective of larval origin. The inclusion of yeast in the adult diet strengthened this shift away from Acetobacteraceae towards Enterobacteriaceae. Interestingly, irradiation increased 16S rRNA gene sequence read volume. Our findings suggest that bacterial populations in fruit flies experience significant bottlenecks during metamorphosis. Gut bacteria in teneral flies were less abundant and less diverse, and impacted by colony origin. In contrast, mature adult flies had selectively increased abundances for some gut bacteria, or acquired these bacteria from the adult diet and environment. Furthermore, irradiation augmented bacterial abundance in mature flies. This implies that either some gut bacteria were compensating for damage caused by irradiation or irradiated flies had lost their ability to regulate bacterial load. Our findings suggest that the adult stage prior to sexual maturity may be ideal to target for probiotic manipulation of fly microbiota to increase fly performance in SIT programmes.

  • Bacterial communities in the gut of wild and mass-reared Zeugodacus cucurbitae and Bactrocera dorsalis revealed by metagenomic sequencing
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Ashok B. Hadapad; Suresh K. G. Shettigar; Ramesh S. Hire

    Insect pests belonging to genus Bactrocera sp. (Diptera: Tephritidae) pose major biotic stress on various fruits and vegetable crops around the world. Zeugodacus and Bactrocera sp. are associated with diverse bacterial communities which play an important role in the fitness of sterile insects. The wild populations of melon fly, Zeugodacus cucurbitae (Coquillett) and Oriental fruit fly, Bactrocera dorsalis (Hendel) were collected from pumpkin and mango fields, respectively. The laboratory populations of Z. cucurbitae and B. dorsalis were mass-reared on bottle gourd and sweet banana, respectively. Bacterial communities present in the gut of wild and mass-reared mature (~ 12 days old) and newly emerged (< 1 h after emergence) male and female adults of Z. cucurbitae and B. dorsalis were assessed. We used Illumina HiSeq next-generation sequencing of 16S rRNA gene to profile the gut bacterial communities of wild and mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. We found diverse bacterial composition in the gut of wild and mass-reared Z. cucurbitae (ZC) and B. dorsalis (BD) with varied relative abundance. Few taxonomic groups were common to both the species. The most dominant phyla in all samples of Z. cucurbitae and B. dorsalis adults were Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The phylum Proteobacteria occurred more in wild Z. cucurbitae (~ 87.72%) and B. dorsalis (~ 83.87%) as compared to mass-reared Z. cucurbitae (64.15%) and B. dorsalis (~ 80.96%). Higher relative abundance of Phylum Firmicutes was observed in mass-reared fruit fly than wild adults. Cyanobacteria/Chloroplast and Actinobacteria were also present with very low relative abundance in both wild as well as mass-reared melon fly and Oriental fruit fly. Enterobacteriaceae (61.21%) was dominant family in the gut of both wild and mass-reared adults. Providencia and Lactococcus were dominant genera with varied relative abundance in wild as well as in mass-reared mature and newly emerged fruit fly adults of both species. Some of the genera like Morganella and Serratia were only detected in mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. Principal Coordinate Analysis (PCoA) showed that fruit fly adult samples were grouped based on species and age of the adults while no grouping was observed on the basis of sex of the adult fruit fly. The gut bacterial communities associated with wild and mass-reared mature and newly emerged adults of Z. cucurbitae and B. dorsalis showed variation that depends on species and age of the insects. Understanding the gut microbiota of wild and mass-reared Z. cucurbitae and B. dorsalis using high throughput technology will help to illustrate microbial diversity and this information could be used to develop efficient mass-rearing protocols for successful implementation of sterile insect technique (SIT).

  • Gut bacterial diversity and physiological traits of Anastrepha fraterculus Brazilian-1 morphotype males are affected by antibiotic treatment
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    María Laura Juárez; Lida Elena Pimper; Guillermo Enrique Bachmann; Claudia Alejandra Conte; María Josefina Ruiz; Lucía Goane; Pilar Medina Pereyra; Felipe Castro; Julieta Salgueiro; Jorge Luis Cladera; Patricia Carina Fernández; Kostas Bourtzis; Silvia Beatriz Lanzavecchia; María Teresa Vera; Diego Fernando Segura

    The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males. AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet. Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.

  • Joint FAO/IAEA coordinated research project on “use of symbiotic bacteria to reduce mass-rearing costs and increase mating success in selected fruit pests in support of SIT application”
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Carlos Cáceres; George Tsiamis; Boaz Yuval; Edouard Jurkevitch; Kostas Bourtzis

    Insects represent the most abundant and speciose group of animals on this planet having established diverse and rather complex interactions with many prokaryotic and eukaryotic organisms. Although the interactions of insects with plants and vertebrates have been extensively studied, their interactions with microorganisms, despite their major significance from the ecological and evolutionary point of view, are still poorly characterized. However, the importance of the microorganisms on insects’ biology and physiology is gradually being unravelled. True fruit flies (Diptera: Tephritidae) are no exception, as they have established sophisticated interactions with microorganisms. There are over 5000 species in this family, many of which are destructive pests of fruits and vegetables. Beyond the direct damage caused by flies ovipositing in ripening fruit, some species are highly invasive, and incur major trade problems across and within national and international borders. These result in strict and costly quarantine regulations. The use of insecticides to control these pests, while effective on the short term, is associated with major environmental and health concerns. Accordingly, The Sterile Insect Technique (SIT) is deployed against several fruit fly species. This is an environment-friendly, species-specific and environment-friendly technology which can be used as an additional tool in integrated pest management programmes. As the efficiency of SIT may be hampered by quality control and cost effectiveness, major research efforts have focussed on ways to improve this process. These focus on the quality of the insects reared for release and the reduction of operational costs. The symbiotic relationships of tephritids were recognized as potential targets for improving sterile fly quality and reducing costs of production, and the papers in this collection describe this research effort. Coordinated Research Projects are a very powerful mechanism to bring together scientists in an attempt to fill knowledge gaps and solve important problems IAEA Member States may be facing. Eight years ago, a coordinated research project (CRP) entitled “Use of Symbiotic Bacteria to Reduce Mass-Rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application” was implemented by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Carlos Cáceres of the FAO/IAEA was the coordinator of this initiative which attracted the participation of 21 scientists from 16 countries, originating from diverse scientific fields, with the common goal of disentangling the symbiotic associations between the fruit flies and various microorganisms. The CRP focused on four key areas of research that could improve the quality management of fruit flies for use in SIT programmes: (1) eliminating the use of expensive ingredients in the larval diet (e.g. brewers / torula yeasts) and chemicals which are used to restrict the presence of unwanted microorganisms thus improving the productivity and quality of mass-reared insect colonies, (2) the use of radiation may disrupt the symbiotic community of mass-reared flies by enabling the presence of some bacterial species while limiting others. Determining the impact of radiation on the symbiotic communities will ultimately result to the development of responses to mitigate these effects in a manner that optimizes SIT efficiency; (3) restoring the symbiotic bacterial community in sterile fruit fly males, before their release, can greatly enhance their mating performance, and (4) it is known that several symbiotic bacteria, such as Wolbachia, are able to manipulate mating behaviour of their hosts and/or to induce reproductive alterations which can be exploited for the population control of insect pests and disease vectors. For example, the incompatible insect technique (IIT) is based on the Wolbachia-induced phenomenon of cytoplasmic incompatibility, which can be used for the population suppression of a target pest by repeated releases of cytoplasmically incompatible males. In some specific cases, SIT and IIT can be used in combination as complementary control tools. In this CRP, we seek to extend these approaches to manipulating the diverse microbiota present in SIT targeted insect pests associated to protect fruit and vegetable crops. In addition, four meetings were organized at about 18-month intervals during which the participating scientists reported their findings, exchanged ideas and coordinated their research plans. This special issue comprises the final research results of the CRP (18 research papers) that summarize the outcomes of the research carried out by participants and collaborators during the CRP. The guest editors are confident that this Cross-Journal Supplement on BMC Microbiology and BMC Biotechnology will provide valuable information regarding the manipulation of insect microbiota in support of sterile insect technique applications against insect fruit pests. Not applicable. Not applicable. About this supplement This article has been published as part of BMC Microbiology Volume 19 Supplement 1, 2019: Proceedings of an FAO/IAEA Coordinated Research Project on Use of Symbiotic Bacteria to Reduce Mass-rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application: microbiology. The full contents of the supplement are available online at https://bmcmicrobiol.biomedcentral.com/articles/supplements/volume-19-supplement-1. Funding This work was funded by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA (CRP No.: D4. 10.24) Vienna, Austria. Affiliations Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, A-1400, Vienna, Austria Carlos Cáceres  & Kostas Bourtzis Department of Environmental Engineering, University of Patras, 2 Seferi St., 30131, Agrinio, Greece George Tsiamis Departments of Entomology and Plant Pathology & Microbiology, Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, POB 12, 76100, Rehovot, Israel Boaz Yuval  & Edouard JurkevitchAuthors Search for Carlos Cáceres in: PubMed • Google Scholar Search for George Tsiamis in: PubMed • Google Scholar Search for Boaz Yuval in: PubMed • Google Scholar Search for Edouard Jurkevitch in: PubMed • Google Scholar Search for Kostas Bourtzis in: PubMed • Google Scholar Contributions CC, GT, BY, EJ and KB. drafted the manuscript and all authors have read and approved the final manuscript. Corresponding author Correspondence to Carlos Cáceres. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This is an open access article distributed under the terms of the Creative Commons Attribution IGO License (https://creativecommons.org/licenses/by/3.0/igo/), which permits unrestricted use, distribution, and reproduction in any medium, provided appropriate credit to the original author(s) and the source is given. Reprints and Permissions Cite this article Cáceres, C., Tsiamis, G., Yuval, B. et al. Joint FAO/IAEA coordinated research project on “use of symbiotic bacteria to reduce mass-rearing costs and increase mating success in selected fruit pests in support of SIT application”. BMC Microbiol 19, 284 (2019) doi:10.1186/s12866-019-1644-y Download citation Published 24 December 2019 DOI https://doi.org/10.1186/s12866-019-1644-y

