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  • Resistance Sniffer: an online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using Next Generation Sequencing data
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-17
    Dillon Muzondiwa; Awelani Mutshembele; Rian E. Pierneef; Oleg N. Reva

    The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up.ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.

    更新日期:2020-01-17
  • The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in clostridium perfringens gas gangrene infections
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-17
    Jackie K. Cheung; Vicki Adams; Danielle D’Souza; Meagan James; Christopher J. Day; Michael P. Jennings; Dena Lyras; Julian I. Rood

    Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4 °C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

    更新日期:2020-01-17
  • A novel type I toxin-antitoxin system modulates persister cell formation in Staphylococcus aureus
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-17
    Gul Habib; Jiade Zhu; Baolin Sun

    A plethora of toxin-antitoxin systems exist in bacteria and has multilateral roles in bacterial pathogenesis and virulence. Toxin-antitoxin systems have been involved in persister cell formation in Escherichia coli and Mycobacterium but have not been reported to be associated with Staphylococcus aureus persistence. Persistence is the ability of bacterial cells to tolerate unfavorable conditions and multiple stresses. There are less known and more unknown factors that either alleviate or aggravate bacterial persistence phenomenon. For the first time, we reported a new chromosomally encoded tripartite toxin-antitoxin system and its role in S. aureus persister cell formation. The toxin gene is bacteriostatic in action and counterbalanced by antitoxin RNA that could basepair with the toxin mRNA and formed a duplex. The transcriptional regulator positively regulates the toxin expression under certain stress conditions. The toxin ectopic induction increased S. aureus susceptibility to norfloxacin, ciprofloxacin, and ofloxacin. Whole-genome RNA sequencing revealed that MDR efflux pump norA is significantly down-regulated by toxin ectopic induction. The deletion of norA from S. aureus genome reduced resistance toward ciprofloxacin, norfloxacin, and ofloxacin, as well as resulted in a decrease in minimal inhibitory concentration while complementation of norA successfully restored the phenotypes. The persistence assay of the norA mutant revealed that deletion of norA increased persister cell survival in S. aureus. Altogether, we have provided insight into the first tripartite type-I TA system and revealed the role of MDR NorA in the persister cell formation of S. aureus.

    更新日期:2020-01-17
  • A snapshot of diversity: Intraclonal variation of Escherichia coli clones as commensals and pathogens
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-17
    Marc Stegger; Rikke Fleron Leihof; Sharmin Baig; Raphael N. Sieber; Karen Rønø Thingholm; Rasmus L. Marvig; Niels Frimodt-Møller; Karen Leth Nielsen

    Whole-genome sequencing has enabled detailed studies on bacterial evolution during infection, but there is limited knowledge on intraclonal variation. In this study, we sought to provide a snapshot of the intraclonal diversity of Escherichia coli as both commensal in the faecal environment and pathogen during urinary tract infection, respectively. This was performed by whole-genome sequencing and analyzing single nucleotide polymorphisms (SNPs) and gene-content variation of ten isolates from rectal swabs or urine samples, which all belonged to the same clone based on random amplification of polymorphic DNA (RAPD) PCR. We identified only one clone in eight of the nine urines sampled (89%). In both the commensal and pathogenic state, the within-host diversity was limited with intraclonal SNP diversity of 0-2 non-synonymous SNPs for each clone. The genetic diversity showed variation in gene content in a range of 2-15 genes in total for all clones, including genes positioned on plasmids, and in the K- and O-antigen cluster. The observed SNP- and gene variation shows that sampling of one colony would be enough for surveillance, outbreak investigations and clonal evolution. However, for studies of adaptation during or between colonization and infection, this variation is relevant to consider.

    更新日期:2020-01-17
  • Discovery of Paenibacillus larvae ERIC V: Phenotypic and genomic comparison to genotypes ERIC I-IV reveal different inventories of virulence factors which correlate with epidemiological prevalences of American Foulbrood
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-16
    Hannes Beims; Boyke Bunk; Silvio Erler; Kathrin I. Mohr; Cathrin Spröer; Silke Pradella; Gabi Günther; Manfred Rohde; Werner von der Ohe; Michael Steinert

    Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious brood disease of honey bees (Apis mellifera). AFB requires mandatory reporting to the veterinary authority in many countries and until now four genotypes, P. larvae ERIC I-IV, have been identified. We isolated a new genotype, ERIC V, from a Spanish honey sample. After a detailed phenotypic comparison with the reference strains of the ERIC I-IV genotypes, including spore morphology, non-ribosomal peptide (NRP) profiling, and in vivo infections of A. mellifera larvae, we established a genomic DNA Macrorestriction Fragment Pattern Analysis (MRFPA) scheme for future epidemiologic discrimination. Whole genome comparison of the reference strains and the new ERIC V genotype (DSM 106052) revealed that the respective virulence gene inventories of the five genotypes corresponded with the time needed to kill 100% of the infected bee larvae (LT100) in in vivo infection assays. The rarely isolated P. larvae genotypes ERIC III-V with a fast-killing phenotype (LT100 3 days) harbor genes with high homology to virulence factors of other insect pathogens. These virulence genes are absent in the epidemiologically prevalent genotypes ERIC I (LT100 12 days) and ERIC II (LT100 7 days), which exhibit slower killing phenotypes. Since killing-retardation is known to reduce the success of hygienic cleaning by nurse bees, the identified absence of virulence factors might explain the epidemiological prevalences of ERIC genotypes. The discovery of the P. larvae ERIC V isolate suggests that more unknown ERIC genotypes exist in bee colonies. Since inactivation or loss of a few genes can transform a fast-killing phenotype into a more dangerous slow-killing phenotype, these rarely isolated genotypes may represent a hidden reservoir for future AFB outbreaks.

    更新日期:2020-01-16
  • Prevalence of Hypermutator Isolates of Achromobacter spp. From Cystic Fibrosis Patients
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-16
    Winnie Ridderberg; Kurt Jensen Handberg; Niels Nørskov-Lauritsen

    Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.

    更新日期:2020-01-16
  • Susceptibility of aging mice to listeriosis: Role of anti-inflammatory responses with enhanced Treg-cell expression of CD39/CD73 and Th-17 cells
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-16
    M. Samiul Alam; Christopher Cavanaugh; Marion Pereira; Uma Babu; Kristina Williams

    Foodborne Listeria monocytogenes (Lm) causes serious illness and death in immunosuppressed hosts, including the elderly population. We investigated Lm susceptibility and inflammatory cytokines in geriatric mice. Young-adult and old mice were gavaged with a Lm strain Lmo-InlAm. Tissues were assayed for Lm burden and splenocytes were analyzed for Th1/Th2/Th17/Treg responses and expression of CD39 and CD73. Old Lm-infected mice lost body-weight dose-dependently, had higher Lm colonization, and showed higher inflammatory responses than Lm-infected young-adult mice. After infection, IL-17 levels increased significantly in old mice whereas IFN-γ levels were unchanged. Levels of IL-10 and Treg cells were increased in infected old mice as compared to infected young-adult mice. Age-dependent enhanced expression of CD39/CD73 was observed in purified Treg prior to infection, suggesting increased baseline adenosine production in old mice. Lm lysate-treated splenocytes from older mice produced significantly higher levels of IL-10, IL17, and IL-1β, produced less IFN-γ and IL-2, and proliferated less than splenocytes from young-adult mice. Data suggests that older mice maybe more susceptible to Lm infection due to an imbalance of Th cell responses with disproportionate and persistent anti-inflammatory responses. Lm infection enhanced differentiation of proinflammatory Th17 cells, which may also exacerbate pathological responses during listeriosis.

    更新日期:2020-01-16
  • Characterization of blaCTX-M-14 transposition from plasmid to chromosome in Escherichia coli experimental strain
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-16
    Kouta Hamamoto; Toshiro Tokunaga; Nobuyoshi Yagi; Itaru Hirai

    Mostly, blaCTX-M is found on transferable plasmids as a component of the blaCTX-M transposition unit containing an insertion sequence, ISEcp1, which exists on the upstream region of blaCTX-Ms. Several recent studies conducted in clinical and community settings have reported the presence of chromosomally located blaCTX-M in extended spectrum β-lactamase (ESBL)-producing bacterial isolates. In this study, we observed the frequency and molecular nature of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome by using an experimental strain of Escherichia coli. We determined 102 different chromosomal transposition sites of blaCTX-M-14 in 126 E. coli isolates following five independent screening procedures. The characterization of the 102 different chromosomal transposition sites of blaCTX-M-14 observed in this study revealed the presence of 5-bp direct repeat (DR) sequences and identical left terminal inverted sequences in 80 E. coli isolates. However, 5'-flanking sequences of the right terminal DR sequences in the 80 E. coli isolates were highly diverse, and consensus sequences of the right terminal inverted repeat sequences were not observed. In case of our E. coli experimental strain, the frequency of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome was determined to be 0.51% (SD = 0.37). Collectively, the molecular nature of ISEcp1 could plausibly be a factor contributing to the high detection rates of E. coli possessing chromosomally located blaCTX-M-14 in both clinical and community settings.

    更新日期:2020-01-16
  • How bacterial pathogens of the gastrointestinal tract use the mucosal glyco-code to harness mucus and microbiota: new ways to study an ancient bag of tricks
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-16
    Christine Josenhans; Johannes Müthing; Lothar Elling; Sina Bartfeld; Herbert Schmidt

    During the last decades, the flourishing scientific field of molecular pathogenesis brought groundbreaking knowledge of the mechanisms of pathogenicity and the underlying bacterial virulence factors to cause infectious diseases. However, a major paradigm shift is currently occurring after it became increasingly evident that bacterial-host and host-host cell interactions including immune responses orchestrated by defined virulence factors are not the sole drivers of infectious disease development. Strong evidence has been collected that information and nutrient flow within complex microbial communities, as well as to and from host cells and matrices are equally important for successful infection. This particularly holds true for gastrointestinal (GI) pathogens and the GI microbiota interacting and communicating with each other as well as with the host GI mucus and mucosa. Gut-adapted pathogens appear to have developed powerful and specific strategies to interact with human GI mucus including the microbiota for nutrient acquisition, mucosal adhesion, inter-species communication and traversing the mucus barrier. This review covers the existing evidence on these topics and explores the mutual dynamics of host GI mucus, the mucosal habitat and incoming acute and chronic pathogens during GI infections. A particular focus is placed on the role of carbohydrates in diverse mucosal interaction, communication and competition processes. Novel techniques to analyze and synthesize mucus-derived carbohydrates and to generate mucus mimetics are introduced. Finally, open questions and future objectives for pathogen - host GI mucus research will be discussed.

