Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Prevalence and multilocus sequence typing of Clostridium perfringens isolated from different stages of a duck production chain.

Clostridium perfringens (C. perfringens) is a zoonotic microorganism and rarely reported in duck production chain. This study aimed to investigate prevalence, serotype distribution, antibiotic resistance and genetic diversity of C. perfringens at different stages of a duck production chain. In total, 319 samples were collected from a large-scale rearing and slaughter one-stop enterprise in Weifang, China, of which 42.95% of samples were positive for C. perfringens. All isolates were genotype A. Cpe and cpb2 genes were found in 2.54% and 24.87% of the isolates, respectively. Antimicrobial susceptibility testing revealed that 55.47% of the isolates resistant to at least 5 classes of commonly used antibiotics. Multilocus sequence typing (MLST) results showed that 65 representative isolates were divided into 47 sequences types (STs), 33.85% of them were included into four clonal complexes (CC). Some of isolates from breeding and slaughtering stages were distributed in the same CC or ST, indicating duck products may be contaminated by C. perfringens originated from the breeding stage. Part of duck isolates were distributed in the same CC as human isolates and systemically close with human isolates. The high contamination rates of duck products, the isolates with multi-drug antibiotic resistance or the cpe gene, and the close relationship between strains from human and ducks, indicated potential public health risks, not only control measures at slaughtering stage but also at rearing stage should be considered to reduce this risks.

A survey of a blown pack spoilage produced by Clostridium perfringens in vacuum-packaged wurstel.

Three hundred Clostridium strains were isolated from spoiled wurstels and were identified by traditional and molecular methods as Clostridium perfringens. The phenotypic characteristics of the strains were studied. All the strains produced acetic and butyric acids and enterotoxin. C. perfringens grew in the spoiled wurstels because it was present in raw meat (Lot 150) at a level of 3.2 log CFU/g due to an unchecked cooling phase that took 28 h to decrease the temperature of the wurstels from 60 to 9-10 °C, which is the lower limit for C. perfringens growth. During the 28 h of cooling, the concentration of C. perfringens increased to 6.5 CFU/g. It was concluded that its presence and the long cooling time were the main factors responsible for the spoilage. Wurstels intentionally made with contaminated meat (3 log CFU/g) but cooled after cooking for 17 h to 9 °C did not support C. perfringens growth; consequently, these wurstels remained unspoiled. The packages of the spoiled wurstels were blown, and the products were soft (soggy), textureless and had the odour of acetic acid, ethanol and sulfur.

Cattle and goats' humoral response to vaccination with Clostridium perfringens type D purified epsilon toxoids.

Herd vaccination is an important preventive measure against enterotoxemia in ruminants. Vaccination in goats should be performed every four months, and recent studies have shown that immunity in cattle lasts for less than one year. One of the mechanisms for increasing the duration of the immune response is to use purified toxoids as immunogens. The aim of the present study was to evaluate the humoral response in cattle and goats after vaccination with purified and semi-purified Clostridium perfringens type D epsilon toxoid. The following three different vaccines were used: vaccine 1 (V1), a semi-purified toxoid adsorbed to aluminum hydroxide; vaccine 2 (V2), a purified toxoid adsorbed to aluminum hydroxide; and vaccine (V3), a purified toxoid adsorbed on chitosan microparticles. Groups of cattle (n = 6-7) and goats (n = 6-7) were vaccinated on days 0 and 30, and serum samples for antitoxin titration were collected every 30 days for one-year post-vaccination. Goats were revaccinated on day 360, and their serum was evaluated on days 367 and 374. The antibody peaks ranged between 6.90 and 11.47 IU/mL in cattle and from 1.11 to 4.40 IU/mL in goats. In cattle administered with the V1 and V2 vaccines, we observed that the antibody titers were maintained above 0.2 IU/mL until the end of the experiment. In goats, V2 elicited long-lasting antibodies, and all animals maintained the protective titers for 210 days after the first dose. In conclusion, the purified toxoid vaccine with aluminum hydroxide adjuvant was able to induce strong and long-lasting humoral responses in both species and could be an alternative for improving the immunization schedule against enterotoxemia in goats and cattle.