  • Assessment of the Bacteria community structure across life stages of the Chinese Citrus Fly, Bactrocera minax (Diptera: Tephritidae)
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Awawing Anjwengwo Andongma; Lun Wan; Yong-Cheng Dong; Yu-Lei Wang; Jin He; Chang-Ying Niu

    Symbiotic bacteria play a critical role in insect’s biology. They also offer great opportunities to improve on current pest management techniques. In order to exploit and integrate the roles played by the gut microbiota on pest management programs, a better understanding of the structural organization of the microbial community in the Chinese citrus fly Bactrocera minax is essential. The results revealed a total of 162 OTUs at 97% similarity interval. The dominant bacteria phyla were Proteobacteria, Bacteroidetes, Antinobacteria and Firmicutes, with the Proteobacteria having the highest relative abundance (more than 80% in all life stages). There was also a shift in the dominant OTUs from the early developmental stages to the late developmental stages and adult stages in B. minax. These OTUs related to Klebsiella pneumoniae, Providencia rettgeri and Enterobacter aerogenes, respectively. Six bacteria OTU were shared by all the life stages. These belonged to the Enterobacteriaceae and the Enterococcaceae families. The common bacteria groups shared by all the life stages and other fruit flies could be important targets for further research. This should aim towards realizing how these bacteria affect the biology of the fly and how their relationship could be exploited in the development of sustainable management strategies against fruit flies.

  • Commensal microbiota modulates larval foraging behaviour, development rate and pupal production in Bactrocera tryoni
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Juliano Morimoto; Binh Nguyen; Shabnam T. Tabrizi; Ida Lundbäck; Phillip W. Taylor; Fleur Ponton; Toni A. Chapman

    Commensal microbes can promote survival and growth of developing insects, and have important fitness implications in adulthood. Insect larvae can acquire commensal microbes through two main routes: by vertical acquisition from maternal deposition of microbes on the eggshells and by horizontal acquisition from the environment where the larvae develop. To date, however, little is known about how microbes acquired through these different routes interact to shape insect development. In the present study, we investigated how vertically and horizontally acquired microbiota influence larval foraging behaviour, development time to pupation and pupal production in the Queensland fruit fly (‘Qfly’), Bactrocera tryoni. Both vertically and horizontally acquired microbiota were required to maximise pupal production in Qfly. Moreover, larvae exposed to both vertically and horizontally acquired microbiota pupated sooner than those exposed to no microbiota, or only to horizontally acquired microbiota. Larval foraging behaviour was also influenced by both vertically and horizontally acquired microbiota. Larvae from treatments exposed to neither vertically nor horizontally acquired microbiota spent more time overall on foraging patches than did larvae of other treatments, and most notably had greater preference for diets with extreme protein or sugar compositions. The integrity of the microbiota early in life is important for larval foraging behaviour, development time to pupation, and pupal production in Qflies. These findings highlight the complexity of microbial relations in this species, and provide insights to the importance of exposure to microbial communities during laboratory- or mass-rearing of tephritid fruit flies.

  • Tephritid-microbial interactions to enhance fruit fly performance in sterile insect technique programs
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Ania T. Deutscher; Toni A. Chapman; Lucas A. Shuttleworth; Markus Riegler; Olivia L. Reynolds

    The Sterile Insect Technique (SIT) is being applied for the management of economically important pest fruit flies (Diptera: Tephritidae) in a number of countries worldwide. The success and cost effectiveness of SIT depends upon the ability of mass-reared sterilized male insects to successfully copulate with conspecific wild fertile females when released in the field. We conducted a critical analysis of the literature about the tephritid gut microbiome including the advancement of methods for the identification and characterization of microbiota, particularly next generation sequencing, the impacts of irradiation (to induce sterility of flies) and fruit fly rearing, and the use of probiotics to manipulate the fruit fly gut microbiota. Domestication, mass-rearing, irradiation and handling, as required in SIT, may change the structure of the fruit flies’ gut microbial community compared to that of wild flies under field conditions. Gut microbiota of tephritids are important in their hosts’ development, performance and physiology. Knowledge of how mass-rearing and associated changes of the microbial community impact the functional role of the bacteria and host biology is limited. Probiotics offer potential to encourage a gut microbial community that limits pathogens, and improves the quality of fruit flies. Advances in technologies used to identify and characterize the gut microbiota will continue to expand our understanding of tephritid gut microbial diversity and community composition. Knowledge about the functions of gut microbes will increase through the use of gnotobiotic models, genome sequencing, metagenomics, metatranscriptomics, metabolomics and metaproteomics. The use of probiotics, or manipulation of the gut microbiota, offers significant opportunities to enhance the production of high quality, performing fruit flies in operational SIT programs.

  • Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Georgios A. Kyritsis; Antonios A. Augustinos; Spyridon Ntougias; Nikos T. Papadopoulos; Kostas Bourtzis; Carlos Cáceres

    Insect species have established sophisticated symbiotic associations with diverse groups of microorganisms including bacteria which have been shown to affect several aspects of their biology, physiology, ecology and evolution. In addition, recent studies have shown that insect symbionts, including those localized in the gastrointestinal tract, can be exploited for the enhancement of sterile insect technique (SIT) applications against major insect pests such as the Mediterranean fruit fly (medfly) Ceratitis capitata. We previously showed that Enterobacter sp. AA26 can be used as probiotic supplement in medfly larval diet improving the productivity and accelerating the development of the VIENNA 8 genetic sexing strain (GSS), which is currently used in large scale operational SIT programs worldwide. Enterobacter sp. AA26 was an adequate nutritional source for C. capitata larvae, comprising an effective substitute for brewer’s yeast. Incorporating inactive bacterial cells in the larval diet conferred a number of substantial beneficial effects on medfly biology. The consumption of bacteria-based diet (either as full or partial yeast replacement) resulted in decreased immature stages mortality, accelerated immature development, increased pupal weight, and elongated the survival under stress conditions. Moreover, neither the partial nor the complete replacement of yeast with Enterobacter sp. AA26 had significant impact on adult sex ratio, females’ fecundity, adults’ flight ability and males’ mating competitiveness. The absence of both yeast and Enterobacter sp. AA26 (deprivation of protein source and possible other important nutrients) from the larval diet detrimentally affected the larval development, survival and elongated the immature developmental duration. Enterobacter sp. AA26 dry biomass can fully replace the brewer’s yeast as a protein source in medfly larval diet without any effect on the productivity and the biological quality of reared medfly of VIENNA 8 GSS as assessed by the FAO/IAEA/USDA standard quality control tests. We discuss this finding in the context of mass-rearing and SIT applications.