    更新日期:2020-01-16
  • Lactobacillus plantarum CBT LP3 ameliorates colitis via modulating T cells in mice
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2020-01-07
    Da Hye Kim; Soochan Kim; Jae Bum Ahn; Jae Hyeon Kim; Hyun Woo Ma; Dong Hyuk Seo; Xiumei Che; Ki Cheong Park; Jeong Yong Jeon; Sang Yong Kim; Han Cheol Lee; Jae-Young Lee; Tae Il Kim; Won Ho Kim; Seung Won Kim; Jae Hee Cheon

    Lactobacillus plantarum has been identified as a probiotic bacterium owing to its role in immune regulation and maintenance of intestinal permeability. Here, we investigated the anti-colitic effects and mechanism of L. plantarum CBT LP3 (LP3). This in vivo study was performed using dextran sodium sulfate (DSS) to induce colitis in mice. Mice were randomly divided into three groups: a control supplied with normal drinking water, a DSS-treated group followed by oral administration of vehicle, and a DSS-treated group gavaged with LP3 daily for 7 days following DSS administration. An analysis of macrophages and T cell subsets harvesting from peritonium cavity cells and splenocytes was performed using a flow cytometric assay. Gene expression and cytokine profiles were measured using quantitative reverse transcriptase polymerase chain reaction. The administration of LP3 significantly attenuated disease activity and histolopathology compared to control. LP3 had anti-inflammatory effects, with increased induction of regulatory T cells and type 2 helper T cells in splenocytes and restoration of goblet cells accompanied by suppression of proinflammatory cytokine expressions. These findings suggest that L. plantarum CBT LP3 can be used as a potent immunomodulator, which has significant implications for IBD treatment.

    更新日期:2020-01-07
  • Tigecycline resistance may be associated with dysregulated response to stress in Mycobacterium abscessus
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-11-18
    Hien Fuh Ng, Yun Fong Ngeow, Sook Fan Yap, Thaw Zin, Joon Liang Tan

    Previously, we characterized 7C, a laboratory-derived tigecycline-resistant mutant of Mycobacterium abscessus ATCC 19977, and found that the resistance was conferred by a mutation in MAB_3542c, which encodes an RshA-like protein. In M. tuberculosis, RshA is an anti-sigma factor that negatively regulates the SigH-dependent heat/oxidative stress response. We hypothesized that this mutation in 7C might dysregulate the stress response which has been generally linked to antibiotic resistance. In this study, we tested this hypothesis by subjecting 7C to transcriptomic dissection using RNA sequencing. We found an over-expression of genes encoding the SigH ortholog, chaperones and oxidoreductases. In line with these findings, 7C demonstrated better survival against heat shock when compared to the wild-type ATCC 19977. Another interesting observation from the RNA-Seq analysis was the down-regulation of ribosomal protein-encoding genes. This highlights the possibility of ribosomal conformation changes which could negatively affect the binding of tigecycline to its target, leading to phenotypic resistance. We also demonstrated that transient resistance to tigecycline could be induced in the ATCC 19977 by elevated temperature. Taken together, these findings suggest that dysregulated stress response may be associated with tigecycline resistance in M. abscessus.

    更新日期:2019-11-18
  • Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-11-11
    Erwin M. Berendsen, Evgeni Levin, René Braakman, Andrei Prodan, Hans C. van Leeuwen, Armand Paauw

    To improve the preparedness against exposure to highly pathogenic bacteria and to anticipate the wide variety of bacteria that can cause bloodstream infections (BSIs), a safe, unbiased and highly accurate identification method was developed. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method can identify highly pathogenic bacteria, their near-neighbors and bacteria that are common causes of BSIs directly from positive blood culture flasks. The developed Peptide-Based Microbe Detection Engine (http://proteome2pathogen.com/app/) relies on a two-step workflow: a genus-level search followed by a species-level search. This strategy enables the rapid identification of microorganisms based on the analyzed proteome. This method was successfully used to identify strains of Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, Yersinia pestis and closely related species from simulated blood culture flasks. This newly developed LC-MS/MS method is a safe and rapid method for accurately identifying bacteria directly from positive blood culture flasks.

    更新日期:2019-11-13
  • Co-existence of Citrobacter freundii exacerbated Pseudomonas aeruginosa infection in vivo
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-11-11
    Qing Peng, Lin Chen, Shuqin Zhou, Haiyan Li, Jun Long, Fen Yao, Yijing Zhuang, Zijie Zhang, Yuanchun Huang, Kangmin Duan

    The presence of bacterial species other than the pathogen at infection site can affect the progression of a bacterial infection. Based on the fact that Citrobacter freundii can coexist during Pseudomonas aeruginosa infection, this study aims to investigate the impact of the co-existing C. freundii on the pathogenesis of P. aeruginosa infection. A murine peritonitis model was used to compare the mortality rates and histopathology of P. aeruginosa PAO1 infection in the presence and absence of a C. freundii clinical isolate C9. We also investigated the intercellular interaction between PAO1 and C9 by examining pyocyanin production and comparing gene expression levels. The results demonstrate that co-infection with C9 significantly increased the mortality rate and tissue damages in PAO1 infected mice. At an inoculum of 106 CFU, no mortality was observed in the C9 infected group at three days post-infection, whereas the mortality rate in the PAO1-C9 co-infection group was 64%, compared with 24% in the PAO1 infected group. Pyocyanin production in P. aeruginosa PAO1 increased 8 folds approximately in the presence of C. freundii C9, and operons associated with phenazine synthesis, phzA1 and phzA2, were also upregulated. Disruption of the phzA1 and phzA2 eliminated the exacerbated pathogenicity in the co-infection group, indicating that the elevated pyocyanin production was the main contributing factor. The results suggest that co-existing C. freundii during P. aeruginosa infection can exacerbate the pathogenicity, which may have significant implications in patients infected with these bacteria.

    更新日期:2019-11-13
  • Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-11-09
    Xi Yang, Xiangning Bai, Ji Zhang, Hui Sun, Shanshan Fu, Ruyue Fan, Xiaohua He, Flemming Scheutz, Andreas Matussek, Yanwen Xiong

    Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity <-- -->in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx2k-containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx2k-containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx2k-containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis.

    更新日期:2019-11-11
  • Over-expression of Tgs1 in Mycobacterium marinum enhances virulence in adult zebrafish
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-11-06
    Ding-qian Liu, Jun-li Zhang, Zhi-fen Pan, Jun-tao Mai, Heng-jun Mei, Yue Dai, Lu Zhang, Qing-zhong Wang

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can persist in the host for decades without causing TB symptoms and can cause a latent infection, which is an intricate challenge of current TB control. The DosR regulon, which contains approximately 50 genes, is crucial in the non-replicating persistence of Mtb. tgs1 is one of the most powerfully induced genes in this regulon during Mtb non-replicating persistence. The gene encodes a triacyl glycerol synthase catalyzing synthesis of triacyl glycerol (TAG), which is proposed as an energy source during bacilli persistence. Here, western blotting showed that the Tgs1 protein was upregulated in clinical Mtb strains. To detect its physiological effects on mycobacterium, we constructed serial recombinant M. marinum including over-expressed Tgs1(Tgs1-H), reduced-expressed Tgs1(Tgs1-L), and wild type M. marinum strains(MmpACT, MmpMV) as controls. Tgs1 over-expression did not influence M. marinum growth under aerobic shaking and in hypoxic cultures, while growth advantages were observed at an early stage under nutrient starvation. Transmission electron microscopy revealed more lipid droplets in Tgs1-H than the other two strains; the droplets filled the cytoplasm. Two-dimensional thin-layer chromatography revealed more phosphatidyl-myo-inositol mannosides in the Tgs1-H cell wall. To assess the virulence of recombinant M. marinum in the natural host, adult zebrafish were infected with Tgs1-H or wild type WT strains. Hypervirulence of Tgs1-H was characterized by markedly increased bacterial load and early death of adult zebrafish. Remarkably, zebrafish infected with Tgs1-H developed necrotizing granulomas much more rapidly and in higher amounts, which facilitated mycobacterial replication and dissemination among organs and eventual tissue destruction in zebrafish. RNA sequencing analysis showed Tgs1-H induced 13 genes differentially expressed under aerobiosis. Among them, PE_PGRS54 (MMAR_5307),one of the PE_PGRS family of antigens, was markedly up-regulated, while 110 coding genes were down-regulated in Tgs1-L.The 110 genes included 22 member genes of the DosR regulon. The collective results indicate an important role for the Tgs1 protein of M. marinumin progression of infection in the natural host. Tgs1 signaling may be involved in a previously unknown behavior of M. marinum under hypoxia/aerobiosis.