Clonal diversity of Clostridium perfringens human clinical isolates with various toxin gene profiles based on multilocus sequence typing and alpha-toxin (PLC) typing.

OBJECTIVES: Clostridium perfringens is a common anaerobic pathogen causing enteritis/enterocolitis and wound infections in humans. We analyzed clonal diversity and toxin gene prevalence in C. perfringens clinical isolates from humans in northern Japan. METHODS: Prevalence of nine toxin genes was analyzed for 585 C. perfringens isolates from patients collected for 20-month period between May 2019 and December 2020 by molecular methods. Sequence type (ST) based on multilocus sequence typing (Xiao's scheme) and alpha-toxin (PLC) sequence type were determined for a total of 124 isolates selected in the present study along with those in our previous study (2017-2018). RESULTS: Toxinotypes A (68.2%) was the most frequent, followed by F (31.6%), and G (0.2%), while additional toxin genes encoding binary enterotoxin (BEC/CPILE) and beta2 toxin were identified in one and six isolates, respectively. Among the 124 isolates with various toxin gene profiles, 62 STs including 53 novel types were identified, revealing the presence of six clonal complexes (CCs) consisting of 27 STs. Most of enterotoxin gene (cpe)-positive isolates belonged to CC36, CC41, and CC117. Based on 22 key amino acids in alpha toxin sequence, four PLC types (I-IV) including 21 subtypes were classified, and their relation to individual STs/CCs was clarified. Two isolates harboring bec/cpile belonged to different STs (ST95, ST131) and PLC types (If, IVb), indicating distribution of this toxin gene to distinct lineages. CONCLUSIONS: The present study revealed the diversity in C. perfringens clones of human origin with various toxin gene profiles represented by ST/CC and PLC type.

Prevalence and characterization of Clostridium perfringens isolated from different chicken farms in China.

Clostridium perfringens (C. perfringens) is a common pathogenic microorganism present in nature, which can cause animal and human diseases, such as necrotizing enteritis (NE) in poultry. Little is known about the current prevalence status of C. perfringens from poultry farms of different types and regions in China. From December 2018 to August 2019, we investigated the prevalence, genotype distribution and drug resistance of C. perfringens from Guangdong, Pingyin, Tai'an and Weifang. A total of 622 samples were collected and processed for C. perfringens isolation, among which 239 (38.42%) samples were determined to be positive for C. perfringens. A total of 312 isolates of C. perfringens were recovered (1-5 strains were isolated for each positive sample), and 98.72% of the isolates were identified as type A, while the others were type F. Antimicrobial susceptibility testing revealed that 47.71% of the isolates were resistant to at least five classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 74 representative isolates were divided into 63 sequence types (STs), and the Simpson's diversity index (Ds) of the STs for the five farms was 0.9799. 37.84% of the isolates were classified into seven clonal complexes (CC1-CC7), and the isolates from the same farm were more concentrated in the minimum spanning tree. In addition, some cloaca isolates and feed isolates were distributed in the same ST or CC; this result indicates that the C. perfringens in chicken can come from the environment (feed etc.).

Evaluation of different antigenic preparations against necrotic enteritis in broiler birds using a novel Clostridium perfringens type G strain.

OBJECTIVE: Keeping in view, the constraints faced by the Indian broiler industry with lack of a suitable vaccine against Necrotic Enteritis (NE), a study has been proposed to explore the prevalence and detail characterization of C. perfringens type G in NE suspected broiler chicken in the process of suitable vaccine development. METHODS: Intestinal scrapings/faecal contents of NE suspected broiler chickens were screened to establish the prevalence of C.perfringens type G in broiler birds. A most pathogenic, highly resistant type G isolate of C. perfringens, bearing both tpeL and gapC gene was selected for preparation of three different vaccine formulations, and to evaluate their immunogenic potential in broiler birds. RESULTS: Screening of clinical samples of NE suspected broiler birds revealed C. perfringens type G, bearing gapC gene in 51.22% samples, of which 47.62% revealed tpeL gene. Seven of the tpeLpos type G isolates were comparatively more pathogenic for mice, of which, one exhibited multidrug resistance towards ciprofloxacin, norfloxacin, tetracycline and levofloxacin. The sonicated supernatant (SS) prepared from the selected tpeL and gapC positive isolate could maintain a significantly higher protective IgG response than toxoid and bacterin preparation from the 21st to 28thday of age in immunized birds. CONCLUSION: The additional TpeL toxin in C. perfringens type G has been proved to be an additional key biological factor in the pathogenesis of NE in broiler chickens. Considering the release of more immunogenic proteins, the SS proved to be a better immunogenic preparation against NE with a multiple immunization dose.

