Toxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and poultry

doi:10.1016/j.anaerobe.2020.102298

Anaerobe

Volume 67, February 2021, 102298

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Highlights

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C. perfringens type A (90.67%), type C (2.67%), type D (4%), type F (2.67%) were recovered.
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Tetracycline (41.33 %), erythromycin (34.66%) and bacitracin (17.33%) resistance genes were predominant among the isolates.
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Weak positive relationship between tetracycline and bacitracin genes based on correlation matrix analysis.

Abstract

The present study was designed to understand the presence of antimicrobial resistance among the prevalent toxinotypes of Clostridium perfringens recovered from different animals of Tamil Nadu, India. A total of 75 (10.76%) C. perfringens were isolated from 697 multi-species fecal and intestinal content samples. C. perfringens type A (90.67%), type C (2.67%), type D (4%) and type F (2.67%) were recovered. Maximum number of isolates were recovered from dog (n = 20, 24.10%) followed by chicken (n = 19, 5.88%). Recovered isolates were resistant to gentamicin (44.00%), erythromycin (40.00%), bacitracin (40.00%), and tetracycline (26.67%), phenotypically and most of the isolates were found to be resistant to multiple antimicrobials. Genotypic characterization revealed that tetracycline (41.33%), erythromycin (34.66%) and bacitracin (17.33%) resistant genes were present individually or in combination among the isolates. Combined results of phenotypic and genotypic characterization showed the highest percentage of erythromycin resistance (26.66%) among the isolates. None of the isolates showed amplification for lincomycin resistance genes. The correlation matrix analysis of genotypic resistance showed a weak positive relationship between the tetracycline and bacitracin resistance while a weak negative relationship between the tetracycline and erythromycin resistance. The present study thus reports the presence of multiple-resistance genes among C. perfringens isolates that may be involved in the dissemination of resistance to other bacteria present across species. Further insights into the genome can help to understand the mechanism involved in gene transfer so that measures can be taken to prevent the AMR spread.

Graphical abstract

Introduction

Clostridium perfringens is a ubiquitous organism found in the normal intestine, sewage, food products, etc. C. perfringens, a gram-positive, anaerobic spore-forming organism is also associated with various diseases in animals and human [1,2]. C. perfringens produces different extracellular enzymes and toxins. Based on the major toxins produced namely α-toxin, β-toxin, ε-toxin and ι-toxin, C. perfringens have been classified into 5 toxinotypes (A to E) [3]. Recently, two more toxinotypes (F and G) has been proposed based on additional toxin repertoire. It was proposed that type F include C. perfringens producing enterotoxin (cpe gene) involved in food poisoning and type G include C. perfringens producing NetB toxin (netB gene) involved in necrotic enteritis in poultry [4].

C. perfringens type A causes enterocolitis in horses, pigs and dogs. Apart from type G, type A is also involved in necrotic enteritis in chickens. Type B causes necrotizing enteritis in cattle, sheep. Type C causes necrohemorrhagic enteritis in sheep, goats, piglets, horses, etc. Type D causes enterotoxaemia in cattle, sheep, goat and type E causes enteritis in cattle, sheep and rabbits. Type F causes hemorrhagic gastroenteritis in dogs and colitis in horse. Type F is mainly responsible for human food poisoning, antibiotic associated diarrhea and sporadic diarrhea. Type G is involved in necrotic enteritis in poultry [5]. Enterotoxaemia caused by type D in sheep and goat is a more prevalent and important disease that can cause sudden mortality [5].

Antimicrobial agents like tetracycline, lincomycin, bacitracin and other agents are commonly used in feed to improve health and production of poultry and other animals [6,7]. Long term use or unintended use of antimicrobial agents has led to the rapid development of resistance among C. perfringens isolates worldwide [8]. Antimicrobial resistance is conferred by various genes and they play a major role in transfer of resistance to other bacteria within the genus or between genera Understanding the role of the resistant genes can aid in the prevention of gene transfer between the microbes. Genes like tetP(B), tet(M), tetA(P), and tetB(P) were linked with tetracycline resistance which has been reported from various parts of the world [9]. Similarly, other genes have been identified and are linked to antimicrobial resistance [10].