    更新日期:2019-11-06
  • Transcriptomic analysis of smooth versus rough Brucella melitensis Rev.1 vaccine strains reveals insights into virulence attenuation
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-10-21
    Mali Salmon-Divon, David Kornspan

    Brucella melitensis Rev.1 is the live attenuated Elberg-originated vaccine strain of the facultative intracellular Brucella species, and is widely used to control brucellosis in small ruminants. However, Rev.1 may cause abortions in small ruminants that have been vaccinated during the last trimester of gestation, it is pathogenic to humans, and it induces antibodies directed at the O-polysaccharide (O-PS) of the smooth lipopolysaccharide, thus making it difficult to distinguish between vaccinated and infected animals. Rough Brucella strains, which lack O-PS and are considered less pathogenic, have been introduced to address these drawbacks; however, as Rev.1 confers a much better immunity than the rough mutants, it is still considered the reference vaccine for the prophylaxis of brucellosis in small ruminants. Therefore, developing an improved vaccine strain, which lacks the Rev.1 drawbacks, is a highly evaluated task, which requires a better understanding of the molecular mechanisms underlying the virulence attenuation of Rev.1 smooth strains and of natural Rev.1 rough strains, which are currently only partly understood. As the acidification of the Brucella-containing vacuole during the initial stages of infection is crucial for their survival, identifying the genes that contribute to their survival in an acidic environment versus a normal environment will greatly assist our understanding of the molecular pathogenic mechanisms and the attenuated virulence of the Rev.1 strain. Here, we compared the transcriptomes of the smooth and natural rough Rev.1 strains, each grown under either normal or acidic conditions. We found 12 key genes that are significantly downregulated in the Rev.1 rough strains under normal pH, as compared with Rev.1 smooth strains, and six highly important genes that are significantly upregulated in the smooth strains under acidic conditions, as compared with Rev.1 rough strains. All 18 differentially expressed genes are associated with bacterial virulence and survival and may explain the attenuated virulence of the rough Rev.1 strains versus smooth Rev.1 strains, thus providing new insights into the virulence attenuation mechanisms of Brucella. These highly important candidate genes may facilitate the design of new and improved brucellosis vaccines.

    更新日期:2019-10-22
  • Elicitation of Th1/Th2 related responses in mice by chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase, outer membrane proteins 10 and 19
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-10-18
    Soojin Shim, Sang Hee Soh, Young Bin Im, Hyun-Eui Park, Chong-Su Cho, Suk Kim, Han Sang Yoo

    Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.

    更新日期:2019-10-19
  • Sub-Inhibitory Concentrations of SOS-Response Inducing Antibiotics Stimulate Integrase Expression and Excision of Pathogenicity Islands in Uropathogenic Escherichia coli Strain 536
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-10-07
    Marco Chittò, Michael Berger, Luisa Klotz, Ulrich Dobrindt

    Urinary tract infections are one of the most common bacterial infections and a major public health problem. The predominant causative agents are uropathogenic Escherichia coli. These strains differ from commensal E. coli by the presence of additional horizontally acquired chromosomal material, so-called pathogenicity islands, which encode traits that promote efficient bacterial colonization of the urinary tract. Uropathogenic model strain E. coli 536 possesses six archetypal pathogenicity islands. Bacteriophage-like integrases encoded by each pathogenicity island contribute to island instability. To learn more about the stability of these six islands and factors controlling their stability we constructed two chromosomal reporter systems for the measurement of island loss, as well as for the measurement of the promoter activity of the six island-associated integrase genes at the population level. We used these reporter gene modules to analyze the role of SOS response in island instability. Tests with subinhibitory concentrations of different antibiotics, including many drugs commonly used for the treatment of urinary tract infection, indicated that only SOS response-inducing antibiotics led to an increased loss of islands which was always associated with an increase in the bacterial subpopulations showing high integrase promoter activity. This suggests that island excision correlates with the expression of the cognate integrase. Our reporter modules are valuable tools to investigate the impact of various growth conditions on genome plasticity. Furthermore, a better understanding of the conditions, which affect bacterial integrase expression may open ways to specifically manipulate the genome content of bacterial pathogens by increasing pathogenicity island deletion rates in infecting or colonizing bacteria, thus leading to the attenuation of bacterial pathogens.

    更新日期:2019-10-08
  • A nanopore array in the septal peptidoglycan hosts gated septal junctions for cell-cell communication in multicellular cyanobacteria
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-03-26
    Ann-Katrin Kieninger, Karl Forchhammer, Iris Maldener

    Some filamentous cyanobacteria are phototrophic bacteria with a true multicellular life style. They show patterned cell differentiation with the distribution of metabolic tasks between different cell types. This life style requires a system of cell-cell communication and metabolite exchange along the filament. During our study of the cell wall of species Nostoc punctiforme and Anabaena sp. PCC 7120 we discovered regular perforations in the septum between neighboring cells, which we called nanopore array. AmiC-like amidases are drilling the nanopores with a diameter of 20 nm, and are essential for communication and cell differentiation. NlpD-like regulators of AmiC activity and septum localized proteins SepJ, FraC and FraD are also involved in correct nanopore formation. By focused ion beam (FIB) milling and electron cryotomography we could visualize the septal junctions, which connect adjacent cells and pass thru the nanopores. They consist of cytoplasmic caps, which are missing in the fraD mutant, a plug inside the cytoplasmic membrane and a tube like conduit. A destroyed membrane potential and other stress factors lead to a conformational change in the cap structure and loss of cell-cell communication. These gated septal junctions of cyanobacteria are ancient structures that represent an example of convergent evolution, predating metazoan gap junctions.

    更新日期:2019-10-07
  • Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-09-19
    Julio C. Carrero, Magda Reyes-López, Jesús Serrano-Luna, Mineko Shibayama, Juan Unzueta, Nidia León-Sicairos, Mireya de la Garza

    Amoebiasis is a parasitic disease caused by Entamoeba histolytica (E. histolytica), an extracellular enteric protozoan. This infection mainly affects people from developing countries with limited hygiene conditions, where it is endemic. Infective cysts are transmitted by the fecal-oral route, excysting in the terminal ileum and producing invasive trophozoites (amoebae). E. histolytica mainly lives in the large intestine without causing symptoms; however, possibly as a result of so far unknown signals, the amoebae invade the mucosa and epithelium causing intestinal amoebiasis. E. histolytica possesses different mechanisms of pathogenicity for the adherence to the intestinal epithelium and for degrading extracellular matrix proteins, producing tissue lesions that progress to abscesses and a host acute inflammatory response. Much information has been obtained regarding the virulence factors, metabolism, mechanisms of pathogenicity, and the host immune response against this parasite; in addition, alternative treatments to metronidazole are continually emerging. An accesible and low-cost diagnostic method that can distinguish E. histolytica from the most nonpathogenic amoebae and an effective vaccine are necessary for protecting against amoebiasis. However, research about the disease and its prevention has been a challenge due to the relationship between E. histolytica and the host during the distinct stages of the disease is multifaceted. In this review, we analyze the interaction between the parasite, the human host, and the colon microbiota or pathogenic microorganisms, which together give rise to intestinal amoebiasis.

    更新日期:2019-09-20
  • Genotypic Differences in CC224, CC363, CC449 and CC446 of Moraxella catarrhalis Isolates Based on Whole Genome SNP, MLST and PFGE Typing
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-09-19
    Ya-Li Liu, Shu-Ying Yu, Xin-Miao Jia, Hai-Jian Zhou, Hong-li Sun, Timothy Kudinha, Fan-Rong Kong, He-Ping Xu, Ying-Chun Xu

    Understanding the evolutionary path of M. catarrhalis from macrolide-susceptible to macrolide-resistant organism, is important for hindering macrolide resistance from propagation. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome SNP typing (WGST), as useful and practical typing tools, have both advantages and disadvantages. We studied the utility of these 3 typing methods, including the level of agreement, consistency and draw backs, in characterizing M. catarrhalis clones and clonal complexes. We focused on four clonal complexes [CC224, CC363, CC449 (CCN10) and CC446 (CCN08)] and found that PFGE and WGST had a high level of agreement and a proper consistency of the same clone or very closely related clones, while MLST is less discriminatory for different clones. Furthermore, we also establish an evolutionary distance cut-off value for “The same clone”. Moreover, we detected macrolide-resistant M. catarrhalis in CC224, which has previously been considered a macrolide-susceptible clonal complex. A higher number of isolates belonged to ST215 compared to ST446, implying that ST215 is more likely to be the primary founder. Our study also demonstrated that all the four clonal complexes belong to the M. catarrhalis lineage 1, which is considered to be related to increased virulence potential and serum resistance. We also observed that copB II was highly related to CC449 and LOS type B was mainly confined in CC224. In conclusion, these findings provide further insight into the evolutionary characteristics of M. catarrhalis.

    更新日期:2019-09-19
  • Staphylococcus petrasii diagnostics and its pathogenic potential enhanced by mobile genetic elements
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-09-05
    Veronika Vrbovská, Vojtěch Kovařovic, Ivana Mašlaňová, Adéla Indráková, Petr Petráš, Ondrej Šedo, Pavel Švec, Lenka Fišarová, Marta Šiborová, Kamil Mikulášek, Ivo Sedláček, Jiří Doškař, Roman Pantůček

    Staphylococcus petrasii is recently described coagulase negative staphylococcal species and an opportunistic human pathogen, still often misidentified in clinical specimens. Four subspecies are distinguished in S. petrasii by polyphasic taxonomical analyses, however a comparative study has still not been done on the majority of isolates and their genome properties have not yet been thoroughly analysed. Here, we describe the phenotypic and genotypic characteristics of 65 isolates and the results of de novo sequencing, whole genome assembly and annotation of draft genomes of five strains. The strains were identified by MALDI-TOF mass spectrometry to the species level and the majority of the strains were identified to the subspecies level by fingerprinting methods, (GTG)5 repetitive PCR and ribotyping. Macrorestriction profiling by pulsed-field gel electrophoresis was confirmed to be a suitable strain typing method. Comparative genomics revealed the presence of new mobile genetic elements carrying antimicrobial resistance factors such as staphylococcal cassette chromosome (SCC) mec, transposones, phage-inducible genomic islands, and plasmids. Their mosaic structure and similarity across coagulase-negative staphylococci and Staphylococcus aureus suggest the possible exchange of these elements. Numerous putative virulence factors such as adhesins, autolysins, exoenzymes, capsule formation genes, immunomodulators, the phage-associated sasX gene, and SCC-associated spermidine N-acetyltransferase, pseudouridine and sorbitol utilization operons might explain clinical manifestations of S. petrasii isolates. The increasing recovery of S. petrasii isolates from human clinical material, the multi-drug resistance including methicillin resistance of S. petrasii subsp. jettensis strains, and virulence factors homologous to other pathogenic staphylococci demonstrate the importance of the species in human disease.