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

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.

Densitometric analysis of rep-PCR data: Insight into genetic variability and transmission of Clostridium perfringens typed with an improved multiplex PCR.

An epidemiological study was conducted in North-East India (part of Indo-Burma biodiversity hotspot) to better understand the distribution, diversity, and transmission of Clostridium perfringens among livestock, pets, wild animals (captive), and humans. A total of 160 C. perfringens isolates were recovered from 642 diarrhoeic faecal samples with an isolation rate of 24.92%. Isolation rate was the highest among captive wild animals (37.5%) followed by dog (34.6%), human (33.8%), pig (32.7%), cattle (20.8%), goat (18.3%) and poultry (9.3%). Isolates were toxin typed using a seven gene multiplex PCR designed for simultaneous detection of cpa, cpb, cpb2, etx, iap, cpe and netB. The majority of isolates, 128 (80%) were of type A, followed by 17 (10.62%), 5 (3.12%), 4 (2.5%), 3 (1.87%), 2 (1.25%) and 1 (0.63%) isolates of type C, D, E, G, F and B, respectively. Beta 2 toxin gene was present in 65 (50%) of type A isolates, followed by 7 (41.2%), 4 (80%), 1(25%), and 1 (100%) of type C, D, G and B isolates, respectively. Beta 2 toxin has a high prevalence among dogs (28.6%), cattle (27.3%), and pig (20.8%) compared to humans, goat, wild animals, and poultry (1.2-14.3%). The prevalence of CPE and NetB toxin-positive strains was low, with only 3 (1.8%) and 5 (3.1%) isolates, respectively. Association of C. perfringens with diarrhoea in Civet Cat, Golden Langur, and Gray Langur has been reported for the first time. The genetic diversity and transmission of isolates were investigated using automated rep-PCR (Diversilab®, bioMérieux) using two densitometry-based matrices: modified Kullback-Leibler (KL) and Pearson's correlation (PC). The PC and modified KL matrices formed three distinct clusters with 59% and 27.2% similarity, respectively. C. perfringens diversity and transmission were best studied using modified KL matrix that placed more emphasis on the presence of bands rather than intensity. However, the PC method was found to be more suitable for differentiating strains within a toxin type, with slightly higher D-values.

Isolation of Clostridium perfringens and Clostridioides difficile in diarrheic and nondiarrheic cats.

The occurrence and characteristics of Clostridioides (previously Clostridium) difficile and Clostridium perfringens in the feces of diarrheic and non-diarrheic cats was investigated. Apparently healthy animals were more likely to be positive for C. perfringens type A (p = 0.009). Two isolates (0.7%), one each from a diarrheic and an apparently healthy cat, were positive for the enterotoxin-encoding gene but negative for the NetF-encoding gene. Six toxigenic C. difficile isolates were isolated, all RT106 and ST42, which is commonly reported in humans with C. difficile infection.

Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China.

