Outer membrane vesicles as nanovaccine candidates against pathogenic Leptospira in experimental Guinea pig model.

Leptospira interrogans serovar Hardjo is a long slender bacterium of size 0.1-0.3 µm × 5-50 µm. It is one of the major causes of bovine leptospirosis and is of economical importance because of the reproductive failure, still birth, abortion, and reduced productivity in cattle. It is also a zoonotic disease-causing infection in humans characterized by headaches, fever, chills, sweats and myalgia, lethargy, aching joints, pulmonary haemorrhages, and death in severe cases. Control of the disease involves antibiotic therapy, management and vaccination, of which immunization is the cheapest and effective means of disease prevention. The present study was developed to isolate and characterize the outer membrane vesicles of Leptospira interrogans serovar Hardjo and to evaluate their vaccine potential in guinea pig model. The OMVs were isolated from the culture by sonication and ultracentrifugation. In transmission electron microscopy, the isolated OMVs appeared as small spherical structures of 50-200 nm size. In Western blot and indirect ELISA, antibodies specific to OMVs were observed as indicative of a good humoral immune response elicited by L. interrogans serovar Hardjo OMV. The OMV-based Leptospira vaccine was able to prevent kidney lesions and renal colonization compared to the control and bacterin vaccinated group as proven by histopathology and PCR.

Polymorphism in the leucine-rich repeats of TLR7 in different breeds of chicken and in silico analysis of its effect on TLR7 structure and function.

Chicken toll-like receptor 7 (chTLR7) is a viral sensing pattern recognition receptor and detects ssRNA. The ligand binding site comprises leucine-rich repeats (LRRs) located in the ectodomain of chTLR7. Hence, any polymorphism in the binding site would modify its functional interaction with the ligand, resulting in varied strength of immune response. This study first aimed to compare the single nucleotide polymorphisms (SNPs) associated with the ligand binding site of TLR7 in three indigenous chicken breeds namely Aseel, Kadaknath, Nicobari along with an exotic breed White Leghorn. Four synonymous SNPs (P123P, I171I, N339N and L421L) and four non-synonymous SNPs (I121V, S135T, F356S and S447G) were identified among various breeds. We employed in silico tools to screen the pathogenic nsSNPs and one nsSNP was identified as having potential impact on chTLR7 protein. Moreover, sequence and structure-based methods were used to determine the effect of nsSNPs on protein stability. It revealed I121V, F356S, and S447G as decreasing the stability while S135T increasing the stability of chTLR7. Additionally, docking analysis confirmed that I121V and F356S reduced the binding affinity of ligands (R-848 and polyU) to chTLR7 protein. The results suggest that the nsSNPs found in this study could alter the ligand binding of chTLR7 and modify the immune response between different breeds further contributing to disease susceptibility or resistance. Further, in vitro and in vivo studies are needed to analyze the effect of these SNPs on susceptibility or resistance against various viral diseases in poultry.

Genome sequence of bubaline herpesvirus-1 associated with pustular vulvovaginitis in Indian water buffalo.

We report here the genome sequence of a bubaline herpesvirus 1 isolated from Indian water buffalo. The bubaline herpesvirus 1 strain S102_1 was isolated in 2021 from a Murrah buffalo heifer with clinical presentation of pustular vulvovaginitis.

Salmonella enterica serovars linked with poultry in India: antibiotic resistance profiles and carriage of virulence genes.