    更新日期:2019-09-05
  • Nucleases of bacterial pathogens as virulence factors, therapeutic targets and diagnostic markers
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-30
    Prince Sharma, Nisha Garg, Anshul Sharma, Neena Capalash, Ravinder Singh

    New frontiers of therapy are being explored against the upcoming bacterial diseases rendered untreatable due to multiple, extreme and pan- antibiotic resistance. Nucleases are ubiquitous in bacterial pathogens performing various functions like acquiring nucleotide nutrients, allowing or preventing uptake of foreign DNA, controlling biofilm formation/dispersal/architecture, invading host by tissue damage, evading immune defence by degrading DNA matrix of neutrophil extracellular traps (NETs) and immunomodulating the host immune response. Secretory nucleases also provide means of survival to other bacteria like iron-reducing Shewanella and such functions help them adapt and survive proficiently. Other than their pro-pathogen roles survival, nucleases can be used directly as therapeutics. One of the powerful armours of pathogens is the formation of biofilms, thus helping them resist and persist in the harshest of environments. As eDNA forms the structural and binding component of biofilm, nucleases can be used against the adhering component, thus increasing the permeability of antimicrobial agents. Nucleases have recently become a model system of intense study for their biological functions and medical applications in diagnosis, immunoprophylaxis and therapy. Rational implications of these enzymes can impact human medicine positively in future by opening new ways for therapeutics which have otherwise reached saturation due to multi drug resistance.

    更新日期:2019-08-31
  • Mycobacterium tuberculosis DosR regulon gene Rv2004c contributes to streptomycin resistance and intracellular survival
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-30
    Sankara Narayana Doddam, Vidyullatha Peddireddy, Priyadarshini Yerra, PV Parvati Sai Arun, Majjid A. Qaria, Ramani Baddam, Nishat Sarker, Niyaz Ahmed

    Tuberculosis (TB) is the deadly infectious disease challenging the public health globally and its impact is further aggravated by co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. In this study, we attempted to characterise the Rv2004c encoded protein, a member of DosR regulon, for its role in drug resistance. In silico docking analysis revealed that Rv2004c binds with streptomycin (SM). Phosphotransferase assay demonstrated that Rv2004c possibly mediates SM resistance through the aminoglycoside phosphotransferase activity. Further, E. coli expressing Rv2004c conferred resistance to 100µM of SM in liquid broth cultures indicating a mild aminoglycoside phosphotransferase activity of Rv2004c. Moreover, we investigated the role of MSMEG_3942 (an orthologous gene of Rv2004c) encoded protein in intracellular survival, its effect on in-vitro growth and its expression in different stress conditions by over expressing it in Mycobacterium smegmatis (M. smegmatis). MSMEG_3942 overexpressing recombinant M. smegmatis strains grew faster in acidic medium and also showed higher bacillary counts in infected macrophages when compared to M. smegmatis transformed with vector alone. Our results are likely to contribute to the better understanding of the involvement of Rv2004c in partial drug resistance, intracellular survival and adaptation of bacilli to stress conditions and point at the possible use of Rv2004c as a target for vaccine development.

    更新日期:2019-08-30
  • Screen for fitness and virulence factors of Francisella sp. strain W12-1067 using amoebae
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-19
    Kristin Köppen, Fan Chen, Kerstin Rydzewski, Rosa Einenkel, Thea Böttcher, Clara Morguet, Roland Grunow, Wolfgang Eisenreich, Klaus Heuner

    Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a “scatter screen”, used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.

    更新日期:2019-08-20
  • Chlamydia pneumoniae infection promotes vascular smooth muscle cell migration via c-Fos/interleukin-17C signaling
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-19
    Ningbo Zheng, Lijun Zhang, Beibei Wang, Guangyan Wang, Jingya Liu, Guolin Miao, Xi Zhao, Changle Liu, Lijun Zhang

    Chlamydia pneumoniae (C. pneumoniae) infection is associated with the initiation and progression of atherosclerosis. The migration of vascular smooth muscle cell (VSMC) from the media to the intima is a key event in the development of atherosclerosis. Interleukin-17C (IL-17C) could enhance cell migration ability. The aim of our study is to investigate the role of IL-17C in C. pneumoniae infection-promoted VSMC migration, thereby possibly accelerating atherosclerosis. We firstly demonstrated that C. pneumoniae infection significantly increased IL-17C expression in VSMCs in the atherosclerotic lesion area from ApoE deficient mice. Our in vitro study further showed that IL-17C is required for C. pneumoniae infection-promoted VSMC migration, and its expression could be regulated by c-Fos through phosphorylating extracellular signal-regulated kinase (ERK). Unexpectedly, in the present study, we also found that IL-17C is critical for C. pneumoniae infection-induced c-Fos activation. c-Fos expression and activation induced by the exposure to recombinant IL-17C were markedly suppressed in the presence of the ERK inhibitor PD98059. These results suggest a possible positive feedback between c-Fos and IL-17C after C. pneumoniae infection. Taken together, our results indicate that C. pneumoniae infection promotes VSMC migration via c-Fos/IL-17C signaling.

    更新日期:2019-08-20
  • Emerging challenges of Whole-Genome-Sequencing–powered epidemiological surveillance of globally distributed clonal groups of bacterial infections, giving Acinetobacter baumannii ST195 as an example
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-19
    Huiqiong Jia, Yan Chen, Jianfeng Wang, Xinyou Xie, Zhi Ruan

    Whole-genome sequencing (WGS) has revolutionized the genotyping of bacterial pathogens and is expected to become the new gold standard for tracing the transmissions of bacterial infectious diseases for public health purposes. However, it is still unexpectedly demanding to employ WGS for global epidemiological surveillance because of the high degree of similarity between the genomes of intercontinental isolates. The aim of this study was to utilize genomically derived bioinformatics analysis to identify globally distributed A. baumannii ST195 lineage and differentiation outbreaks to address this issue. The genomic sequences and their related epidemiological metadata of 2850 A. baumannii isolates were recruited from NCBI Genbank database. Assignment into sequence type (Oxford scheme) and lineage (global clone 2/CC92) were performed. A total of 91 ST195 A. baumannii isolates were subsequently classified to perform the bacterial source tracking analysis by implementing both core genome MLST (cgMLST) and core genome SNP (cgSNP) strategy that were integrated in our recently updated BacWGSTdb 2.0 server. Antibiotic resistance genes were identified using the ResFinder database. The ST195 A. baumannii isolates distributed widely in eight countries and harboured multiple antimicrobial resistance genes simultaneously. In most cases, the bacterial isolates recovered from geographically distant sources may present less genomic sequence similarity, i.e., the phylogenetic relationship between these ST195 isolates worldwide was roughly congruent with their country of isolation. However, a few isolates collected from distant geographic regions were revealed to possess smaller genetic distances (less than 8 loci or 20 SNPs) than the threshold without an observable epidemiological link. Our study highlights the emerging challenges entailed in the WGS-powered epidemiological surveillance of globally distributed clonal groups. Standardization is urgently required before WGS can be routinely applied to infectious diseases outbreak investigations.

    更新日期:2019-08-20
  • ADH1 promotes Candida albicans pathogenicity by stimulating oxidative phosphorylation
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-17
    Yanjun Song, Shuixiu Li, Yajing Zhao, Yishan Zhang, Yan Lv, Yuanying Jiang, Yan Wang, Dongmei Li, Hong Zhang

    Objective Alcohol dehydrogenase I is encoded by ADH1 in Candida albicans, and is one of the key enzymes in fungal metabolism by which it catalyzes the conversion from acetaldehyde to ethanol. The role of the associated protein Adh1p, encoded by ADH1 in fungal pathogenicity has not been thoroughly studied despite its near ubiquity in the fungal kingdom. Using C. albicans as a model, this study proposes to determine the possible pathogenic roles for ADH1 and its possible underlying mechanisms. Methods The SAT1 flipper strategy was used to construct the ADH1 deletion mutant. Growth curves and spot assay were used to compare growth and cell viability of the mutant to wild type C. albicans. Three host model systems (infected mice, C. elegans, and G. mellonella) were used to investigate the effects of ADH1 deletion in vivo on C. albicans pathogenicity. Then, adhesion, hyphal formation, biofilm formation, cell surface hydrophobicity (CSH) and RT-qPCR were performed to investigate the effects of ADH1 deletion in vitro on C. albicans virulence. Finally, Xfe 96 seahorse assay, ROS level, mitochondrial membrane potential, and intracellular ATP content were used to determine the effects of ADH1 deletion on bioenergetics. Results ADH1 deletion has no effects on the growth and cell viability of C. albicans, but significantly prolongs survival time in each of the three host models, decreases fungal burden in kidney and liver, and lessens pathological tissue damage (P < 0.05). In addition, ADH1 deletion significantly increases CSH and reduces C. albicans virulence in terms of adhesion, hyphal formation and biofilm formation in accord with the downregulation of virulence-related genes such as ALS1, ALS3, HWP1, and CSH1 (P < 0.05). For bioenergetics, ADH1 deletion has no obvious effect on glycolysis, but a lack of ADH1 significantly increases ROS levels and decreases mitochondrial membrane potential and intracellular ATP content even through the mitochondrial oxygen consumption rate and NADH/NAD+ ratio are elevated (P < 0.05). Conclusion Our results suggest that the fermentative enzyme ADH1 is required for the pathogenicity of C. albicans under one of the presumed mechanisms via its effects on oxidative phosphorylation activities in mitochondria.

    更新日期:2019-08-18
  • Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-16
    Regina Selb, Vidmante Fuchs, Barbara Graf, Axel Hamprecht, Michael Hogardt, Ludwig Sedlacek, Roman Schwarz, Evgeny A. Idelevich, Sören L. Becker, Jürgen Held, Claus P. Küpper-Tetzel, Ilka McCormick-Smith, Daniela Heckmann, Jasmin Gerkrath, Chang-Ok Han, Dunja Wilmes, Volker Rickerts

    Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients’ samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.