Clostridium perfringens is an important zoonotic microorganism, which can cause diseases in animal and human under certain conditions. Contamination of C. perfringens in chicken and pork meat has been reported worldwide, but it is rarely reported in duck products. The current study was undertaken to investigate C. perfringens contamination in duck products from a large retail market in Tai'an region, China and the serotype distribution, antimicrobial resistance and genetic relatedness of the isolates. In total, 173 samples of duck products, 10 samples of environmental origins and 7 samples of fresh faeces from healthy shopkeepers were collected between March and November 2018, of which, 58 (31.69%), 10 (100%) and 7 (100%) samples were determined to be positive for C. perfringens, respectively. Ninety-nine isolates of C. perfringens were recovered, all of which were identified as type A. Beta2 (cpb2) toxin gene was found in 54.30% and 33.30% of the isolates from duck products and healthy shopkeepers, respectively. Antimicrobial susceptibility testing revealed that 90.10% of the isolates from duck products and environment showed multiple antibiotic resistance, among which, 49.40% were resistant to at least 6 classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 58 representative isolates were divided into 41 sequences types (STs), among which, ST11 (8.60%) was the most common; 37.90% of all isolates were classified into four clonal complexes (CC1-CC4). The most prolific clonal complex (CC1), accounting for 24.13% of all isolates, contained isolates mainly from carcass, animal intestinal contents and environment of four retail stores. A portion of human isolates and duck isolates was distributed in the same CC or ST. In conclusion, C. perfringens contamination in some duck products in Tai'an retail market was relatively high, and most of the isolates exhibited broad-spectrum antimicrobial resistance. Although all the isolates belong to type A, considerable genetic diversity was observed, and a portion of the strains from human and duck was found to be phylogenetically close. The results indicated that antimicrobial-resistance strains of duck origin pose a potential threat to humans by spreading through the food chain.

Expansion of the Clostridium perfringens toxin-based typing scheme.

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, ß-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not ß-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

Development and evaluation of isothermal amplification assay for the rapid and sensitive detection of Clostridium perfringens from chevon.

Loop-mediated isothermal amplification (LAMP) is a promising, simple, rapid and sensitive molecular detection method. In the present study, LAMP assay was developed for detecting Clostridium perfringens in chevon. Primers were designed to detect the cpa gene of C. perfringens. A panel of 19 bacterial strains, including 3 C. perfringens and 16 other strains, were included in this study to standardize and evaluate the LAMP assay. No false positive amplification was observed indicating 100% specificity of the assay. The detection limit of LAMP and conventional PCR in the DNA extracted from pure C. perfringens was 0.34 pg and 3.4 pg, respectively. This revealed that LAMP assay is 10 times more sensitive than conventional PCR. The sensitivity of the LAMP assay for the detection of C. perfringens in raw chevon was found to be 1.2 × 102 CFU/g after 6-h enrichment and 1.2 × 105 CFU/g without enrichment in artificial spiking studies. Improved C. perfringens detection of 12 CFU/g within 12 h was obtained proving that LAMP assay is significantly faster than traditional methods that take >2 d. The developed LAMP assay also detected the targeted organism in clinical and environmental samples with the sensitivity and specificity of 97% and 84%, respectively with Kappa agreement of 0.824 respects to PCR assay. This method shows immense potential for routine diagnosis and monitoring of C. perfringens in food, environment and clinical samples. This is the first report in which the LAMP assay was optimized for the detection of C. perfringens in chevon.

Diversity of toxin-genotypes among Clostridium perfringens isolated from healthy and diarrheic neonatal cattle and buffalo calves.

The diversity of toxin-genotypes of C. perfringens in neonatal calves was determined in this study. A total of 682 fresh faecal samples comprising 559 healthy and 123 diarrheic neonatal calves (cattle and buffalo) were collected from various farms in Northern India. The samples were processed for isolation of C. perfringens and toxin-genotyping by multiplex PCR. The overall prevalence of C. perfringens was 37.2%. The most predominant toxin-genotype was type A (59.7%) and the least prevalent was type C. There was no association between toxin genotypes and diarrhea of cattle and buffalo neonatal calves (P > .05). Also, 38 (14.6%) and 16 (6.1%) isolates out of the 259 carried enterotoxin (cpe) and beta 2 toxin (cpb2) genes, respectively. Ten different toxin-genotypes were identified, and iota toxin gene was not detected in any of the sample.

Identification of epsilon toxin-producing Clostridium perfringens strains in American retail food.