Antimicrobial resistance among C. perfringens isolates recovered from India has been documented by various researchers [11,12]. Most of the studies from India on antimicrobial resistance among C. perfringens were restricted to phenotypic testing. Reports on genotypic characterization of antimicrobial resistance genes among C. perfringens from India are scarce. A comprehensive study on the prevalence of C. perfringens from different animal species, its antimicrobial resistance determination both by phenotypic and genotypic testing can help to understand the role of the genes in resistance transfer. Therefore, the present study was designed to know the prevalence of C. perfringens among different animal species and its antimicrobial resistance both by phenotypic and genotypic methods.

Section snippets

Sample collection

A total of 697 samples were collected randomly from four different districts of Tamil Nadu (Table 1). Fecal swabs were collected from Kanchipuram (n = 100), Vellore (n = 91), Chennai (n = 83, dog samples), Tiruvallur (n = 207) while intestinal contents were collected during post-mortem (PM) from Chennai (n = 116, chicken samples) and Tiruvallur (n = 100, chicken samples). Sterile swabs were used for the collection of feces sample and intestinal contents were collected in sterile containers

Isolation and toxinotyping

Out of 697 samples collected, 75 samples (10.76%) were positive for C. perfringens cpa gene by PCR. Among the 75 isolates, 67 (89.33%) isolates belong to Type A (cpa gene positive) and one (1.33%) isolate was positive for cpa and cpb2 gene. Two isolates (2.67%) belonged to type C (cpa and cpb gene), 3 (4.0%) isolates belonged to type D (cpa and etx gene) and 2 (2.67%) isolates belonged to type F (cpa and cpe gene) (Fig. 1). iap and netB genes were found negative in all the isolates tested.

Discussion

In the present study, an overall prevalence of 10.76% has been reported which is lesser compared to other earlier multi-species studies from India [12]. Samples were collected randomly and fecal samples from healthy animals constituted a major part of this survey. Multi-species (pig, goat, fish and human) studies from India involving mostly samples from diarrheic cases reported a higher prevalence of 36.09% [12]. Most of the C. perfringens recovered in the present investigation were type A

Conclusions

Antimicrobial resistance is a burning problem worldwide and resistance is recorded among various anaerobic organisms including C. perfringens. The present study documented the phenotypic and genotypic antimicrobial resistance among C. perfringens isolates of multi-species origin. C. perfringens type A, C, D and F were isolated during the study and multidrug resistance was recorded among several isolates. Multidrug-resistant genes were also present in the study showing the impact of

Declaration of competing interest

•
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
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It is to specifically state that “No Competing interests are at stake and there is No Conflict of Interest” with other people or organizations that could inappropriately influence or bias the content of the paper.

Acknowledgements

Authors like to acknowledge the funding received from Department of Biotechnology (DBT) for the Twinning project on “Genetic “Characterization of Antibiotic Resistance in Clostridium perfringens and Clostridium difficile of food animal origin and its Public Health Significance” (BT/PR25444/NER/95/1200/2017, May 23, 2018).