  • Wolbachia infection in Argentinean populations of Anastrepha fraterculus sp1: preliminary evidence of sex ratio distortion by one of two strains
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Claudia Alejandra Conte; Diego Fernando Segura; Fabian Horacio Milla; Antonios Augustinos; Jorge Luis Cladera; Kostas Bourtzis; Silvia Beatriz Lanzavecchia

    Wolbachia, one of the most abundant taxa of intracellular Alphaproteobacteria, is widespread among arthropods and filarial nematodes. The presence of these maternally inherited bacteria is associated with modifications of host fitness, including a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, host feminization and male-killing. Wolbachia has attracted much interest for its role in biological, ecological and evolutionary processes as well as for its potential use in novel and environmentally-friendly strategies for the control of insect pests and disease vectors including a major agricultural pest, the South American fruit fly, Anastrepha fraterculus Wiedemann (Diptera: Tephritidae). We used wsp, 16S rRNA and a multilocus sequence typing (MLST) scheme including gatB, coxA, hcpA, fbpA, and ftsZ genes to detect and characterize the Wolbachia infection in laboratory strains and wild populations of A. fraterculus from Argentina. Wolbachia was found in all A. fraterculus individuals studied. Nucleotide sequences analysis of wsp gene allowed the identification of two Wolbachia nucleotide variants (named wAfraCast1_A and wAfraCast2_A). After the analysis of 76 individuals, a high prevalence of the wAfraCast2_A variant was found both, in laboratory (82%) and wild populations (95%). MLST analysis identified both Wolbachia genetic variants as sequence type 13. Phylogenetic analysis of concatenated MLST datasets clustered wAfraCast1/2_A in the supergroup A. Paired-crossing experiments among single infected laboratory strains showed a phenotype specifically associated to wAfraCast1_A that includes slight detrimental effects on larval survival, a female-biased sex ratio; suggesting the induction of male-killing phenomena, and a decreased proportion of females producing descendants that appears attributable to the lack of sperm in their spermathecae. We detected and characterized at the molecular level two wsp gene sequence variants of Wolbachia both in laboratory and wild populations of A. fraterculus sp.1 from Argentina. Crossing experiments on singly-infected A. fraterculus strains showed evidence of a male killing-like mechanism potentially associated to the wAfraCast1_A - A. fraterculus interactions. Further mating experiments including antibiotic treatments and the analysis of early and late immature stages of descendants will contribute to our understanding of the phenotypes elicited by the Wolbachia variant wAfraCast1_A in A. fraterculus sp.1.

  • Detection and characterization of bacterial endosymbionts in Southeast Asian tephritid fruit fly populations
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-24
    Elias D. Asimakis; Vangelis Doudoumis; Ashok B. Hadapad; Ramesh S. Hire; Costas Batargias; Changying Niu; Mahfuza Khan; Kostas Bourtzis; George Tsiamis

    Various endosymbiotic bacteria, including Wolbachia of the Alphaproteobacteria, infect a wide range of insects and are capable of inducing reproductive abnormalities to their hosts such as cytoplasmic incompatibility (CI), parthenogenesis, feminization and male-killing. These extended phenotypes can be potentially exploited in enhancing environmentally friendly methods, such as the sterile insect technique (SIT), for controlling natural populations of agricultural pests. The goal of the present study is to investigate the presence of Wolbachia, Spiroplasma, Arsenophonus and Cardinium among Bactrocera, Dacus and Zeugodacus flies of Southeast Asian populations, and to genotype any detected Wolbachia strains. A specific 16S rRNA PCR assay was used to investigate the presence of reproductive parasites in natural populations of nine different tephritid species originating from three Asian countries, Bangladesh, China and India. Wolbachia infections were identified in Bactrocera dorsalis, B. correcta, B. scutellaris and B. zonata, with 12.2–42.9% occurrence, Entomoplasmatales in B. dorsalis, B. correcta, B. scutellaris, B. zonata, Zeugodacus cucurbitae and Z. tau (0.8–14.3%) and Cardinium in B. dorsalis and Z. tau (0.9–5.8%), while none of the species tested, harbored infections with Arsenophonus. Infected populations showed a medium (between 10 and 90%) or low (< 10%) prevalence, ranging from 3 to 80% for Wolbachia, 2 to 33% for Entomoplasmatales and 5 to 45% for Cardinium. Wolbachia and Entomoplasmatales infections were found both in tropical and subtropical populations, the former mostly in India and the latter in various regions of India and Bangladesh. Cardinium infections were identified in both countries but only in subtropical populations. Phylogenetic analysis revealed the presence of Wolbachia with some strains belonging either to supergroup B or supergroup A. Sequence analysis revealed deletions of variable length and nucleotide variation in three Wolbachia genes. Spiroplasma strains were characterized as citri–chrysopicola–mirum and ixodetis strains while the remaining Entomoplasmatales to the Mycoides–Entomoplasmataceae clade. Cardinium strains were characterized as group A, similar to strains infecting Encarsia pergandiella. Our results indicated that in the Southeast natural populations examined, supergroup A Wolbachia strain infections were the most common, followed by Entomoplasmatales and Cardinium. In terms of diversity, most strains of each bacterial genus detected clustered in a common group. Interestingly, the deletions detected in three Wolbachia genes were either new or similar to those of previously identified pseudogenes that were integrated in the host genome indicating putative horizontal gene transfer events in B. dorsalis, B. correcta and B. zonata.

  • Exploring the antimicrobial resistance profiles of WHO critical priority list bacterial strains
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-23
    Benjamin Havenga; Thando Ndlovu; Tanya Clements; Brandon Reyneke; Monique Waso; Wesaal Khan

    The antimicrobial resistance of clinical, environmental and control strains of the WHO “Priority 1: Critical group” organisms, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa to various classes of antibiotics, colistin and surfactin (biosurfactant) was determined. Acinetobacter baumannii was isolated from environmental samples and antibiotic resistance profiling was performed to classify the test organisms [A. baumannii (n = 6), P. aeruginosa (n = 5), E. coli (n = 7) and K. pneumoniae (n = 7)] as multidrug resistant (MDR) or extreme drug resistant (XDR). All the bacterial isolates (n = 25) were screened for colistin resistance and the mobilised colistin resistance (mcr) genes. Biosurfactants produced by Bacillus amyloliquefaciens ST34 were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI–MS). The susceptibility of strains, exhibiting antibiotic and colistin resistance, to the crude surfactin extract (cell-free supernatant) was then determined. Antibiotic resistance profiling classified four A. baumannii (67%), one K. pneumoniae (15%) and one P. aeruginosa (20%) isolate as XDR, with one E. coli (15%) and three K. pneumoniae (43%) strains classified as MDR. Many of the isolates [A. baumannii (25%), E. coli (80%), K. pneumoniae (100%) and P. aeruginosa (100%)] exhibited colistin resistance [minimum inhibitory concentrations (MICs) ≥ 4 mg/L]; however, only one E. coli strain isolated from a clinical environment harboured the mcr-1 gene. UPLC-MS analysis then indicated that the B. amyloliquefaciens ST34 produced C13–16 surfactin analogues, which were identified as Srf1 to Srf5. The crude surfactin extract (10.00 mg/mL) retained antimicrobial activity (100%) against the MDR, XDR and colistin resistant A. baumannii, P. aeruginosa, E. coli and K. pneumoniae strains. Clinical, environmental and control strains of A. baumannii, P. aeruginosa, E. coli and K. pneumoniae exhibiting MDR and XDR profiles and colistin resistance, were susceptible to surfactin analogues, confirming that this lipopeptide shows promise for application in clinical settings.

  • The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-21
    Se Yeon Kim; Mi Hyun Kim; Seung Il Kim; Joo Hee Son; Shukho Kim; Yoo Chul Lee; Minsang Shin; Man Hwan Oh; Je Chul Lee

    Acinetobacter baumannii is an important opportunistic pathogen responsible for various nosocomial infections. The BfmRS two-component system plays a role in pathogenesis and antimicrobial resistance of A. baumannii via regulation of bacterial envelope structures. This study investigated the role of the sensor kinase, BfmS, in localization of outer membrane protein A (OmpA) in the outer membrane and production of outer membrane vesicles (OMVs) using wild-type A. baumannii ATCC 17978, ΔbfmS mutant, and bfmS-complemented strains. The ΔbfmS mutant showed hypermucoid phenotype in the culture plates, growth retardation under static culture conditions, and reduced susceptibility to aztreonam and colistin compared to the wild-type strain. The ΔbfmS mutant produced less OmpA in the outer membrane but released more OmpA via OMVs than the wild-type strain, even though expression of ompA and its protein production were not different between the two strains. The ΔbfmS mutant produced 2.35 times more OMV particles and 4.46 times more OMV proteins than the wild-type stain. The ΔbfmS mutant OMVs were more cytotoxic towards A549 cells than wild-type strain OMVs. The present study demonstrates that BfmS controls production of OMVs in A. baumannii. Moreover, BfmS negatively regulates antimicrobial resistance of A. baumannii and OMV-mediated host cell cytotoxicity. Our results indicate that BfmS negatively controls the pathogenic traits of A. baumannii via cell envelope structures and OMV production.