    更新日期:2019-08-16
  • Immunogenic potential and protective efficacy of a sptP deletion mutant of Salmonella Enteritidis as a live vaccine for chickens against a lethal challenge
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-12
    Yaxin Guo, Ying Xu, Xilong Kang, Dan Gu, Yang Jiao, Chuang Meng, Peipei Tang, Xiaohai Wang, Cuiying Huang, Shizhong Geng, Xinan Jiao, Zhiming Pan

    Salmonella Enteritidis (SE) is a highly adapted pathogen causing severe economic losses in the poultry industry worldwide. Chickens infected by SE are a major source of human food poisoning. Vaccination is an effective approach to control SE infections. This study evaluated the immunogenicity and protective efficacy of a SE sptP deletion mutant (C50336ΔsptP) as a live attenuated vaccine (LAV) candidate in chickens. 14 day-old specific pathogen-free (SPF) chickens were intramuscularly immunized with various doses of C50336ΔsptP. Several groups of chickens were challenged with the virulent wild-type SE strain Z-11 via the same route at 14 days post vaccination. Compared to the control group, the groups vaccinated with 1 × 106, 1 × 107 and 1 × 108 colony-forming units (CFU) of C50336ΔsptP exhibited no clinical symptoms after immunization. Only slight pathological changes occurred in the organs of the 1 × 109 CFU vaccinated group. C50336ΔsptP bacteria were cleared from the organs of immunized chickens within 14 days after vaccination. Lymphocyte proliferation and serum cytokine analyses indicated that significant cellular immune responses were induced after the vaccination of C50336ΔsptP. Compared to the control group, specific IgG antibody levels increased significantly in vaccinated chickens, and the levels increased markedly after the challenge. The 1 × 107, 1 × 108, and 1 × 109 CFU vaccinated chickens groups showed no clinical symptoms or pathological changes, and no death after the lethal challenge. Whereas severe clinical signs of disease and pathological changes were observed in the control group chickens after the challenge. These results suggest that a single dose of C50336ΔsptP could be an effective LAV candidate to against SE infection in chickens.

    更新日期:2019-08-13
  • Rapid Diagnosis of Bacterial Meningitis by Nanopore 16S Amplicon Sequencing: A pilot study
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-08-12
    Jangsup Moon, Narae Kim, Tae-Joon Kim, Jin-Sun Jun, Han Sang Lee, Hye-Rim Shin, Soon-Tae Lee, Keun-Hwa Jung, Kyung-Il Park, Ki-Young Jung, Manho Kim, Sang Kun Lee, Kon Chu

    Early administration of antibiotics is crucial in the management of bacterial meningitis. Rapid pathogen identification helps to make a definite diagnosis of bacterial meningitis and enables tailored antibiotic treatment. We investigated if the 16S amplicon sequencing performed by MinION, a nanopore sequencer, was capable of rapid pathogen identification in bacterial meningitis. Six retrospective cases of confirmed bacterial meningitis and two prospective cases were included. The initial cerebrospinal fluid (CSF) samples of these patients were used for the experiments. DNA was extracted from the CSF, and PCR was performed on the 16S ribosomal DNA (16S rDNA). Sequencing libraries were prepared using the PCR products, and MinION sequencing was performed for up to 3 h. The reads were aligned to the bacterial database, and the results were compared to the conventional culture studies. Pathogenic bacteria were successfully detected from the CSF by 16S sequencing in all retrospective cases. 16S amplicon sequencing was more sensitive than conventional diagnostic tests and worked properly even in antibiotics-treated samples. MinION sequencing significantly reduced the turnaround time, and even 10 minutes of sequencing was sufficient for pathogen detection in certain cases. Protocol adjustment could further increase the sensitivity and reduce the turnaround time for MinION sequencing. Finally, the prospective application of MinION 16S sequencing was successful. Nanopore 16S amplicon sequencing is capable of rapid bacterial identification from the CSF of the bacterial meningitis patients. It may have many advantages over conventional diagnostic tests and should therefore be applied in a larger number of patients in the future.

    更新日期:2019-08-13
  • Function and regulation of Staphylococcus aureus wall teichoic acids and capsular polysaccharides
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-18
    Daniela Keinhörster, Shilpa Elizabeth George, Christopher Weidenmaier, Christiane Wolz

    Staphylococcus aureus produces different secondary cell wall glycopolymers such as wall teichoic acids (WTA) and capsular polysaccharides (CP). These structures play an important role in S. aureus colonization, pathogenesis and bacterial evasion of the host immune defences. To fulfil their diverse functions, biosynthesis of both glycopolymers has to be tightly controlled. Regulation of WTA biosynthesis and modification is only partially understood. The transcription factor MgrA and the two-component systems (TCS) Agr, GraRS, and ArlRS control WTA export, chain-length and modification. CP synthesis is determined by transcriptional and post-transcriptional regulatory circuits. On the transcriptional level expression of the capA-P operon is mainly driven by the alternative Sigma factor B and modulated by several transcriptional factors and TCS. Post-transcriptional mechanisms are in place to avoid conflict between precursor usage by the CP synthesis machinery and the synthesis machinery of other cell wall glycopolymers. The complex interplay of these regulatory systems determines the peculiar, strictly temporal expression of CP in the late growth phase and the high degree of phenotypic heterogeneity. Differential expression of CP, WTA and its modification systems during infection and colonisation are likely important for disease development, immune escape and survival within the host.

    更新日期:2019-07-18
  • Environmental and cellular factors affecting the localization of T6SS proteins in Burkholderia thailandensis
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-18
    Jan Lennings, Munira Makhlouf, Przemyslaw Olejnik, Christian Mayer, Heike Brötz-Oesterhelt, Sandra Schwarz

    The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.

    更新日期:2019-07-18
  • Unraveling the Mechanism of Peptidoglycan Amidation by the Bifunctional Enzyme Complex GatD/MurT: a Comparative Structural Approach
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-18
    Erik R. Nöldeke, Thilo Stehle

    The bacterial cell wall provides structural integrity to the cell and protects the cell from internal pressure and the external environment. During the course of the twelve-year funding period of the Collaborative Research Center 766, our work has focused on conducting structure-function studies of enzymes that modify (synthesize or cleave) cell wall components of a range of bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Nostoc punctiforme. Several of our structures represent promising targets for interference. In this review, we highlight a recent structure-function analysis of an enzyme complex that is responsible for the amidation of Lipid II, a peptidoglycan precursor, in S. aureus.

    更新日期:2019-07-18
  • Diversity of peptidoglycan structure – Modifications and their physiological role in resistance in antibiotic producers
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-17
    Sandra Unsleber, Wolfgang Wohlleben, Evi Stegmann

    Peptidoglycan (PG) is a bacteria specific cell surface layer that ensures the bacterial shape and integrity. The two actinomycetes Amycolatopsis balhimycina and Microbispora sp. PTA-5024 are producers of PG targeting antibiotics. To prevent the binding of their secreted product to their own PG, they developed specific self-resistance mechanisms. Modifications of PG, which are applied by both strains, are the introduction of amide-residues at the PG precursors and the alternative crosslinks within the nascent PG. The PG modifications found in Microbispora sp. PTA-5024 seemed to be an intrinsic characteristic of the genus Microbispora, rather than a specific mechanism of NAI-107 resistance. In contrast, the modifications in A. balhimycina represent an alternative way to avoid suicide specific for glycopeptide producers. The different PG modifications reflect the fact that antibiotic producing organisms contain not only one but multiple mechanisms to ensure protection against biologically active molecules produced by themselves.

    更新日期:2019-07-17
  • Investigating the assembly of the bacterial type III secretion system injectisome by in vivo photocrosslinking
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-15
    Nidhi Singh, Samuel Wagner

    Virulence-associated type III secretion systems serve the injection of bacterial effector proteins into eukaryotic host cells. These effector proteins modulate host cell biology in order to promote colonization and infection, hence type III secretion systems are often essential bacterial pathogenicity factors. The core of type III secretion systems is a cell envelope-spanning macromolecular machine called injectisome. It consists of almost twenty different components in a stoichiometry of one to more than one hundred. Assembly of this 6 MDa complex requires the coordinated integration of components from the cytoplasm, the inner membrane, the periplasm, the outer membrane and even the extracellular space of Gram-negative bacteria. Here, we review injectisome assembly with an emphasis on the techniques that were employed towards its investigation. In particular, we focus on in vivo photocrosslinking, a technique that exploits the encoding of the artificial UV-inducible crosslinking amino acid p-benzoyl-phenylalanine to identify protein-protein interactions and to delineate assembly pathways.

    更新日期:2019-07-15
  • Consequences of dosing and timing on the antibacterial effects of ADEP antibiotics
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-14
    Christian Mayer, Peter Sass, Heike Brötz-Oesterhelt

    Antibiotic acyldepsipeptides (ADEPs) exert potent antibacterial activity in rodent models of bacterial infection and exceptional efficacy against persister cells of methicillin-resistant Staphylococcus aureus (MRSA). The mechanism of ADEP action is unusual in that the antibiotic releases the destructive capacity of over-activated ClpP, the proteolytic core of the bacterial Clp protease. The essential bacterial cell division protein FtsZ had emerged in a previous study as a preferred protein substrate of ADEP-activated ClpP but it is definitely not the only cellular substrate. In the current study, we set out to follow the morphological changes that lead to ADEP-mediated bacterial death in S. aureus and Bacillus subtilis, differentiating between antibacterial effects at low and high ADEP concentrations. Here, fluorescence and time-lapse microscopy data show that cells adopt a characteristic phenotype of cell division inhibition at ADEP levels close to the MIC, but retain the capacity to form viable daughter cells for a substantial period of time when transferred to ADEP-free growth medium. After extended exposure to low ADEP concentrations, nucleoids of B. subtilis started to disorganize and upon compound removal many cells failed to re-organize nucleoids, re-initiate cytokinesis and consequently died. Survival versus cell death of filamentous cells attempting recovery depended on the timing of completion of new septa in relation to the loss of cell envelope integrity. We show that the potential to recover after ADEP removal depends on the antibiotic concentration as well as the treatment duration. When exposed to ADEP at concentrations well above the MIC, biomass production ceased rapidly as did the potential to recover. In time-kill studies both long-time exposure to low ADEP levels as well as short-time exposure to high concentrations proved highly effective, while intermittent concentrations and time frames were not. We here provide new insights into the antimicrobial activity of ADEP antibiotics and the consequences of dosing and timing for bacterial physiology which should be considered in view of a potential therapeutic application of ADEPs.