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Clostridium perfringens associated with dairy farm systems show diverse genotypes
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The presence of this resistance marker is also associated with resistance to macrolides and streptogramin B, and explains the phenotypic resistance to erythromycin these 3 isolates also showed. Other studies have also noted relatively higher prevalence of resistance to clindamycin and erythromycin among isolate cohorts, and collectively this suggests these may be sub-optimal choices in treatment where the antibiogram is unknown (Anju et al., 2021; Li et al., 2020; Slavić et al., 2011). In animals, treatment of type D-related infection is problematic.
Clostridium perfringens is a bacterial species of importance to both public and animal health. Frequently found in food system environments, it presents a risk to food animal health such as dairy herds, and may cross contaminate associated ingredients or food products, with potential to cause sporadic and outbreaks of disease in human populations, including gastroenteric illness. In this study, we characterized C. perfringens isolated from bovine, caprine, and ovine dairy farm systems (n = 8, 11 and 4, respectively). Isolates were phenotypically screened for antimicrobial sensitivity profiling, and subjected to whole genome sequencing to elucidate related genetic markers, as well as examine virulence gene markers, mobile genetic elements, and other features. Both toxin type A and type D isolates were identified (78 % and 22 % of isolates, respectively), including 20 novel sequence types. Resistance to clindamycin was most prevalent among antibiotics screened (30 %), followed by erythromycin (13 %), then penicillin and tetracycline (4 %), although an additional 3 isolates were non-susceptible to tetracycline. Most isolates harboured plasmids, which mobilised virulence markers such as etx, cpb2, and resistance markers tetA(P), tetB(P), and erm(Q), on conjugative plasmids. The presence of type D isolates on caprine farms emphasizes the need for control efforts to prevent infection and potential enterotoxemia. Clostridium perfringens enterotoxin (cpe) was not identified, suggesting lower risk of gastrointestinal illness from contaminated foods, the presence of other virulence and antimicrobial resistance markers suggests farm hygiene remains an important consideration to help ensure food safety of associated dairy foods produced.
Prevalence, molecular characterization, and antimicrobial resistance profile of Clostridium perfringens from India: A scoping review
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Clostridium perfringens is one of the most important foodborne pathogens that causes histotoxic diseases and intestinal infections in both humans and animals. The present scoping review has been designed to analyze the literature published during 2000–2021 from India on the prevalence, molecular characterization, and antimicrobial resistance profile of C. perfringens isolates recovered from humans, animals, animal-based foods, and associated environmental samples. The peer-reviewed articles retrieved from four electronic databases (Google Scholar, PubMed, Science Direct, and Web of Science) were assessed using PRISMA-ScR guidelines. A total of 32 studies from India were selected on the basis of their relevance and inclusion criteria. The overall prevalence of C. perfringens among domestic animals having history of clinical symptoms and among healthy animals was found to be 65.8% (508/772) and 42.8% (493/1152), respectively. The pathogen was also detected in clinically affected wild animals (75%), healthy wild animals (35.4%), and captive birds (24.5%). The detection of C. perfringens among poultry having necrotic enteritis and among healthy birds was found to be 66.8% (321/480) and 25.6% (80/312), respectively. The detection of pathogen among animal-based foods (i.e., meat, milk, and fish and their products) and environmental samples depicted a prevalence of 20.8% (325/1562) and 30.2% (23/76), respectively. However, the prevalence of C. perfringens among humans having history of diarrhea and among healthy humans was found to be 25% (70/280) and 23.2% (36/155), respectively. The genotyping of C. perfringens isolates revealed that toxin type A was found to be the most prevalent genotype. Along with the alpha toxin gene (cpa), beta (cpb), epsilon (etx), iota (itx), enterotoxin (cpe), beta-2 toxin (cpb2), and NetB (netB) toxins were also detected in different combinations. Antimicrobial resistance profile of C. perfringens isolates recovered from different sources demonstrated that the highest resistance was detected against sulphonamides (76.8%) and tetracycline (41.3%) by phenotypic and genotypic detection methods, respectively. Comprehensive scientific studies covering different geographical areas at the human-animal-environment interface are crucial to generalize the real magnitude of C. perfringens-associated problem in India and for establishing a reliable database.
Prevalence, toxinotyping, antimicrobial susceptibility and biofilm-forming ability of Clostridium perfringens isolated from free-living rodents and shrews
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Citation Excerpt :