  • Pathogenic fungi-induced susceptibility is mitigated by mutual Lactobacillus plantarum in the Drosophila melanogaster model
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-21
    Wanzhen Su; Jialin Liu; Peng Bai; Baocang Ma; Wei Liu

    Since animals frequently encounter a variety of harmful fungi in nature, their ability to develop sophisticated anti-fungal strategies allows them to flourish across the globe. Extensive studies have highlighted the significant involvement of indigenous microbial communities in human health. However, the daunting diversity of mammalian microbiota and host genetic complexity are major obstacles to our understanding of these intricate links between microbiota components, host immune genotype, and disease phenotype. In this study, we sought to develop a bacterium-fungus-Drosophila model to systematically evaluate the anti-fungal effects of commensal bacteria. We isolated the pathogenic fungal strain, Diaporthe FY, which was detrimental to the survival and development of Drosophila upon infection. Using Drosophila as a model system, Drosophila-associated Lactobacillus plantarum functioned as a probiotic, and protected the flies from mortality induced by Diaporthe FY. Our results show that L. plantarum hindered the growth of Diaporthe FY in vitro, and decreased the mortality rate of Diaporthe FY-infected flies in vivo, consequently mitigating the toxicity of Diaporthe FY to the hosts. Additionally, the presence of L. plantarum overrode the avoidance of oviposition on Diaporthe FY-associated substrates. Diaporthe FY was identified as a potential Drosophila pathogen. Commensal L. plantarum mitigated the susceptibility of Drosophila to pathogenic fungi, providing insight into the natural interplay between commensal and pathogenic microbial communities that contribute to animal health and pathogenesis.

  • The dishwasher rubber seal acts as a reservoir of bacteria in the home environment
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-19
    Jerneja Zupančič; Martina Turk; Miha Črnigoj; Jerneja Ambrožič Avguštin; Nina Gunde-Cimerman

    In modern lifestyles, people make their everyday tasks easier by using household appliances, for example dishwashers. Previous studies showed massive contamination of dishwasher rubber seals with fungi, thus bacterial community, able to survive under harsh conditions, remain undetermined. Bacteria that colonise the extreme environment of household dishwasher rubber seals were investigated using cultivation-dependent and metagenomic approaches. All bacterial isolates were tested for resistance to seven selected antibiotics. Same time bacterial diversity of tap water, connected to the dishwashers was investigated. All 30 dishwashers investigated were colonised by various bacteria. Cultivation approaches resulted in 632 bacterial isolates in total, belonging to four phyla, eight classes, 40 genera and 74 species. The majority were Gram-positive, as solely Firmicutes (dominated by the Bacillus cereus group) and Actinobacteria. Gammaproteobacteria were primarily represented by Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Escherichia coli. Metagenomic assessment of the bacterial biodiversity of the dishwasher rubber seals confirmed the predominance of Gram-positive bacteria, as primarily Actinobacteria, followed by Proteobacteria dominated by Gammaproteobacteria, and by pathogenic species such as Escherichia sp., Acinetobacter baumannii, Pseudomonas sp., Stenotrophomonas maltophilia, and Enterobacter sp.. Metagenomic assessment of bacterial biodiversity in the tap water connected to dishwashers revealed predominance of Gram-negative bacteria, in particular Proteobacteria, mainly represented by Tepidimonas sp.. Actinobacteria showed low numbers while no Firmicutes were detected in the tap water. The bacterial diversity of tap water was also lower, 23 genera compared to 39 genera on dishwasher rubber seals. Only 13 out of 49 genera identified by metagenomics approach was found in both environments, of those Gordonia was enriched while half of 13 genera were depleted in dishwashers compared to tap water. These data indicate that colonisation of dishwasher rubber seals probably depends primarily on the bacterial input from the dirty vessels, and much less on the bacteria in the tap water. Based on the antibiotic resistance data, the dishwasher rubber seal bacterial isolates do not represent a serious threat for the spread of antibiotic resistance into the household environment. Nevertheless dishwashers cannot be ignored as potential sources of human infections, in particular for immuno-compromised individuals.

  • Genetic diversity of Listeria monocytogenes strains in ruminant abortion and rhombencephalitis cases in comparison with the natural environment
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-18
    Bojan Papić; Mateja Pate; Benjamin Félix; Darja Kušar

    Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both animals and humans. Here, multilocus sequence typing (MLST) was used to characterize the genetic diversity of Listeria monocytogenes strains isolated from the natural environment and animal clinical cases in Europe. The prevalence of clonal complexes (CCs) obtained was compared according to (i) the origin of isolation – clinical cases vs. natural environment – and (ii) the clinical form of animal listeriosis – rhombencephalitis vs. abortion. To this aim, two datasets were constructed. The clinical dataset consisted of 350 animal clinical isolates originating from France and Slovenia and supplemented with isolates from Switzerland and Great Britain. The natural environment dataset consisted of 253 isolates from the natural environment originating from Slovenia and supplemented with isolates from nine other European countries. For the clinical cases, CC1, CC4-CC217 and CC412 were the most prevalent in rhombencephalitis and CC1, CC37 and CC4-CC217 in abortion. The hypervirulent CC1 and CC4-CC217 prevailed in both datasets. These results indicated that livestock is constantly exposed to hypervirulent CCs. CC1 was significantly associated with a clinical origin, whereas CC9, CC29 and CC14 were associated with the natural environment. CC1 was predominant among rhombencephalitis cases both in cattle and small ruminants, and its prevalence did not differ significantly between these two groups. A novel association of CC37 and CC6 with abortion cases was revealed. Here, we show that CC1 and CC4-CC217 are prevalent in isolates of environmental and animal clinical origin, suggesting that ruminants are frequently exposed to hypervirulent CCs. The presence of CC4 in two mastitis cases calls for further attention due to direct threat to the consumer. We showed several associations between CCs and the origin of isolation or clinical form of listeriosis, e.g. CC37 and CC6 with abortion. This study improves our understanding of the population structure of L. monocytogenes isolates from the natural environment and animal clinical cases. Moreover, it provides a basis for future studies aiming to determine the underlying mechanisms of phenotypic traits of interest.

  • ShiF acts as an auxiliary factor of aerobactin secretion in meningitis Escherichia coli strain S88
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-17
    Mathieu Genuini; Philippe Bidet; Jean-François Benoist; Dimitri Schlemmer; Chloé Lemaitre; André Birgy; Stéphane Bonacorsi

    The neonatal meningitis E. coli (NMEC) strain S88 carries a ColV plasmid named pS88 which is involved in meningeal virulence. Transcriptional analysis of pS88 in human serum revealed a strong upregulation of an ORF of unknown function: shiF, which is adjacent to the operon encoding the siderophore aerobactin. The aim of this work is to investigate the role of shiF in aerobactin production in strain S88. Study of the prevalence of shiF and aerobactin operon in a collection of 100 extra-intestinal pathogenic E. coli strains (ExPEC) and 50 whole genome-sequenced E. coli strains revealed the colocalization of these two genes for 98% of the aerobactin positive strains. We used Datsenko and Wanner’s method to delete shiF in two S88 mutants. A cross-feeding assay showed that these mutants were able to excrete aerobactin meaning that shiF is dispensable for aerobactin excretion. Our growth assays revealed that the shiF-deleted mutants grew significantly slower than the wild-type strain S88 in iron-depleted medium with a decrease of maximum growth rates of 23 and 28% (p < 0.05). Using Liquid Chromatography-Mass Spectrometry, we identified and quantified siderophores in the supernatants of S88 and its shiF deleted mutants after growth in iron-depleted medium and found that these mutants secreted significantly less aerobactin than S88 (− 52% and - 49%, p < 0.001). ShiF is physically and functionally linked to aerobactin. It provides an advantage to E. coli S88 under iron-limiting conditions by increasing aerobactin secretion and may thus act as an auxiliary virulence factor.

  • Investigation of the molecular characteristics of Brucella isolates from Guangxi Province, China
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-16
    Zhi-guo Liu; Miao Wang; Hong-yan Zhao; Dong-ri Piao; Hai Jiang; Zhen-jun Li

    Human brucellosis has become a severe public health problem in China’s Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates. A total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012–2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group. Our investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region.