    更新日期:2019-07-15
  • Role of the Streptomyces spore wall synthesizing complex SSSC in differentiation of Streptomyces coelicolor A3(2)
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-11
    B. Vollmer, N. Steblau, N. Ladwig, C. Mayer, B. Macek, L. Mitousis, S. Sigle, A. Walter, W. Wohlleben, G. Muth

    A crucial stage of the Streptomyces life cycle is the sporulation septation, a process were dozens of cross walls are synchronously formed in the aerial hyphae in a highly coordinated manner. This process includes the remodeling of the spore envelopes to make Streptomyces spores resistant to detrimental environmental conditions. Sporulation septation and the synthesis of the thickened spore envelope in S. coelicolor A3(2) involves the Streptomyces spore wall synthesizing complex SSSC. The SSSC is a multi-protein complex including proteins directing peptidoglycan synthesis (MreBCD, PBP2, Sfr, RodZ) and cell wall glycopolymer synthesis (PdtA). It also includes two eukaryotic like serin/threonine protein kinases (eSTPK), PkaI and PkaH, which were shown to phosphorylate MreC. Since unbalancing phosphorylation activity by either deleting eSTPK genes or by expressing a second copy of an eSTPK gene affected proper sporulation, a model was developed, in which the activity of the SSSC is controlled by protein phosphorylation.

    更新日期:2019-07-12
  • Role of α-glucan-induced oxygen species in dendritic cells and its impact in immune response against tuberculosis
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-11
    María Mercedes Romero, Alejandra Duarte, Mercedes Pastorini, Mercedes Alemán

    With 10 million new cases and three million deaths estimated to occur yearly ̶ more than any time in history ̶ tuberculosis (TB) remains the single most widespread and deadly infectious disease. Until recently, it was thought that both latent and active TB was primarily related to host factors. Nonetheless, the participation of bacterial factors is becoming increasingly evident. Minimal variations in genes related to Mycobacterium tuberculosis (Mtb) virulence and pathogenesis can lead to marked differences in immunogenicity. Dendritic cells (DC) are professional antigen presenting cells whose maturation can vary depending on the cell wall composition of each particular Mtb strain being critical for the onset of the immune response against Mtb. Here we evaluated the role played by α-glucan, in the endogenous production of reactive oxygen species, ROS, and the impact on DC maturation and function. Results showed that α-glucans on Mtb induce ROS production leading to DC maturation and lymphocyte proliferation. Even more, α-glucans induced Syk activation but were not essential in non-opsonized phagocytosis. In summary, α-glucans of Mtb participates in ROS production and the subsequent DC maturation and antigen presentation, suggesting a relevant role of α-glucans for the onset of the protective immune response against TB.

    更新日期:2019-07-12
  • Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-02-19
    Cinthia Alves-Barroco, Catarina Roma-Rodrigues, Natesan Balasubramanian, Marcia Aparecida Guimarães, Bernadete T. Ferreira-Carvalho, Jayaraman Muthukumaran, Daniela Nunes, Elvira Fortunato, Rodrigo Martins, Teresa Santos-Silva, Agnes M.S. Figueiredo, Alexandra R. Fernandes, Ilda Santos-Sanches

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

    更新日期:2019-07-06
  • Carriage meningococcal isolates with capsule null locus dominate among high school students in a non-endemic period, Italy, 2012–2013
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-03-06
    Arianna Neri, Cecilia Fazio, Luigina Ambrosio, Paola Vacca, Annamaria Barbui, Laura Daprai, Caterina Vocale, Iolanda Santino, Marco Conte, Lucia Rossi, Andrea Ciammaruconi, Anna Anselmo, Florigio Lista, Paola Stefanelli

    Meningococcal disease incidence in Italy remains quite low in the overall population except for infants. Within a study on carriage isolates among high school students we aimed to define: i) the prevalence of carriage isolates, ii) the phenotypic and iii) the molecular features of meningococci by Whole Genome Sequencing (WGS). A total of 1697 pharyngeal samples from undergraduate students (age range 14–19 years) were collected from 2012 to 2013 from six larger cities in Italy. One hundred and twenty culture positive meningococci (7%) were analyzed. Carriage isolates were sent to the National Reference Laboratory for invasive meningococcal disease (IMD) for PCR-based serogroup identification, Multilocus Sequence Typing, PorA and FetA typing. Moreover, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were typed. Core genome MLST (cgMLST) was performed on a subsample of 75 carriage isolates. Capsule null locus (cnl) predominated (47%), followed by serogroup B (27%). The antimicrobial susceptibility profile revealed an high prevalence of reduced susceptibility to penicillin G (54%) and a full susceptibility to ceftriaxone, ciprofloxacin and rifampicin. Carriage isolates presented a high genetic diversity: the clonal complexes (ccs) cc1136, cc198 and cc41/44, were the predominant. An high heterogeneity was also observed for PorA and FetA types. The fhbp and nhba genes were identified in all the carriage isolates; only 5% of the carriage isolates presented the nadA gene. The core genome MLST analysis revealed that the majority of the cnl isolates clustered in a distinct group. The evidence gathered during this study provides the estimate of carriage isolates in high school students in a non-epidemic period in Italy that was lower than expected. Moreover, the highest proportion of carriage isolates were cnl and, overall, they were molecular heterogeneous.

    更新日期:2019-07-06
  • Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-03-05
    Fabian K. Berger, Sabine Gfrörer, Sören L. Becker, Rossella Baldan, Daniela Maria Cirillo, Martinique Frentrup, Matthias Steglich, Pit Engling, Ulrich Nübel, Alexander Mellmann, Markus Bischoff, Barbara Gärtner, Lutz von Müller

    Clostridium (Clostridioides) difficile is the main cause of nosocomial diarrhoea. Ribotype 018 (RT018) has been recognized as the predominant strain responsible for C. difficile infection (CDI) in Italy, whereas in most other European countries only sporadic RT018 cases occur. Between August and October 2015, a suspected C. difficile outbreak at two associated hospitals in Southern Germany was investigated by comprehensive molecular typing. Surprisingly, RT018 was detected in 9/82 CDI patients, which has never been described before in a German outbreak. Phenotypic analysis revealed fluoroquinolone and macrolide resistance. Genetic subtyping using multiple-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS) was performed and outbreak isolates were directly compared to sporadic German RT018 isolates and to epidemic ones from Milan, Northern Italy. Molecular typing confirmed a hospital outbreak with closely related RT018 isolates. Both, MLVA and WGS revealed high similarity of outbreak strains with epidemic isolates from Italy, but low similarity to other German isolates. Comparison between both typing strategies showed that ribotyping in combination with MLVA was appropriate to identify related isolates and clonal complexes, whereas WGS provided a better discrimination with more detailed information about the phylogenetic relationship of isolates. This is the first hospital outbreak in Germany presumably caused by cross-national transmission of an Italian epidemic RT018 strain.

    更新日期:2019-07-06
  • Evolution of Klebsiella pneumoniae with mucoid and non-mucoid type colonies within a single patient
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-03-05
    Haejeong Lee, Juyoun Shin, Yeun-Jun Chung, Jin Yang Baek, Doo Ryeon Chung, Kyong Ran Peck, Jae-Hoon Song, Kwan Soo Ko

    We obtained nine Klebsiella pneumoniae isolates successively isolated from a single patient. Four pairs (M1–M4 and NM1–NM4) obtained simultaneously from the same site showed different colony types, mucoid and non-mucoid, while the final isolate (M5) was isolated alone from the blood and showed a mucoid phenotype. The whole genome of isolate M5 was sequenced de novo using the PacBio RSII system, while the others were sequenced with an Illumina Hiseq4000 and mapped to the genome sequences of M5. To identify insertions or deletions in the cps locus, we amplified and sequenced cps locus genes. We identified insertion sequence (IS) elements in several genes of the cps locus or one amino acid substitution in WcaJ in all non-mucoid isolates. Five additional amino acid alterations in RpsJ, LolE, Lon-2, PpsE, and a hypothetical protein were detected in some mucoid and non-mucoid isolates. Based on the genome data and cps locus sequences, the mucoid phenotype may have been lost or converted into the non-mucoid phenotype because of the insertion of IS elements or amino acid alterations at this locus. We inferred a within-host evolutionary scenario, in which non-mucoid variants emerged repeatedly from mucoid isolates, but may be short-lived because of their low fitness.

    更新日期:2019-07-06
  • Bacteria's different ways to recycle their own cell wall
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-07-04
    Christoph Mayer, Robert Kluj, Maraike Mühleck, Axel Walter, Sandra Unsleber, Isabel Hottmann, Marina Borisova

    The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6 P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This “anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6 P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.

    更新日期:2019-07-05
  • Assessing capreomycin resistance on tlyA deficient and point mutation (G695A) Mycobacterium tuberculosis strains using multi-omics analysis
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-24
    Jiao Zhao, Wenjing Wei, Huimin Yan, Ying Zhou, Zhenyan Li, Yanmei Chen, Chenchen Zhang, Jincheng Zeng, Tao Chen, Lin Zhou

    Capreomycin (CAP), a cyclic peptide antibiotic, is considered to be an ideal second-line drug for tuberculosis (TB). However, in the past few years, the emergence of more CAP-resistant (CAPr) TB patients has limited its use. Although it has been reported that CAP resistance to Mycobacterium tuberculosis (Mtb) is associated with rrs or tlyA mutation, the exact mechanism of CAPr Mtb strains, especially the mechanism associated with tlyA deficient or mutation, is not fully understood. Herein, we utilized a multi-omics (genome, proteome, and metabolome) approach to assess CAP resistance on tlyA deficient CAPr Mtb strains (CAPr1) and tlyA point mutation CAPr Mtb strains (CAPr2) that we established for the first time in vitro to investigate the CAP-resistant mechanism. Our results showed that the CAPr1 strains (> 40 μg/ml) was more resistant to CAP than the CAPr2 strains (G695A, 10 μg/ml). Furthermore, multi-omics analysis indicated that the CAPr1 strains exhibited greater drug tolerance than the CAPr2 strains may be associated with the weakening of S-adenosyl-L-methionine-dependent methyltransferase (AdoMet-MT) activity and abnormal membrane lipid metabolism such as suppression of fatty acid metabolism, promotion of glycolipid phospholipid and glycerolipid metabolism. As a result, these studies reveal a new mechanism for CAP resistance to tlyA deficient or mutation Mtb strains, and may be helpful in developing new therapeutic approaches to prevent Mtb resistance to CAP.