In the current study, C. perfringens type A (59.2%) was the most prevalent followed by Type A cpb2+ (33.3%), Type C (3.7%), and Type C cpb2+ (3.7%). Almost all earlier studies have reported the predominance of type A in their samples [16,26–28,34]. Among the 7 toxigenic types, C. perfringens type A is the most implicated type in foodborne outbreaks throughout the world [35].
Clostridium perfringens (C. perfringens), is a spore-forming and toxin-producing pathogenic Gram-positive rod-shaped bacterium with immense public health/zoonotic concern. Rodents are well-known reservoirs and vectors for a large number of zoonoses and strong links have been recognized between synanthropic rodents and foodborne disease outbreaks throughout the world. To date, no study has been conducted for studying the prevalence of C. perfringens in rodents and shrews. In this study, we investigated faecal samples from free-living rodents and shrews trapped in Meghalaya, a North-eastern hill state of India for the presence of virulent and antimicrobial-resistant C. perfringens.
A total of 122 animals comprising six species of rodents and one species of shrews were trapped: Mus musculus (n = 15), Mus booduga (n = 7), Rattus rattus (n = 9), Rattus norvegicus (n = 3), Bandicota indica (n = 30), Bandicota bengalensis (n = 32) and Suncus murinus (n = 26). The faecal swabs were collected and processed for the isolation of C. perfringens. Toxinotyping was done using PCR. Antimicrobial susceptibility testing and biofilm forming ability testing were done using Kirby Bauer disc diffusion method and crystal violet assay.
C. perfringens was isolated from 27 of the 122 faecal swabs (22.1%), from six species of rodents and shrews. Five of the host species were rodents, Bandicota bengalensis (25%), Bandicota indica (16.7%), Rattus norvegicus (33.3%), Mus musculus (13.3%), Mus booduga (42.8%) and Suncus murinus (shrew) (29.6%). The common toxinotype was type A (59.2%) followed by Type A with beta2 toxin (33.3%), Type C (3.7%) and Type C with beta2 toxin (3.7%). None of the isolates harboured cpe, etx, iap, and NetB genes and therefore none was typed as either B, D, E, F, or G. Nine isolates (33.3%) turned out to be multi-drug resistant (MDR), displaying resistance to three or more categories of antibiotics tested. Twenty-three out of twenty-seven isolates (85.2%) were forming biofilms.
Globally, this is the first study to report the prevalence of C. perfringens and its virulence profile and antimicrobial resistance in free-living rodents and shrews. The rodents and shrews can potentially contaminate the food and environment and can infect humans and livestock with multi-drug resistant/virulent Type A and Type C C. perfringens.
Tracking Clostridium perfringens strains from breeding duck farm to commercial meat duck farm by multilocus sequence typing
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Citation Excerpt :
The results of toxin typing showed that not only C. perfringens type A was isolated, but also type F and type G were isolated. Type A was the most prevalent in this study, which was also the main prevalent toxin type of C. perfringens in poultry around the world (Anju et al., 2020). Secondly, strains belonging to type F were closely associated not only with antibiotic-associated diarrhea but also with food poisoning (Kiu and Hall, 2018).
Clostridium perfringens (C. perfringens) is an important zoonotic food-borne pathogenic microorganism. Currently, there are many reports on the prevalence of C. perfringens in poultry farms, while few studies on the prevalence and infection source of C. perfringens in egg hatcheries. The present study was undertaken to investigate and track C. perfringens from one breeding duck farm, one duck egg hatchery and one commercial meat duck farm along the production chain. A total of 334 isolates were obtained from 788 samples, including 316 type A strains (94.61 %), 11 type F strains (3.29 %) and seven type G strains (2.10 %). Antimicrobial susceptibility testing revealed that 61.87 % of the tested isolates were multidrug-resistant. Multilocus sequence typing showed that 66 representative isolates encompassed 60 different sequence types (STs), clustered in ten clonal complexes (CCs) and 20 singletons. CC2 was the most popular CC, accounting for 58.82 % (10/17) of the strains from breeding duck farm. Some strains of duck embryos were distributed in the same ST or CC as strains from breeding duck farm and duck egg hatchery, indicating close genetic relationship between them. The study showed that isolates from breeding duck farm had formed a dominant CC through long-term evolution, some duck embryos had been infected with C. perfringens during the incubation period and the infected strains should be from the duck egg hatchery or breeding duck farm with the possibility of vertical transmission. The close relationship between strains from breeding ducks and duck embryos, the high antibiotic resistance of isolates and the presence of cpe-positive or netB-positive isolates indicated that alert should be paid to such risks associated with these.
Removal of tetracycline from livestock wastewater by positive single pulse current electrocoagulation: Mechanism, toxicity assessment and cost evaluation
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Although the use of antibiotics has made livestock farming more economically profitable, it has also created widespread antibiotic pollution, which continues to be difficult to resolve (Antonio et al., 2021). Previous studies have revealed that the discharge of livestock wastewater has contributed to the presence of bacterial pathogens in the aquatic environment (Anju et al., 2021). Accordingly, the discharge of such wastewater may present a significant threat to human health.