  • Antimicrobial efficacy and toxicity of novel CAMPs against P. aeruginosa infection in a murine skin wound infection model
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-16
    Ming Yang; Chunye Zhang; Sarah A. Hansen; William J. Mitchell; Michael Z. Zhang; Shuping Zhang

    Treatment of P. aeruginosa wound infection is challenging due to its inherent and acquired resistance to many conventional antibiotics. Cationic antimicrobial peptides (CAMPs) with distinct modes of antimicrobial action have been considered as the next-generation therapeutic agents. In the present study, a murine skin surgical wound infection model was used to evaluate the in vivo toxicity and efficacy of two newly designed antimicrobial peptides (CAMP-A and CAMP-B), as chemotherapeutic agents to combat P. aeruginosa infection. In the first trial, topical application of CAMPs on the wounds at a dose equivalent to 4 × MIC for 7 consecutive days did not cause any significant changes in the physical activities, hematologic and plasma biochemical parameters, or histology of systemic organs of the treated mice. Daily treatment of infected wounds with CAMP-A and CAMP-B for 5 days at a dose equivalent to 2× MIC resulted in a significant reduction in wound bacterial burden (CAMP-A: 4.3 log10CFU/g of tissue and CAMP-B: 5.8 log10CFU/g of tissue), compared to that of the mock-treated group (8.1 log10CFU/g of tissue). Treatment with CAMPs significantly promoted wound closure and induced epidermal cell proliferation. Topical application of CAMP-A on wounds completely prevented systemic dissemination of P. aeruginosa while CAMP-B blocked systemic infection in 67% of mice and delayed the onset of systemic infection by at least 2 days in the rest of the mice (33%). In a second trial, daily application of CAMP-A at higher doses (5× MIC and 50× MIC) didn’t show any significant toxic effect on mice and the treatments with CAMP-A further reduced wound bacterial burden (5× MIC: 4.5 log10CFU/g of tissue and 50× MIC: 3.8 log10CFU/g of tissue). The data collectively indicated that CAMPs significantly reduced wound bacterial load, promoted wound healing, and prevented hepatic dissemination. CAMP-A is a promising alternative to commonly used antibiotics to treat P. aeruginosa skin infection.

  • Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-16
    Fatemah Al-Kandari; Rabeah Al-Temaimi; Arnoud H. M. van Vliet; Martin J. Woodward

    Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thyr). The phenotype was stable after multiple sub-cultures without thymol. Cell morphology studies by scanning electron microscopy (SEM) suggest that thymol renders bacterial cell membranes permeable and disrupts cellular integrity. 1H Nuclear magnetic resonance (NMR) data showed an increase in lactate and the lactic acid family amino acids in the wild type and JM109-Thyr in the presence of thymol, indicating a shift from aerobic respiration to fermentation. Sequencing of JM109-Thyr defined multiple mutations including a stop mutation in the acrR gene resulting in a truncation of the repressor of the AcrAB efflux pump. AcrAB is a multiprotein complex traversing the cytoplasmic and outer membrane, and is involved in antibiotic clearance. Our data suggests that thymol tolerance in E. coli induces morphological, metabolic and genetic changes to adapt to thymol antimicrobial activity.

  • Role of membrane compartment occupied by Can1 (MCC) and eisosome subdomains in plant pathogenicity of the necrotrophic fungus Alternaria brassicicola
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-16
    Justine Colou; Guillaume Quang N’Guyen; Ophélie Dubreu; Kévin Fontaine; Anthony Kwasiborski; Franck Bastide; Florence Manero; Bruno Hamon; Sophie Aligon; Philippe Simoneau; Thomas Guillemette

    MCC/eisosomes are membrane microdomains that have been proposed to participate in the plasma membrane function in particular by regulating the homeostasis of lipids, promoting the recruitment of specific proteins and acting as provider of membrane reservoirs. Here we showed that several potential MCC/eisosomal protein encoding genes in the necrotrophic fungus A. brassicicola were overexpressed when germinated spores were exposed to antimicrobial defence compounds, osmotic and hydric stresses, which are major constraints encountered by the fungus during the plant colonization process. Mutants deficient for key MCC/eisosome components did not exhibit any enhanced susceptibility to phytoalexins and to applied stress conditions compared to the reference strain, except for a slight hypersensitivity of the ∆∆abpil1a-abpil1b strain to 2 M sorbitol. Depending on the considered mutants, we showed that the leaf and silique colonization processes were impaired by comparison to the wild-type, and assumed that these defects in aggressiveness were probably caused by a reduced appressorium formation rate. This is the first study on the role of MCC/eisosomes in the pathogenic process of a plant pathogenic fungus. A link between these membrane domains and the fungus ability to form functional penetration structures was shown, providing new potential directions for plant disease control strategies.

  • Whole-genome sequence-based comparison and profiling of virulence-associated genes of Bacillus cereus group isolates from diverse sources in Japan
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-16
    Akiko Okutani; Satoshi Inoue; Akira Noguchi; Yoshihiro Kaku; Shigeru Morikawa

    The complete genome sequences of 44 Bacillus cereus group isolates collected from diverse sources in Japan were analyzed to determine their genetic backgrounds and diversity levels in Japan. Multilocus sequence typing (MLST) and core-genome single-nucleotide polymorphism (SNP) typing data from whole-genome sequences were analyzed to determine genetic diversity levels. Virulence-associated gene profiles were also used to evaluate the genetic backgrounds and relationships among the isolates. The 44 B. cereus group isolates, including soil- and animal-derived isolates and isolates recovered from hospitalized patients and food poisoning cases, were genotyped by MLST and core-genome SNP typing. Genetic variation among the isolates was identified by the MLST and core-genome SNP phylogeny comparison against reference strains from countries outside of Japan. Exploratory principal component analysis and nonmetric multidimensional scaling (NMDS) analyses were used to assess the genetic similarities among the isolates using gene presence and absence information and isolate origins as the metadata. A significant correlation was seen between the principal components and the presence of genes encoding hemolysin BL and emetic genetic determinants in B. cereus, and the capsule proteins in B. anthracis. NMDS showed that the cluster of soil isolates overlapped with the cluster comprising animal-derived and clinical isolates. Molecular and epidemiological analyses of B. cereus group isolates in Japan suggest that the soil- and animal-derived bacteria from our study are not a significant risk to human health. However, because several of the clinical isolates share close genetic relationships with the environmental isolates, both molecular and epidemiological surveillance studies could be used effectively to estimate virulence in these important pathogens.

  • Comparative study on the effects of different feeding habits and diets on intestinal microbiota in Acipenser baeri Brandt and Huso huso
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-16
    Guanling Xu; Wei Xing; Tieliang Li; Min Xue; Zhihong Ma; Na Jiang; Lin Luo

    Siberian sturgeon (Acipenser baeri Brandt) and Beluga sturgeon (Huso huso) are two important commercial fish in China, and the feeding habits of them are very different. Diets and feeding habits are two significant factors to affect the gastrointestinal microbiota in fish. The intestinal microbiota has been reported to play a key role in nutrition and immunity. However, it is rarely reported about the relationship between the intestinal microbiota and feeding habits/diets on different Acipenseridae fish. This study is to comparative analysis of gut microbial community in Siberian sturgeon and Beluga sturgeon fed with the same diet/Beluga sturgeon fed with different diets in order to determine the effects of different feeding habits/diets on the fish intestinal microbiota. According to the experimental objectives, BL and BH groups were Beluga sturgeon (Huso huso) fed with low fishmeal diet and high fishmeal diet, respectively. SH group represented Siberian sturgeon (Acipenser baeri Brandt) fed with the same diet as BH group. After 16 weeks feeding trial, the intestinal microbiota was examined by 16S rRNA high-throughput sequencing technology. On the phylum level, Proteobacteria and Bacteroidetes were significantly higher in BL group than BH group, and Cyanobacteria showed the opposite trend. Compared with BH group, Proteobacteria and Firmicutes were significantly increased in SH group, whereas Cyanobacteria were clearly decreased. At the genus level, Pseudomonas and Citrobacter in BL group were significantly higher comparing with BH group, while Bacillus, Luteibacter, Staphylococcus and Oceanobacillus was lower in BH group than SH group. Alpha and beta diversities indicated that the intestinal microflora were significant difference between Siberian sturgeon and Beluga sturgeon when they fed with the same diet. Meanwhile, Beluga sturgeon fed with low fishmeal diet can increase the species diversity of intestinal microbiota than it fed high fishmeal diet. Therefore, feeding habits clearly affected the gastrointestinal microbiota of sturgeons. Moreover, the impact of changes in food on the gut microbiota of sturgeons should be taken into consideration during the process of sturgeon aquaculture.