    更新日期:2019-06-24
  • Identification of two abundant Aerococcus urinae cell wall-anchored proteins
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-24
    Erik Senneby, Torgny Sunnerhagen, Björn Hallström, Rolf Lood, Johan Malmström, Christofer Karlsson, Magnus Rasmussen

    Aerococcus urinae is an emerging pathogen that causes urinary tract infections, bacteremia and infective endocarditis. The mechanisms through which A. urinae cause infection are largely unknown. The aims of this study were to describe the surface proteome of A. urinae and to analyse A. urinae genomes in search for genes encoding surface proteins. Two proteins, denoted Aerococcal surface protein (Asp) 1 and 2, were through the use of mass spectrometry based proteomics found to quantitatively dominate the aerococcal surface. The presence of these proteins on the surface was also shown using ELISA with serum from rabbits immunized with the recombinant Asp. These proteins had a signal sequence in the amino-terminal end and a cell wall-sorting region in the carboxy-terminal end, which contained an LPATG-motif, a hydrophobic domain and a positively charged tail. Twenty-three additional A. urinae genomes were sequenced using Illumina HiSeq technology. Six different variants of asp genes were found (denoted asp1-6). All isolates had either one or two of these asp-genes located in a conserved locus, designated Locus encoding Aerococcal Surface Proteins (LASP). The 25 genomes had in median 13 genes encoding LPXTG-proteins (range 6-24). For other Gram-positive bacteria, cell wall-anchored surface proteins with an LPXTG-motif play a key role for virulence. Thus, it will be of great interest to explore the function of the Asp proteins of A. urinae to establish a better understanding of the molecular mechanisms by which A. urinae cause disease.

    更新日期:2019-06-24
  • The Multicopper Oxidase of Mycobacterium tuberculosis (MmcO) Exhibits Ferroxidase Activity and Scavenges Reactive Oxygen Species in Activated THP-1 Cells
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-24
    Eyad Kinkar, Ayat Kinkar, Mazen Saleh

    The MmcO protein of Mycobacterium tuberculosis is a membrane-associated multicopper oxidase. Its natural substrate(s) and its role in pathogenesis are not well characterized. A recent report proposes that MmcO contributes to copper resistance in M. tuberculosis during infection. We have expressed and reconstituted the active enzyme from inclusion bodies in E. coli. MmcO exhibits maximal activity against the experimental substrate 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) or ABTS, at pH 4. The enzyme also exhibits ferroxidase activity at pH 4. Most notable was the finding that MmcO is able to scavenge the reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase enzyme system. This ROS scavenging activity of MmcO was also evident against ROS generated by THP-1 cells. We propose that MmcO protects M. tuberculosis during infection against ROS attack in addition to providing copper resistance to the pathogen.

    更新日期:2019-06-24
  • Assembly and targeting of Secretins in the bacterial outer membrane
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-19
    Janani Natarajan, Nidhi Singh, Doron Rapaport

    In Gram-negative bacteria, secretion of toxins ensure the survival of the bacterium. Such toxins are secreted by sophisticated multiprotein systems. The most conserved part in some of these secretion systems are components, called secretins, which form the outer membrane ring in these systems. Recent structural studies shed some light on the oligomeric organization of secretins. However, the mechanisms by which these proteins are targeted to the outer membrane and assemble there into ring structures are still not fully understood. This review discusses the various species-specific targeting and assembly pathways that are taken by secretins in order to form their functional oligomers.

    更新日期:2019-06-19
  • Cryopreservation of the human gut microbiota: current state and perspectives
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-04
    Daria V. Smirnova, Ljubov V. Zalomova, Angelika V. Zagainova, Valentin V. Makarov, Ludmila M. Mezhevikina, Eugeny E. Fesenko, Sergey M. Yudin

    The human intestinal microbiota is a complex ecosystem that consists of thousands of bacterial species that are responsible for human health and disease. The intestinal microbiota is a natural resource for production of therapeutic and preventive medicals, such as probiotics and fecal transplants. Modern lifestyles have resulted in the extinction of evolutionally selected microbial populations upon exposure to environmental factors. Therefore, it is very important to preserve the human gut microbiota to have the opportunity for timely restoration with minimal safety risks. Cryopreservation techniques that are suitable for the preservation of viable, mixed microbial communities and a biobanking approach are currently under development in different countries. However, the number of studies in this area is very limited. The variety of morphological and physiological characteristics of microbes in the microbiota, the different cryopreservation goals, and the criteria for the evaluation of cryopreservation effectiveness are the main challenges in the creation of a universal and standardized cryopreservation protocol. In this review, we summarized the current progress of the main cryopreservation techniques for gut microbiota communities and the methods for the assessment of the effectiveness of these techniques in the context of practical application.

    更新日期:2019-06-04
  • MprF-mediated daptomycin resistance
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-04
    Christoph M. Ernst, Andreas Peschel

    Daptomycin has become an important antibiotic for the treatment of serious Methicillin-Resistant Staphylococcus aureus (MRSA) infections. Unlike other approved antibiotics, its mode of action is still under active investigation, as well as the molecular basis of daptomycin resistance, which emerges in some cases during daptomycin treatment. Small nucleotide polymorphisms (SNPs) in the Multiple Peptide Resistance Factor (MprF) appear to play a major role in the resistance mechanism. Until recently, the impact of the SNPs on MprF activity has remained unclear, which is due to conflicting reports on resistance-associated phenotypes and an incomplete understanding of the mode of action of MprF. However, recent structural insights into MprF and studies with isogenic mutants have now led to a new model of MprF-mediated daptomycin resistance, which harmonizes most of the observed phenotypes and provides a basis for challenging biochemical investigations.

    更新日期:2019-06-04
  • Bacterial Adhesion and Host Cell Factors Leading to Effector Protein Injection by Type III Secretion System
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-04
    Erwin Bohn, Michael Sonnabend, Kristina Klein, Ingo B. Autenrieth

    Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-β-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed.

    更新日期:2019-06-04
  • Structural diversity of coiled coils in protein fibers of the bacterial cell envelope
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-03
    Birte Hernandez Alvarez, Jens Bassler, Andrei N. Lupas

    The cell envelope of bacteria shows great diversity in architecture and composition, to a large extent due to its proteome. Proteins localized to the cell envelope, whether integrally embedded in the membrane, membrane-anchored, or peripherally associated as part of a macromolecular complex, often form elongated fibers, in which coiled coils represent a prominent structural element. These coiled-coil segments show a surprising degree of structural variability, despite being shaped by a small number of simple biophysical rules, foremost being their geometry of interaction referred to as 'knobs-into-holes'. Here we will review this diversity, particularly as it has emerged over the last decade.

    更新日期:2019-06-04
  • Type I interferon induced by TLR2-TLR4-MyD88-TRIF-IRF3 controls Mycobacterium abscessus subsp. abscessus persistence in murine macrophages via nitric oxide
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-01
    Nanthapon Ruangkiattikul, Doris Rys, Ketema Abdissa, Manfred Rohde, Torsten Semmler, Pia-K. Tegtmeyer, Ulrich Kalinke, Carsten Schwarz, Astrid Lewin, Ralph Goethe

    Mycobacterium abscessus (MAB) is an emerging, rapidly growing non-tuberculous Mycobacterium causing therapy-resistant pulmonary disease especially in patients with cystic fibrosis (CF). Smooth and rough colony type MAB can be isolated from infected patients whereby rough colony type MAB are more often associated with severe disease. Disease severity is also associated with an alternated type I interferon (IFN-I) response of the MAB-infected patients. However the relevance of this response for the outcome of MAB infection is still unknown. In this study, we analyzed the IFNβ expression of murine macrophages infected with a MAB rough colony strain (MAB-R) isolated from a patient with progressive CF and compared it to macrophages infected with the MAB smooth colony type reference strain (MAB-S). We found that MAB-R infected macrophages expressed significantly more IFNβ mRNA and protein than MAB-S infected macrophages. Higher IFNβ induction by MAB-R was associated with higher TNF expression and intracellular killing while low IFNβ induction was associated with lower TNF expression and persistence of MAB-S. IFNβ induction was independent of the intracellular cGAS-STING recognition pathway. MAB appeared to be recognized extracellularly and induced IFNβ expression via TLR2-TLR4-MyD88-TRIF-IRF3 dependent pathways. By using macrophages lacking the IFN-I receptor we demonstrate that MAB induced IFN-I response essentially contributed to restricting MAB-R and MAB-S infections by activating macrophage Nos2 expression and nitric oxide production. Thus IFN-I seem to influence the intrinsic ability of macrophages to control MAB infections. As MAB persists over long time periods in susceptible patients, our findings suggest that virulence of MAB strains is promoted by an insufficient IFN-I response of the host.

    更新日期:2019-06-03
  • Biogenesis and function of the autotransporter adhesins YadA, intimin and invasin
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-06-01
    Karolin Leibiger, Jonas Malte Schweers, Monika Schütz

    Bacteria often express numerous virulence factors. These virulence factors make them successful pathogens, by e.g. mediating attachment to host cells and thereby facilitating persistence or invasion, or by contributing to the evasion of the host immune system to allow proliferation and spread within the host and in the environment. The site of first contact of Gram negative bacteria with the host is the bacterial outer membrane (OM). Consisting of an asymmetrical lipid bilayer with phospholipids forming the inner, and lipopolysaccharides forming the outer leaflet, the OM harbors numerous integral membrane proteins that are almost exclusively β-barrel proteins. One distinct family of OM β-barrel proteins strongly linked to bacterial virulence are the autotransporter (AT) proteins. During the last years huge progress has been made to better understand the mechanisms underlying the insertion of AT proteins into the OM and also AT function for interaction with the host. This review shortly summarizes our current knowledge about outer membrane protein (OMP) and more specifically AT biogenesis and function. We focused on the AT proteins that we haved studied in most detail: i.e. the Yersinia adhesin A (YadA) and invasin of Yersinia enterocolitica (Ye) as well as its homolog intimin (Int) expressed by enteropathogenic Escherichia coli. In addition, this review provides a short outlook about how we could possibly use this knowledge to fight infection.