Salmonella is an important poultry pathogen with zoonotic potential. Being a foodborne pathogen, Salmonella-contaminated poultry products can act as the major source of infection in humans. In India, limited studies have addressed the diversity of Salmonella strains of poultry origin. This study represented 26 strains belonging to Salmonella serovars Typhimurium, Infantis, Virchow, Kentucky, and Agona. The strains were tested for resistance to 14 different antimicrobial agents using the Kirby-Bauer disk-diffusion assay. The presence of the invA, hilA, agfA, lpfA, sopE, and spvC virulence genes was assessed by polymerase chain reaction (PCR), and the genetic diversity was assessed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR). The highest resistance to tetracycline (n = 17; 65.38%) followed by nalidixic acid (n = 16; 61.53%) was detected among the strains. Among the strains (n = 17) phenotypically resistant to tetracycline, 94% (n = 16) were also positive for the tetA gene. Based on the presence of virulence genes, the strains were characterized into three virulence profiles (PI, P2, and P3). Among the investigated virulence genes, invA, hilA, agfA, and lpfA were present in all strains. The sopE gene was mostly associated with serovars Virchow (n = 3; 100%) and Typhimurium (n = 8; 80%), whereas spvC gene was exclusive for two Typhimurium strains that lacked sopE gene. ERIC-PCR profiling indicated clusters correlating their serovar, geographical, and farm origins. These results demonstrate that Salmonella isolates with a wide genetic range, antibiotic resistance, and virulence characteristics can colonize poultry. The presence of such strains is crucial for both food safety and public health.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains.

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.

In vitro virulotyping, antifungal susceptibility testing and DNA fingerprinting of Microsporum canis strains of canine and feline origin.

Microsporum canis is considered the common dermatophyte agent associated with ringworm in felines and canines. In the present study, we sampled n = 548 felines and canines for the probable isolation of M. canis. The rate of isolation from the cats and dogs was 70.27 % (52/74) and 1.68 % (8/474), respectively and Persian cats were found to be highly susceptible to M. canis infection. The strains were evaluated for their production of phospholipase, lipase, catalase, and hemolysis and their ability to grow at 35 â„ƒ. All the strains were identified as low producers of catalase and n = 17 strains exhibited high thermotolerance ability. Terbinafine was found to be the most effective antifungal drug and fluconazole was the least effective, in vitro. AFLP analysis revealed three genotypes of M. canis with 15 sub-clusters showing ≥ 90 % similarity and 7 sub-clusters exhibiting 100 % similarity. However, the phenotypic characters cannot be attributed based on the AFLP profiles.

Emerging porcine Enterovirus G infections, epidemiological, complete genome sequencing, evolutionary and risk factor analysis in India.

The current study reports the in-depth analysis of the epidemiology, risk factors, and molecular characterization of a complete genome of Enterovirus G (EV-G) isolated from Indian pigs. We analysed several genes of EV-G isolates collected from various provinces in India, using phylogenetic analysis, recombination detection, SimPlot, and selection pressure analyses. Our analysis of 534 porcine faecal samples revealed that 11.61% (62/534) of the samples were positive for EV-G. While the G6 genotype was the most predominant, our findings showed that Indian EV-G strains also clustered with EV-G types G1, G6, G8, and G9. Furthermore, Indian EV-G strains exhibited the highest nucleotide similarity with Vietnamese (81.3%) and Chinese EV-G isolates (80.3%). Moreover, we identified a recombinant Indian EV-G strain with a putative origin from a Japanese isolate and South Korean EV-G isolate. In summary, our findings provide significant insights into the epidemiology, genetic diversity, and evolution of EV-G in India.

Pathology, virulence-associated gene profiling, antimicrobial susceptibility, and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India.

Pasteurella multocida is widely distributed in all pig-rearing countries, affecting the economic viability and profitability of pig production. The present research highlights the molecular characterization and pathology of untypeable capsular serotypes of P. multocida in slaughtered pigs from prominent pig-rearing states of India. The prevalence of Pasteurellosis was 27.17% by Pasteurella multocida specific Pasteurella multocida specific PCR (PM-PCR). assay, while isolation rate was 7.62%. The microscopic lesions of bronchopneumonia, tonsillitis, and the presence of bacterial antigens in immunohistochemistry confirmed P. multocida with pathologies. In capsular typing, the majority of the isolates were untypeable with prevalence of 52.15% and 43.58% in molecular and microbiological methods, respectively. All the isolates showed the uniform distribution of virulence genes such as exbB, nanB, sodC, plpB, and oma87 (100%), while the variations were observed in ptfA, hasR, ptfA, pfhA, hsf-1, and plpE genes. The untypeable isolates showed higher prevalence of hsf-1 gene as compared to others. The untypeable serotypes showed a higher degree of resistance to ampicillin, amoxicillin, and penicillin antibiotics. The mouse pathogenicity testing of untypeable capsular isolates confirmed its pathogenic potential. The higher frequency of pathogenic untypeable isolates with antibiotic resistance profile might pose a serious threat to the pigs, and therefore, preventive measures should be adopted for effective control.