  • The efficacy of lyticase and β-glucosidase enzymes on biofilm degradation of Pseudomonas aeruginosa strains with different gene profiles
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-12
    Maryam Banar; Mohammad Emaneini; Reza Beigverdi; Rima Fanaei Pirlar; Narges Node Farahani; Willem B. van Leeuwen; Fereshteh Jabalameli

    Pseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and β-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated. This study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa. In contrast to the lyticase enzyme, β-glucosidase reduced the ceftazidime MBECs significantly (P < 0.05). Both enzymes had no cytotoxic effect on the A-549 human lung carcinoma epithelial cell lines and A-431 human epidermoid carcinoma cell lines. Considering the characteristics of the β-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed.

  • iTRAQ-based proteomic analysis reveals the mechanisms of Botrytis cinerea controlled with Wuyiencin
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-11
    Liming Shi; Beibei Ge; Jinzi Wang; Binghua Liu; Jinjin Ma; Qiuhe Wei; Kecheng Zhang

    Grey mould is an important plant disease worldwide, caused by Botrytis cinerea, resulting in serious economic loss. Wuyiencin, a low toxicity, high efficiency, and broad-spectrum agricultural antibiotic, has been demonstrated effectiveness against B. cinerea. Wuyiencin treatment inhibited growth and sporulation of B. cinerea, specifically altering hypha morphology and intracellular structures. These changes were accompanied by differential expression (fold change > 2.0) of 316 proteins identified by iTRAQ-labelling LC-MS/MS analysis (P < 0.05). Up-regulation of 14 proteins, including carbohydrate metabolism proteins and cell wall stabilization proteins, was validated by parallel reaction monitoring (PRM). Down-regulation of 13 proteins was validated by PRM, including regulators of energy metabolism, nucleotide/protein synthesis, and the biosynthesis of mediators of plant stress and decay. Our results confirm the inhibitory biological effects of wuyiencin on B. cinereal and elaborate on the differentially expressed proteins and associated pathways implicated in the capacity of wuyiencin to debilitate the growth and pathogenicity of grey mould. This study provides validated candidates for further targeted exploration with the goal of optimizing wuyiencin as a safe, low-toxicity agent for biological control.

  • The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-09
    Luke V. Blakeway; Aimee Tan; Joseph A. Jurcisek; Lauren O. Bakaletz; John M. Atack; Ian R. Peak; Kate L. Seib

    Moraxella catarrhalis is a leading cause of otitis media (OM) and chronic obstructive pulmonary disease (COPD). M. catarrhalis contains a Type III DNA adenine methyltransferase (ModM) that is phase-variably expressed (i.e., its expression is subject to random, reversible ON/OFF switching). ModM has six target recognition domain alleles (modM1–6), and we have previously shown that modM2 is the predominant allele, while modM3 is associated with OM. Phase-variable DNA methyltransferases mediate epigenetic regulation and modulate pathogenesis in several bacteria. ModM2 of M. catarrhalis regulates the expression of a phasevarion containing genes important for colonization and infection. Here we describe the phase-variable expression of modM3, the ModM3 methylation site and the suite of genes regulated within the ModM3 phasevarion. Phase-variable expression of modM3, mediated by variation in length of a 5′-(CAAC)n-3′ tetranucleotide repeat tract in the open reading frame was demonstrated in M. catarrhalis strain CCRI-195ME with GeneScan fragment length analysis and western immunoblot. We determined that ModM3 is an active N6-adenine methyltransferase that methylates the sequence 5′-ACm6ATC-3′. Methylation was detected at all 4446 5′-ACATC-3′ sites in the genome when ModM3 is expressed. RNASeq analysis identified 31 genes that are differentially expressed between modM3 ON and OFF variants, including five genes that are involved in the response to oxidative and nitrosative stress, with potential roles in biofilm formation and survival in anaerobic environments. An in vivo chinchilla (Chinchilla lanigera) model of otitis media demonstrated that transbullar challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to a modM3 ON variant. In addition, co-infection experiments with NTHi and M. catarrhalis modM3 ON or modM3 OFF variants revealed that phase variation of modM3 altered survival of NTHi in the middle ear during early and late stage infection. Phase variation of ModM3 epigenetically regulates the expression of a phasevarion containing multiple genes that are potentially important in the progression of otitis media.

  • Severe gut microbiota dysbiosis caused by malnourishment can be partly restored during 3 weeks of refeeding with fortified corn-soy-blend in a piglet model of childhood malnutrition
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-10
    Bingfeng Leng; Maria B. Sørensen; Witold Kot; Thomas Thymann; Lukasz Krych; Dennis S. Nielsen

    Childhood malnutrition is a global health challenge associated with multiple adverse consequences, including delayed maturation of the gut microbiota (GM) which might induce long-term immune dysfunction and stunting. To understand GM dynamics during malnutrition and subsequent re-feeding, we used a piglet model with a malnutrition-induced phenotype similar to humans. Piglets were weaned at the age of 4 weeks, fed a nutritionally optimal diet for 1 week post-weaning before being fed a pure maize diet for 7 weeks to induce symptoms of malnutrition. After malnourishment, the piglets were re-fed using different regimes all based on general food aid products, namely Corn-Soy blend (CSB) fortified with phosphorus (CSB+), CSB fortified with phosphorus and skim milk powder (CSB++) and CSB fortified with phosphorus and added whey permeate (CSB + P). Malnourishment had profound impact on the GM of the piglets leading to a less diverse GM dominated especially by Akkermansia spp. as determined by 16S rRNA gene amplicon sequencing. All three re-feeding regimes partly restored GM, leading to a more diverse GM compositionally closer to that of well-nourished piglets. This effect was even more pronounced for CSB++ compared to CSB+ and CSB + P. The GM of piglets were profoundly disturbed by malnourishment resulting in significantly increased abundance of Akkermansia spp. CSB++ may have superior effect on recovering GM diversity compared to the two other food aid products used in this study.

  • Diversity and bioactivities of fungal endophytes from Distylium chinense, a rare waterlogging tolerant plant endemic to the Three Gorges Reservoir
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-10
    Xiaoxiang Duan; Fangfang Xu; Dan Qin; Tiancong Gao; Weiyun Shen; Shihao Zuo; Baohong Yu; Jieru Xu; Yajun Peng; Jinyan Dong

    The present study involves diversity and biological activities of the endophytic fungal community from Distylium chinense, a rare waterlogging tolerant plant endemic to the Three Gorges Reservoir. This study has been conducted hypothesizing that the microbial communities in the TGR area would contribute to the host plant tolerating a range of abiotic stress such as summer flooding, infertility, drought, salinity and soil erosion etc., and they may produce new metabolites, which may possess plentiful bioactive property, especially antioxidant activity. Therefore in the current study, the antioxidant, antimicrobial and anticancer activities of 154 endophytes recovered from D. chinense have been investigated. Furthermore, the active metabolites of the most broad-spectrum bioactive strain have also been studied. A total of 154 fungal endophytes were isolated from roots and stems. They were categorized into 30 morphotypes based on cultural characteristics and were affiliated with 27 different taxa. Among these, the most abundant fungal orders included Diaporthales (34.4%) and Botryosphaeriales (30.5%), which were predominantly represented by the species Phomopsis sp. (24.7%) and Neofusicoccum parvum (23.4%). Fermentation extracts were evaluated, screening for antioxidant, antimicrobial and anticancer activities. Among the 154 isolates tested, 99 (64.3%) displayed significant antioxidant activity, 153 (99.4%) exhibited inclusive antimicrobial activity against at least one tested microorganism and 27 (17.5%) showed exclusive anticancer activity against one or more cancer cell lines. Specifically, the crude extract of Irpex lacteus DR10–1 exhibited note-worthy bioactivities. Further chemical investigation on DR10–1 strain resulted in the isolation and identification of two known bioactive metabolites, indole-3-carboxylic acid (1) and indole-3-carboxaldehyde (2), indicating their potential roles in plant growth promotion and human medicinal value. These results indicated that diverse endophytic fungal population inhabits D. chinense. One of the fungal isolate DR10–1 (Irpex lacteus) exhibited significant antioxidant, antimicrobial and anticancer potential. Further, its active secondary metabolites 1 and 2 also showed antioxidant, antimicrobial and anticancer potential.