Food samples (n = 216) from New York city were tested for the presence of C. perfringens via PCR for specific toxin genes. Thirty-four (16%) samples were positive for C. perfringens. Of these 34, 31 (91.2%) were type A or E, one (2.9%) was type B, and two (5.9%) were type D.

Prevalence and characterization of Clostridium perfringens in broiler chickens and retail chicken meat in central China.

We report that Clostridium perfringens was present in 23.1% (130/562) of broiler chickens and 15.1% (38/252) of retail chicken meat samples in central China. Among 168 isolates, type A was the preponderant genotype, and 3% of isolates were positive C. perfringens enterotoxin (cpe). Among different sources, the prevalence was higher in free-range chickens compared to chickens from intensive poultry farms, but with lower proportions of antimicrobial resistance.

Genetic variation among Clostridium perfringens isolated from food and faecal specimens in Lagos.

BACKGROUND: Clostridium perfringens is an anaerobic Gram-positive bacterium which is commonly present in the gastrointestinal tract of man and animals and causes enteritic diseases in animals and food poisoning in humans. Previous studies have looked at the epidemiological relationship between C. perfringens isolates from outbreak source. In this study, the genetic diversity of C. perfringens strains from non-outbreak food and faecal specimens was investigated for epidemiological purposes. METHODS: We analyzed thirty-eight (38) Clostridium perfringens strains isolated from food and faecal specimens in Lagos State. Bacterial identification was done using colonial morphology, Gram stain reaction, conventional biochemical tests and PCR. Genetic analysis was performed using arbitrary primed polymerase chain reaction (AP-PCR) technique with oligonucleotide primer of random sequences (OPA-3) to determine the genetic diversity of C. perfringens. The distance between the different bands produced were analyzed using numerical taxonomy and multivariate system software (NTSYS). RESULTS: Seventeen (44.7%) C. perfringens strains showed at least one polymorphic DNA patterns when genotyped. However, this method identified polymorphisms among the C. perfringens species from which four genetic groups (1, 2, 3 and 4) were established. CONCLUSIONS: Our findings suggest that there may be faecal contamination of food products and similar clones of Clostridium perfringens may be incriminated.

Association between bacterial strain type and host biomarkers in Clostridium perfringens infected goats.

The study project was designed to determine the effects of Clostridium perfringens type D infection on hematological and biochemical parameters in goats. Purposive blood samples were collected from 6 healthy and 12 diseased goats positive for C. perfringens infection. Neither the animals nor their mother were vaccinated against Clostridium perfringens from whom samples were obtained. Study was carried out in two different topographic areas; hilly (district Swat) and plain (district Mardan) of Khyber Pakhtunkhwa, Pakistan but nonsignificant (P > 0.05) statistical difference was recorded between the prevalence of Clostridium perfringens infected goats. Mean erythrocytes count (RBC) and hemoglobin level decreased significantly (P < 0.05) while the white blood cells (WBC) increased significantly (P < 0.05) in diseased animals compared to the healthy animals. However non-significant differences (P > 0.05) were observed in packet cell volume (PCV) and platelets count in healthy and diseased animals. According to biochemical analysis, a significant increase (P < 0.05) in liver enzymes, total bilirubin, serum creatinine, blood urea and glucose was recorded in diseased goats. . The results demonstrated that fluctuation in most of the mean hematological values remained within the normal range however the mean liver enzymes, total bilirubin, serum creatinine, blood urea and glucose levels gone beyond the normal levels which demonstrated severe damages to liver and kidneys.

Prevalence and molecular typing of Clostridium perfringens in captive wildlife in India.

The prevalence of Clostridium perfringens in captive wildlife in India has not been reported. The objective of the study was to determine the fecal prevalence of C. perfringens in captive wildlife in India. The prevalence in captive wild ruminants, non-ruminants, birds and caretakers were 34.1%, 36%, 22.5% and 6.7%, respectively. Toxinotyping of C. perfringens indicated that the predominant type was type A with a prevalence rate of 69.7%, followed by type A with cpb2 gene (28.3%) and type B (2.%).

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.