Draft Genome Sequence of a Pasteurella multocida Strain Isolated from a Spotted Deer (Axis axis) in India.

Here, we report the genome sequence of a Pasteurella multocida strain isolated from the heart blood of a spotted deer (Bareilly, India). The 2.44-Mbp genome has 2,227 coding sequences, with a G+C content of 40.7%.

Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India.

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63-1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum.

Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug-resistant Salmonella enterica serovar Kentucky.

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens which is isolated in high frequency from poultry meat in the recent decades and is known for its multidrug resistance. The current study was aimed to isolate and characterize a bacteriophage against S. enterica serovar Kentucky isolate, 5925, which showed resistance to at least seven antibiotics and to study its efficiency to decontaminate S. Kentucky from chicken skin. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2 representing the place, source, and host. Electron microscopy revealed that the phage possesses isometric head and contractile tail, indicative of Siphoviridae family. Molecular detection of major capsid protein E gene yielded 511 bp, and NCBI blast analysis revealed that the phage belonged to the genus chivirus. The optimum temperature and pH for phage survival and multiplication were found to be - 20 to 42 °C and 6-10, respectively. One-step growth curve experiment of vB_SenS_Ib_psk2 revealed a latent period of 20 min and burst size of 253 phages/bacterial cell. The host susceptibility studies revealed that 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Artificial spiking studies on chicken skin revealed that high multiplicity of infection (MOI) of phages of 106 pfu/mL is required for significant reduction (p ≤ 0.01) of bacterial concentration (0.14 ± 0.04) after 24-h incubation at 8 °C compared to group 1 (2.55 ± 0.89 cfu/mL).

Isolation and characterization of bacteriophage Ib_pec2 against shigatoxigenic Escherichia coli.

This study was aimed to isolate and characterize bacteriophage against drug-resistant, shigatoxigenic Escherichia coli (STEC), one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n = 35) from neonatal calves, dairy workers, and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multidrug resistant to important antibiotics like ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin, and tetracycline. Bacteriophage namely Ib_pec2 was isolated against one of the STEC isolates and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophages belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. Proteomic analysis was able to identify five proteins identical to Tequatrovirus of myoviridae family. One-step growth curve experiment revealed a latency period of 40 min and a burst size of 893 pfu/bacteria. Temperature and pH ranging from 40°C to 50°C, pH 6-8, respectively. Phage could able to lyse majority of the STEC isolates. STEC are commensal organisms in the gastrointestinal tract of ruminants but are pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control bacterial growth in ruminants and prevent its further spillage in the environment.

Molecular epidemiology of Trichophyton infections among canines from Northern India.

Dermatophytes are keratinophilic fungi that cause skin infections in both humans and animals. Recently, the incidence rates of fungal infections associated with Trichophyton spp. have been considered endemic in many locations. The aim of this study was to isolate and characterize Trichophyton spp. from canines and felines. In the present study, screened 442 canine (n = 386) and feline (n = 56) samples for dermatophytes. Among all the samples, ten isolates were identified as Trichophyton spp. based on micro-morphological features. For comparative analysis, we included three human strains of Trichophyton mentagrophytes complex. In vitro susceptibility of antifungal drugs indicated the highest sensitivity except for fluconazole. The canine and human strains were genetically characterized by sequencing three genes: the internal transcribed spacer region of rDNA, translation elongation factor 1- gene, and beta-tubulin. Based on sequence homology and phylogenetic analysis, the ten canine strains belonged to four different species/ genotypes such as T. mentagrophytes genotype VIII (T. indotineae) (n = 5), T. interdigitale (n = 2), T. simii (n = 2) and T. quinckeanum (n = 1). The three human strains used for comparative analysis were identified as T. mentagrophytes genotype VIII (n = 2) and T. benhamiae (n = 1). The study hence indicates that the T. mentagrophytes genotype VIII, considered as an endemic and emerging human pathogenic clone in India, is also the prevalent in animals.