  • CtcS, a MarR family regulator, regulates chlortetracycline biosynthesis
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-10
    Lingxin Kong; Jia Liu; Xiaoqing Zheng; Zixin Deng; Delin You

    Chlortetracycline (CTC) is one of the commercially important tetracyclines (TCs) family product and is mainly produced by Streptomyces. CTC is still in a great demand due to its broad-spectrum activity against pathogens. Engineering transcriptional control allows the cell to allocate its valuable resources towards protein production and provides an important method for the build-up of desired metabolites. Despite extensive efforts concerning transcriptional regulation for increasing the productivities of TCs, the regulatory mechanisms of the CTC biosynthesis remain poorly understood. In this study, the possible regulatory function of CtcS, a potential member of MarR (multiple antibiotic resistance regulator) family of transcriptional regulators in S. aureofaciens F3, was demonstrated. Knockdown of ctcS altered the transcription of several biosynthesis-related genes and reduced the production of tetracycline (TC) and CTC, without obvious effect on morphological differentiation and cell growth. Especially, CtcS directly repressed the transcription of the adjacent divergent gene ctcR (which encodes a putative TC resistance efflux protein). A CtcS-binding site was identified within the promoter region of ctcR by DNase I footprinting and an inverted repeat (5′-CTTGTC-3′) composed of two 6-nt half sites in the protected region was found. Moreover, both CTC and TC could attenuate the binding activity of CtcS with target DNA. ctcS regulated the production of TC and CTC in S. aureofaciens F3 and the overexpression of it could be used as a simple approach for the construction of engineering strain with higher productivity. Meanwhile, CtcS was characterized as a TC- and CTC-responsive MarR family regulator. This study provides a previously unrecognized function of CtcS and will benefit the research on the regulatory machinery of the MarR family regulators.

  • Change of the duodenal mucosa-associated microbiota is related to intestinal metaplasia
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-09
    Jian Gong; Lixiang Li; Xiuli Zuo; Yanqing Li

    In this study, we aimed to investigate the characteristics of the duodenal mucosal microbiota of patients with intestinal metaplasia (IM) and compare it with those of the gastric mucosal microbiota. We collected the duodenal and gastric mucosal samples from 10 adult patients with IM and 10 healthy controls (HC). The V3-V4 region of the bacterial 16S rRNA gene was examined by high throughput sequencing method. The diversity of the HC duodenal microbiota was higher than that of IM patient based on the Shannon and Simpson index while the Chao indices of IM duodenal mucosal microbiota was significantly higher than that of gastric mucosal microbiota of patients with IM. There was a marked difference in the duodenal microbiota structure between patients with IM and HC (ANOSIM, R = 1, P = 0.001). We also found that the Helicobacter pylori infection in gastric mucosa did not influence the structure of duodenal mucosal microbiota. The gastric mucosal microbiota structure significantly differed between patients with IM and HC who were H. pylori-negative (ANOSIM, R = 0.452, P = 0.042) or H. pylori-positive (ANOSIM, R = 0.548, P = 0.003), respectively. For duodenal mucosal microbiota, genera Lactococcus, Flavobacterium, Psychrobacter, Mysroides, Enhydrobacter, Streptococcus, and Leuconostoc were enriched in patients with IM. In contrast, genera Bacillus, Solibacillus, Lysinibacillus, Exiguobacterium, Oceanobacillus, and Paenibacillus were enriched in HC. A marked dysbiosis duodenal mucosal microbiota in patients with IM was observed, and this dysbiosis might be responsible for IM pathogenesis.

  • Antiviral activity of mitoxantrone dihydrochloride against human herpes simplex virus mediated by suppression of the viral immediate early genes
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-07
    Qiang Huang; Jue Hou; Peng Yang; Jun Yan; Xiaoliang Yu; Ying Zhuo; Sudan He; Feng Xu

    HSV-1 is a common pathogen that infects 50–90% of the human population worldwide. HSV-1 causes numerous infection-related diseases, some of which are severely life-threatening. There are antiviral medications with activity against HSV-1. However, with the emergence of drug-resistant mutant strains of HSV-1, there is an urgent need to develop new effective anti-HSV-1 agents. Therefore, we screened a chemical library of approximately 1500 compounds to identify inhibitors of HSV-1-induced toxicity for further drug development. Moreover, we performed several experiments, including western blot analysis, Q-PCR analysis and luciferase activity assay, to explore the antiviral mechanism of the candidates. Here, we identified a small molecule, mitoxantrone dihydrochloride, with potency against HSV-1-induced toxicity. Furthermore, the viral titers and expression levels of HSV-1 viral proteins were potently reduced by the presence of MD in many cell lines. Using Q-PCR analysis, we found that MD efficiently reduced the transcription of viral genes that are essential for DNA synthesis, namely, UL5, UL9, UL29, UL30, UL42 and UL52. Notably, MD also significantly inhibited the transcription of the immediate early genes ICP0, ICP22, ICP27 and ICP47, all of which are required for the expression of early and late viral gene products. Using immunofluorescence and western blot analysis, we found that the antiviral effect of MD was independent of the activation of the NF-κB and MAPK pathways. Furthermore, we found that the reduction in the transcription of viral immediate early genes was not related to the promoter activities of ICP0. Therefore, the identification of compound MD as an inhibitor of toxicity induced by HSV-1 highlights its potential use in the development of novel anti-HSV-1 drugs.

  • Household cockroaches carry CTX-M-15-, OXA-48- and NDM-1-producing enterobacteria, and share beta-lactam resistance determinants with humans
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-04
    Noah Obeng-Nkrumah; Appiah-Korang Labi; Harriet Blankson; Georgina Awuah-Mensah; Daniel Oduro-Mensah; Judelove Anum; James Teye; Solomon Dzidzornu Kwashie; Evariste Bako; Patrick Ferdinand Ayeh-Kumi; Richard Asmah

    This study was designed to investigate whether household cockroaches harbor cephalosporin-resistant enterobacteria that share resistance determinants with human inhabitants. From February through July 2016, whole cockroach homogenates and human fecal samples from 100 households were cultured for cephalosporin-resistant enterobacteria (CRe). The CRe were examined for plasmid-mediated AmpC, ESBL, and carbapenemase genes; antibiotic susceptibility patterns; and conjugative transfer of antibiotic resistance mechanisms. Clonal associations between CRe were determined by multi-locus sequence typing (MLST). Twenty CRe were recovered from whole cockroach homogenates from 15 households. The prevalence of households with cockroaches that harbored CRe, AmpC- (based on phenotype, with no identifiable blaAmpC genes), ESBL-, and carbapenemase-producers were 15, 4, 5%(2 blaCTX-M-15/TEM-1; 1 blaCTX-M-15/TEM-4; 1 blaTEM-24; 1 blaSHV-4) and 3%(2 blaNDM-1 genes and 1 blaOXA-48 gene), respectively. Overall, 20 CRe were recovered from 61 fecal samples of inhabitants from all 15 households that had cockroach samples positive for CRe. Of these, 5CRe (1 per household) were positive for ESBLs (blaTEM-24, blaTEM-14, blaCTX-M-15/TEM-4, blaSHV-3, blaCTX-M-15/TEM-1) and none carried AmpCs or carbapenemases. From 4% of households, the pair of cockroach and human CRe shared the same sequence type (ST), clonal complex (CC), antibiogram, and conjugable bla gene sequence (house 34, E. coli ST9/CC20-blaTEM-4; house 37, E. coli ST44/CC10-blaCTX-15/TEM-4; house 41, E. coli ST443/CC205-blaCTX-15/TEM-1; house 49, K. pneumoniae ST231/CC131-blaSHV-13). The findings provide evidence that household cockroaches may carry CTX-M-15-, OXA-48- and NDM-1-producers, and share clonal relationship and beta-lactam resistance determinants with humans.