The widespread use of veterinary antibiotics has led to the significant problem of contamination of livestock wastewater with significant amount of antibiotics. Electrocoagulation (EC) has become a prominent research topic because of the technique's ability to remove antibiotics from livestock wastewater. However, an urgent solution is needed to reduce the high operating costs associated with the process. Therefore, in this study, we developed a positive single pulse current (PSPC)-EC system to remove tetracycline (TC) from synthetic and actual livestock wastewater. Influential factors were investigated, and the optimal PSPC-EC operating parameters were identified as follows: duty ratio = 60%, pH = 4, electrode spacing = 1 cm, current intensity = 0.2 A, and conductivity = 2 mS cm⁻¹. The mechanism of PSPC-EC was characterised using techniques including scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. The TC decomposition pathway was proposed based on the generation of its intermediate products. A toxicity estimation software tool (TEST) model was used to evaluate the toxicity of TC and its main degradation products, and most of its intermediates were found to be less toxic than TC. The contribution ratios of floc adsorption and electrochemical oxidation for removing TC were 74.17% and 21.48%, respectively. The highest TC removal rate reached 95% with an operating cost of 0.011 USD/m³. Finally, under the optimum conditions identified, actual livestock wastewater was treated by PSPC-EC. Compared with conventional EC and coagulation treatment techniques that consume electricity and produce pollution, the results indicate that the PSPC-EC technique with changing current operation mode is a more cost-effective and attractive option for removing TC from livestock wastewater.
Isolation, toxinotyping and antimicrobial susceptibility testing of Clostridium perfringens isolated from Pakistan poultry
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Citation Excerpt :
Although it is among the microbiota of chicken's intestine, various predisposing factors cause rapid growth of C. perfringens that results in necrotic enteritis (NE) [7]. In poultry, C. perfringens toxinotype A and G are mostly linked with pathogenesis of NE [8]. NE is a clinical form of clostridiosis, characterized by hemorrhage, inflammation, and necrosis in broiler intestine.
Clostridium perfringens is a causative agent of enteric infections in animals including poultry by producing twenty different types of toxins. A single strain produces only a subset of these toxins, which form the basis of its classification into seven toxinotypes (A-G). C. perfringens toxinotype A is a widespread cause of necrotic enteritis (NE) in poultry. The current study was conducted to determine the prevalence of different toxins and antimicrobial susceptibility of C. perfringens isolated from Pakistan NE affected poultry.
A total of 134 intestinal samples of the diseased birds were collected postmortem and processed for isolation of C. perfringens using tryptose sulphite cycloserine (TSC) agar supplemented with d-cycloserine. Isolates were confirmed by Gram's staining, biochemical and molecular analyses. Toxinotyping was performed by multiplex PCR. Antimicrobial susceptibility profile of isolates was performed by Kirby Bauer disc diffusion method.
A total of 34 strains of C. perfringens were isolated from 134 samples with prevalence rate of 25.37%. All the isolated strains were toxinotype A, as they were positive for alpha toxin (CPA) and negative for other tested toxins such as beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), toxin perfringens large (TpeL) and necrotic B-like toxin (NetB). Interestingly, all the isolated strains of C. perfringens were multidrug resistant. The highest resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin which are routinely used at Pakistan poultry production.
C. perfringens toxinotype A is prevalent in Pakistan poultry. Incidence of C. perfringens with prevalence rate of 25.37% can pose serious threat to Pakistan's poultry industry given that all the isolated strains were multidrug resistant. Our findings highlight the need for new antibiotics and antibiotic alternatives to overcome multidrug resistance.

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¹: Both authors contributed equally to this work.

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Anaerobes in animal diseaseToxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and poultry

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Sample collection

Isolation and toxinotyping

Discussion

Conclusions

Declaration of competing interest

Acknowledgements

Trends Microbiol.

Anaerobe

Poultry Sci.

Vet. Microbiol.

Anaerobe

Vet. Microbiol.

Gene

Anaerobe

Anaerobe

Anim. Nutr.

Plasmid

Vet. Microbiol.

Clostridial enteric diseases of domestic animals

Clin. Microbiol. Rev.

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

PLoS Pathog.

Clostridium perfringens toxins involved in mammalian veterinary diseases

Open Toxinol. J.

Antibiotic in Canadian poultry productions and anticipated alternatives

Front. Microbiol.

Spread of antibiotic resistance with food-borne pathogens

Cell. Mol. Life Sci.

Anaerobes in animal disease
Toxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and poultry