Dermatophytosis caused by Nannizzia nana (Microsporum nanum): a comprehensive review on a novel pathogen.

Keratinophilic fungi are mostly soil-inhabiting organisms with occasional infections in humans and animals. Even though most dermatophytes are host-adapted, cross-species infections are common by zoophilic and geophilic dermatophytes. N. nana is considered an etiological agent of ringworm in pigs but has also been isolated from other animals, including humans. However, it also possesses many characteristics of geophilic dermatophytes including the ability to grow in soil. N. nana produces characteristic pear-shaped macroconidia and usually exhibits an ectothrix pattern of hair infection. It has been isolated from dermatitis lesions as well as from soil. N. nana infections in pigs are not of much concern as far as economy or health is concerned. But it has been associated with onychomycosis and gonathritis in humans, which are significant in human medicine. The shift in the predominance of dermatophytes in humans and the ability to evolve into a potential tinea pathogen necessitates more understanding of the physiology and genetics of N. nana. In this review, we have attempted a detailed analysis of the studies about N. nana, emphasizing growth and cultural characters, physiology, isolation, infection in humans and animals, molecular characterization and antifungal susceptibility.

Whole genome sequencing and comparative genomics of Mycobacterium orygis isolated from different animal hosts to identify specific diagnostic markers.

Introduction: Mycobacterium orygis, a member of MTBC has been identified in higher numbers in the recent years from animals of South Asia. Comparative genomics of this important zoonotic pathogen is not available which can provide data on the molecular difference between other MTBC members. Hence, the present study was carried out to isolate, whole genome sequence M. orygis from different animal species (cattle, buffalo and deer) and to identify molecular marker for the differentiation of M. orygis from other MTBC members. Methods: Isolation and whole genome sequencing of M. orygis was carried out for 9 samples (4 cattle, 4 deer and 1 buffalo) died due to tuberculosis. Comparative genomics employing 53 genomes (44 from database and 9 newly sequenced) was performed to identify SNPs, spoligotype, pangenome structure, and region of difference. Results: M. orygis was isolated from water buffalo and sambar deer which is the first of its kind report worldwide. Comparative pangenomics of all M. orygis strains worldwide (n= 53) showed a closed pangenome structure which is also reported for the first time. Pairwise SNP between TANUVAS_2, TANUVAS_4, TANUVAS_5, TANUVAS_7 and NIRTAH144 was less than 15 indicating that the same M. orygis strain may be the cause for infection. Region of difference prediction showed absence of RD7, RD8, RD9, RD10, RD12, RD301, RD315 in all the M. orygis analyzed. SNPs in virulence gene, PE35 was found to be unique to M. orygis which can be used as marker for identification. Conclusion: The present study is yet another supportive evidence that M. orygis is more prevalent among animals in South Asia and the zoonotic potential of this organism needs to be evaluated.

Core Genome Multilocus Sequence Typing Scheme for Improved Characterization and Epidemiological Surveillance of Pathogenic Brucella.

Brucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.

A comparative genomics approach for identifying genetic factors in Escherichia coli isolates associated with bovine diseases.

AIMS: E. coli are ubiquitously present bacterial pathogens that cause septicaemia, diarrhoea and other clinical illness in farm animals. Many pathogen factors can be associated with disease conditions. Currently, studies inferring E. coli genetic factors associated with infection in bovines are limited. Hence, the present study envisaged to determine the pathogen genetic factors associated with bovine disease conditions. METHOD AND RESULTS: The comparative genomic analysis involved genome sequence data of 135 diseased and 145 healthy bovine origin E. coli strains. Phylogroups A and C, as well as pathotypes ExPEC and EPEC, were found to have a strong connection with bovine disease strains. STEC strains, including EHEC, seem to play a less important role in bovine disease. Sequence types (STs) predominant among strains from diarrhoeal origin were ST 301 (CC 165) and ST 342. Correlation of core genome phylogeny with accessory gene-based clustering, phylogroups and pathotypes indicated lineage-specific virulence factors mostly associated with disease conditions. CONCLUSIONS: Comparative genomic analysis was applied to infer genetic factors significant in bovine disease origin E. coli strains. Isolates from bovine disease origin were enriched for the phylogroups A and C, and for the pathotypes ExPEC and EPEC. However, there was minimal evidence of STEC involvement. The study also indicated predominant genetic lineages and virulence genes (pap, sfa and afa) associated with disease origin strains. SIGNIFICANCE AND IMPACT OF STUDY: The study revealed significant pathotypes, phylogroups, serotypes and sequence types associated with bovine disease conditions. These identified genetic factors can be applied for disease diagnosis, implementing vaccines and therapeutic measures. In addition, E. coli isolates from the bovine species revealed a complex pattern of disease epidemiology.