  • Spatial structure of the microbiome in the gut of Pomacea canaliculata
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-05
    Lan-Hua Li; Shan Lv; Yan Lu; Ding-Qi Bi; Yun-Hai Guo; Jia-Tong Wu; Zhi-Yuan Yue; Guang-Yao Mao; Zhong-Xin Guo; Yi Zhang; Yun-Feng Tang

    Gut microbes can contribute to their hosts in food digestion, nutrient absorption, and inhibiting the growth of pathogens. However, only limited studies have focused on the gut microbiota of freshwater snails. Pomacea canaliculata is considered one of the worst invasive alien species in the world. Elucidating the diversity and composition of the microbiota in the gut of P. canaliculata snails may be helpful for better understanding the widespread invasion of this snail species. In this study, the buccal masses, stomachs, and intestines were isolated from seven P. canaliculata snails. The diversity and composition of the microbiota in the three gut sections were then investigated based on high-throughput Illumina sequencing targeting the V3-V4 regions of the 16S rRNA gene. The diversity of the microbiota was highest in the intestine but lowest in the buccal mass. A total of 29 phyla and 111 genera of bacteria were identified in all of the samples. In general, Ochrobactrum, a genus of putative cellulose-degrading bacteria, was the most abundant (overall relative abundance: 13.6%), followed by Sediminibacterium (9.7%), Desulfovibrio (7.8%), an unclassified genus in the family Aeromonadaceae (5.4%), and Cloacibacterium (5.4%). The composition of the microbiota was diverse among the different gut sections. Ochrobactrum (relative abundance: 23.15% ± 7.92%) and Sediminibacterium (16.95 ± 5.70%) were most abundant in the stomach, an unclassified genus in the family Porphyromonadaceae (14.28 ± 7.29%) and Leptotrichia (8.70 ± 4.46%) were highest in the buccal mass, and two genera in the families Aeromonadaceae (7.55 ± 4.53%) and Mollicutes (13.47 ± 13.03%) were highest in the intestine. The diversity and composition of the microbiome vary among different gut sections of P. canaliculata snails. Putative cellulose-degrading bacteria are enriched in the gut of P. canaliculata.

  • Genomic and evolutionary features of two AHPND positive Vibrio parahaemolyticus strains isolated from shrimp (Penaeus monodon) of south-west Bangladesh
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-03
    Shawon Ahmmed; Md. Abdullah-Al-Kamran Khan; Md. Mostavi Enan Eshik; Nusrat Jahan Punom; Abul Bashar Mir Md. Khademul Islam; Mohammad Shamsur Rahman

    Due to its rapid lethal effect in the early development stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) has been causing great economic losses, since its first outbreak in southeast China in 2009. Vibrio parahaemolyticus, carrying the pirA and pirB toxin genes is known to cause AHPND in shrimp. The overall objective of this study was to sequence the whole genome of AHPND positive V. parahaemolyticus strains isolated from shrimp (Peneaus monodon) of the south-west region of Bangladesh in 2016 and 2017 and characterize the genomic features and emergence pattern of this marine pathogen. Two targeted AHPND positive V. parahaemolyticus strains were confirmed using PCR with 16S rRNA, ldh, AP3 and AP4 primers. The assembled genomes of strain MSR16 and MSR17 were comprised of a total of 5,393,740 bp and 5,241,592 bp, respectively. From annotation, several virulence genes involved in chemotaxis and motility, EPS type II secretion system, Type III secretion system-1 (T3SS-1) and its secreted effectors, thermolabile hemolysin were found in both strains. Importantly, the ~ 69 kb plasmid was identified in both MSR16 and MSR17 strains containing the two toxin genes pirA and pirB. Antibiotic resistance genes were predicted against β-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains. The findings of this research may facilitate the tracking of pathogenic and/or antibiotic-resistant V. parahaemolyticus isolates between production sites, and the identification of candidate strains for the production of vaccines as an aid to control of this devastating disease. Also, the emergence pattern of this pathogen can be highlighted to determine the characteristic differences of other strains found all over the world.

  • Prevalence of fluoroquinolone resistance and mutations in the gyrA, parC and parE genes of Riemerella anatipestifer isolated from ducks in China
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-03
    Dekang Zhu; Mingyu Zheng; Jinge Xu; Mingshu Wang; Renyong Jia; Shun Chen; Mafeng Liu; Xinxin Zhao; Qiao Yang; Ying Wu; Shaqiu Zhang; Juan Huang; Yunya Liu; Ling Zhang; Yanling Yu; Leichang Pan; Xiaoyue Chen; Anchun Cheng

    Riemerella anatipestifer is one of the most serious infectious disease-causing pathogens in the duck industry. Drug administration is an important method for prevention and treatment of infection in duck production, leading to widespread drug resistance in R. anatipestifer. For a total of 162 isolates of R. anatipestifer, the MICs were determined for a quinolone antimicrobial agent, namely, nalidixic acid, and three fluoroquinolones, namely, ciprofloxacin, enrofloxacin and ofloxacin. The gyrA, parC, and parE gene fragments were amplified by PCR to identify the mutation sites in these strains. Site-directed mutants with mutations that were detected at a high frequency in vivo were constructed (hereafter referred to as site-directed in vivo mutants), and the MICs of these four drugs for these strains were determined. In total, 100, 97.8, 99.3 and 97.8% of the 137 R. anatipestifer strains isolated between 2013 and 2018 showed resistance to nalidixic acid, ciprofloxacin, enrofloxacin, and ofloxacin, respectively. The high-frequency mutation sites were detected in a total of 162 R. anatipestifer strains, such as Ser83Ile and Ser83Arg, which are two types of substitution mutations of amino acid 83 in GyrA; Val799Ala and Ile811Val in ParC; and Val357Ile, His358Tyr, and Arg541Lys in ParE. MIC analysis results for the site-directed in vivo mutants showed that the strains with only the Ser83Ile mutation in GyrA exhibited an 8–16-fold increase in MIC values, and all mutants showed resistance to ampicillin and ceftiofur. The resistance of R. anatipestifer to quinolone agents is a serious problem. Amino acid 83 in GyrA is the major target mutation site for the fluoroquinolone resistance mechanism of R. anatipestifer.

  • Effect of tetracycline treatment regimens on antibiotic resistance gene selection over time in nursery pigs
    BMC Microbiol. (IF 3.287) Pub Date : 2019-12-02
    Kaare Græsbøll; Inge Larsen; Julie Clasen; Anna Camilla Birkegård; Jens Peter Nielsen; Lasse Engbo Christiansen; John Elmerdahl Olsen; Øystein Angen; Anders Folkesson

    The majority of antimicrobials given during the production of pigs are given to nursery pigs. The influence of antimicrobial use on the levels of antimicrobial resistant (AMR) genes is important to quantify to be able to assess the impact of resistance on the food chain and risk to human and animal health. This study investigated the response on the levels of nine AMR genes to five different treatment strategies with oxytetracycline, and the dynamics of gene abundance over time by following 1167 pigs from five different farms in Denmark. The results showed no significant difference between treatments and an increase in abundance for the efflux pump encoding tet(A) gene and the genes encoding the ribosomal protection proteins tet(O) and tet(W) tetracycline resistant genes following treatment, while tet(M) showed no response to treatment. However, it was also observed that the levels of tet(O), tet(W), and ermB in some farms would drift more over time compared to a single treatment-course with antibiotic. This study underlines the large variation in AMR levels under natural conditions and the need for increased investigation of the complex interactions of antimicrobial treatment and other environmental and managerial practices in swine production on AMR gene abundance.

  • A new culture-based method for rapid identification of microorganisms in polymicrobial blood cultures by MALDI-TOF MS
    BMC Microbiol. (IF 3.287) Pub Date : 2019-11-29
    Walter Florio; Susanna Cappellini; Cesira Giordano; Alessandra Vecchione; Emilia Ghelardi; Antonella Lupetti

    The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to microbial identification has allowed the development of rapid methods for identification of microorganisms directly in positive, blood cultures (BCs). These methods can yield accurate results for monomicrobial BCs, but often fail to identify multiple microorganisms in polymicrobial BCs. The present study was aimed at establishing a rapid and simple method for identification of bacteria and yeast in polymicrobial BCs from patients with bloodstream infection. The rapid method herein proposed is based on short-term culture in liquid media allowing selective growth of microorganisms recovered from polymicrobial BCs, followed by rapid identification by MALDI-TOF MS. To evaluate the accuracy of this method, 56 polymicrobial BCs were comparatively analyzed with the rapid and routine methods. The results showed concordant identification for both microbial species in 43/50 (86%) BCs containing two different microorganisms, and for two microbial species in six BCs containing more than two different species. Overall, 102/119 (85.7%) microorganisms were concordantly identified by the rapid and routine methods using a cut-off value of 1.700 for valid identification. The mean time to identification after BC positivity was about 4.2 h for streptococci/enterococci, 8.7 h for staphylococci, 11.1 h for Gram-negative bacteria, and 14.4 h for yeast, allowing a significant time saving compared to the routine method. The proposed method allowed rapid and reliable microbial identification in polymicrobial BCs, and could provide clinicians with timely, useful information to streamline empirical antimicrobial therapy in critically ill patients.

Contents have been reproduced by permission of the publishers.