    更新日期:2019-06-03
  • Live-cell imaging of Streptomyces conjugation
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-05-31
    L. Thoma, Bernd Vollmer, F. Oesterhelt, G. Muth

    Time-lapse imaging of conjugative plasmid transfer in Streptomyces revealed intriguing insights into the unique two-step conjugation process of this Gram+ mycelial soil bacterium. Differentially labelling of donor and recipient strains with distinct fluorescent proteins allowed the visualization of plasmid transfer in living mycelium. In nearly all observed matings, plasmid transfer occurred when donor and recipient hyphae made intimate contact at the lateral walls. Plasmid transfer does not involve a complete fusion of donor and recipient hyphae, but depends on a pore formed by the FtsK-like DNA translocase TraB. Following the initial transfer at the contact site of donor and recipient, the plasmids spread within the recipient mycelium by invading neighboring compartments, separated by cross walls. Intra-mycelial plasmid spreading depends on a septal cross wall localized multi-protein DNA translocation apparatus consisting of TraB and several Spd proteins and is abolished in a spd mutant. The ability to spread within the recipient mycelium is a crucial adaptation to the mycelial life style of Streptomyces, potentiating the efficiency of plasmid transfer.

    更新日期:2019-05-31
  • Caprazamycins: biosynthesis and structure activity relationship studies
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-05-24
    Franziska Wiker, Nils Hauck, Stephanie Grond, Bertolt Gust

    Cell wall biosynthesis represents a valid target for antibacterial action but only a limited number of chemical structure classes selectively interact with specific enzymes or protein structures like transporters of the cell envelope. The integral membrane protein MraY translocase is essential for peptidoglycan biosynthesis catalysing the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate, thereby generating the cell wall intermediate lipid I. Not present in eukaryotic cells, MraY is a member of the superfamily of yet not well-understood integral membrane enzymes which involve proteins for bacterial lipopolysaccharide and teichoic acid or eukaryotic N-linked saccharides biosynthesis. Different natural nucleoside antibiotics as inhibitors of MraY translocase have been discovered comprising a glycosylated heterocyclic pyrimidin base among other potential lipid-, peptidic- or sugar moieties. Caprazamycins are liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2. They possess activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including M. intracellulare, M. avium and M. tuberculosis. Structural elucidation revealed the (+)-caprazol core skeleton as a unique moiety, the caprazamycins share with other MraY inhibitors such as the liposidomycins, A-90289 and the muraminomicins. They also share structural features such as uridyl-, aminoribosyl- and fatty acyl-moieties with other MraY translocase inhibitors like FR-900493 and the muraymycins. Intensive studies on their biosynthesis during the last decade identified not only common initial biosynthetic steps, but also revealed possible branching points towards individual biosynthesis of the respective compound. Structural diversity of caprazamycins was generated by feeding experiments, genetic engineering of the biosynthetic gene clusters and chemical synthesis for structure activity relationship studies with its target, MraY translocase.

    更新日期:2019-05-24
  • IP-10 contributes to the inhibition of mycobacterial growth in an ex vivo whole blood assay
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-05-22
    Ivana Palucci, Basem Battah, Alessandro Salustri, Flavio De Maio, Linda Petrone, Fabiola Ciccosanti, Michela Sali, Vincent Bondet, Darragh Duffy, Gian Maria Fimia, Delia Goletti, Giovanni Delogu

    Interferon-γ inducible protein 10 (IP-10), is a potent chemoattractant that promotes migration of monocytes and activated T-cells to inflammation foci. IP-10 is elevated in serum of patients with chronic hepatitis C virus (HCV) and tuberculosis (TB) infections, although it remains to be determined the contribution of IP-10 in restricting Mycobacterium tuberculosis (Mtb) replication. Here, we investigated the impact of IP-10 on mycobacteria replication using the ex vivo model of human whole-blood (WB) assay. In particular, we compared the levels of IP-10 upon infection with different Mtb clinical strains and species of non-tuberculous mycobacteria (NTM) and evaluated how IP-10 may contain bacterial replication. Interestingly, we observed that the inhibition of the host enzyme dipeptidyl peptidase IV (DPP-IV), which inactivates IP-10 through cleavage of two amino acids at the chemokine N-terminus, restricted mycobacterial persistence in WB, supporting the critical role of full length IP-10 in mediating an anti-Mtb response. Addition of recombinant IP-10 expressed in eukaryotic cells enhanced the anti-mycobacterial activity in WB, although no differences were observed when IP-10 containing different proportions of cleaved and non-cleaved forms of the chemokine were added. Moreover, recombinant IP-10 did not exert a direct anti-mycobacterial effect. Our results underscore the clinical relevance of IP-10 in mycobacteria pathogenesis and support the potential outcomes that may derive by targeting the IP-10/CXCR3 pathway as host directed therapies for the treatment of Mtb or NTM infections.

    更新日期:2019-05-23
  • The prevalence of Staphylococcus aureus with mucoid phenotype in the airways of patients with cystic fibrosis – A prospective study
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-05-11
    Farina E. Lennartz, Bianca Schwartbeck, Angelika Dübbers, Jörg Große-Onnebrink, Christina Kessler, Peter Küster, Holger Schültingkemper, Georg Peters, Barbara C. Kahl

    Background Staphylococcus aureus is one of the most frequently isolated pathogens in the respiratory tract of CF patients. Recently, we characterized peculiar mucoid S. aureus isolates, which are excessive biofilm formers and which carried a 5bp-deletion within the intergenic region of the ica operon. In this prospective study, we determined the prevalence of mucoid S. aureus-isolates in the airways of CF-patients during a 3-months period. Methods We analyzed specimens (sputa, throat swabs) from 81 CF patients who attended two CF centers in Münster, Germany. Ten S. aureus isolates were randomly picked from every S. aureus-positive airway specimen and evaluated for mucoidy using Congo Red agar and phenotypic tests. Mucoid isolates were characterized by spa sequence typing, biofilm production and sequencing of the intergenic region of the ica operon to screen for the 5bp-deletion. Results In 7 of 81 examined patients (8.6%), we detected mucoid S. aureus phenotypes (37 out of 1050 isolates; 3.5%). Twenty-five mucoid isolates carried the 5bp-deletion. Mucoid isolates produced excessive biofilm and were significantly more resistant to certain antibiotics. Conclusions In our prospective study, mucoid S. aureus was present in 8.6% of S. aureus–positive CF-patients. In 6 of 7 patients, mucoid isolates carried the 5bp-deletion, indicating that also other so far not identified mechanisms cause excessive biofilm formation. Further studies are necessary to ascertain the clinical impact of mucoid S. aureus phenotypes on the severity of the CF disease.

    更新日期:2019-05-12
  • Genomic investigation of a sequence type 67 Clostridium difficile causing community-acquired fulminant colitis in Hong Kong
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-05-11
    Huiluo Cao, Sally Cheuk-Ying Wong, Wing-Cheong Yam, Melissa Chun-Jiao Liu, Kin-Hung Chow, Alan Ka-Lun Wu, Pak-Leung Ho

    In 2017, we identified a Clostridium difficile strain HKCD4 that caused community-acquired fulminant colitis in a previously healthy child. Phylogenetically, it belonged to clade 2, sequence type 67 and was resistant to fluoroquinolone and tetracycline. The strain was pathogenicity locus and binary toxin positive. It has a mutation in the trehalose repressor treR leading to the L172I substitution that was previously reported in the epidemic ribotype 027 lineage. HKCD4 has a tcdB sequence that shared very high identities with 3 highly virulent reference strains. It has a CpG depleted genome that is characteristic of hypervirulent C. difficile. The emergence of ST67 lineage with molecular feature of hypervirulence in the community is concerning and emphasizes the need for full characterization of strains causing severe disease in patients without classical risk factors.

    更新日期:2019-05-12
  • Effect of amyloid curli fibrils and curli CsgA monomers from Escherichia coli on in vitro model of intestinal epithelial barrier stimulated with cytokines
    Int. J. Med. Microbiol. (IF 3.362) Pub Date : 2019-05-11
    Beata Sobieszczańska, Barbara Pawłowska, Anna Duda-Madej, Krzysztof Pawlik, Jerzy Wiśniewski, Jędrzej Grzegrzółka, Michał Turniak, Urszula Walczuk, Andrzej Gamian

    Amyloid curli fibrils produced by Escherichia coli are well-known virulence factor influencing E. coli adhesion and biofilm formation. However, the impact of curli on intestinal epithelial barrier stimulated with proinflammatory cytokines is unknown. In the study, we examined the effect of curli produced by nonpathogenic E. coli K-12 and wild-type E. coli EC32 strains, and purified CsgA proteins on differentiated Caco-2 cell monolayers stimulated with a mixture of IL-1β, TNF-α, and INFγ cytokines as a model of ‘inflamed intestinal epithelial barrier’ in vitro. The results of the study indicated that curliated E. coli adhered better to polarized Caco-2 cells than their curli-deficient mutants and the adherence was further augmented by stimulation of epithelial cells with proinflammatory cytokines. Interestingly, curli reduced internalization but enhanced intracellular survival of the wild-type E. coli strain EC32 within intestinal epithelial cells. Curli-expressing E. coli, as well as purified CsgA proteins, attenuated IL-8 secretion by unstimulated Caco-2 cells, although the effect was barely observed on cytokine-stimulated cells. The findings of the study revealed that curli fibrils are an important virulence factor enabling curliated E. coli to effectively colonize intestinal epithelium especially in individuals with inflammatory intestinal disorders.

    更新日期:2019-05-12
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