Pathotyping and antimicrobial susceptibility testing of Escherichia coli isolates from neonatal calves.

Neonatal calf mortality is a major concern to livestock sector worldwide. Neonatal calf diarrhoea (NCD), an acute severe condition causes morbidity and mortality in calves. Amongst various pathogens involved in NCD, E. coli is considered as one of the major causes. The study was targeted to characterize E. coli isolates from neonatal calves for diarrhoeagenic Escherichia coli (DEC) types (pathotyping), antimicrobial resistance (AMR) profiling and to correlate with epidemiological parameters. From neonates, a total of 113 faecal samples were collected, out of that 308, lactose fermenting colonies were confirmed as E. coli. Pathotypable isolates (12.3%) were represented by STEC (6.1%), EPEC (2.9%), ETEC (1.9%), EAEC (0.9%) and EHEC (0.3%). Occurrence of STEC was more in non-diarrhoeic calves, whereas ETEC was observed more in diarrhoeic calves. EPEC occurrence was observed in both diarrhoeic and non-diarrhoeic calves. Fishers extract test showed no significant association for occurrence of DEC types to type of dairies, health status, species, breed, age and sex of neonatal calves. Two hundred and eighty isolates were tested for antimicrobial susceptibility. The isolates showed maximum resistance towards ampicillin (55.4%) followed by tetracycline (54.3%), while minimum resistance was observed towards meropenem (2.5%). Multidrug resistant E. coli isolates were found to be 139 (49.6%), and Extended-spectrum beta-lactamase (ESBL) producers were 120 (42.9%). DEC pathotypes like STEC, ETEC, EHEC and EAEC that are also multidrug resistant present in neonatal calves have zoonotic potential and hence are of public health significance.

Antimicrobial resistance in Indian isolates of non typhoidal Salmonella of livestock, poultry and environmental origin from 1990 to 2017.

A retrospective antimicrobial resistance study of nontyphoidal Salmonella enterica isolates from India during 1990-2017 was conducted to study the microbial susceptibility to antibiotics. A total of 271 Salmonella enterica isolates from poultry (n = 146), farm animals (n = 55) and environmental sources (n = 70) were tested for susceptibility using 15 antimicrobial drugs. The drug classes include aminoglycosides, phenicols, cephalosporins, penicillins, carbapenems, fluoroquinolones, and sulphonamide-trimethoprim. Study revealed that overall, 133 (49.08%) of 271 isolates were resistant to ≥ 1 antimicrobial drugs and 81 (29.89%) out of 271 isolates were multidrug resistant (resistance to ≥ 3 drugs). Majority (68.96%) of Typhimurium serovars (n = 87) were susceptible to all antibiotics tested, whereas only 5% Kentucky serovars (n = 40) were pan susceptible. All the drugs revealed decreasing trend of susceptibility from 1990 towards 2017 except cephalosporins and carbapenems. Statistical analysis of association between time period and antimicrobial resistance revealed a significance of < 0.05. Molecular detection of genetic determinants associated with antimicrobial resistance revealed the presence of genes like class I integrons, sul1, sul2, catIII, cmlA, dfrA, blaTEM, blaAmpC in the resistant isolates. Furthermore, plasmid mediated quinolone resistant determinants like qnrD and qnrS were also reported in the current study.

First Comparative Analysis of Clostridium septicum Genomes Provides Insights Into the Taxonomy, Species Genetic Diversity, and Virulence Related to Gas Gangrene.

Clostridium septicum is a Gram-positive, toxin-producing, and spore-forming bacterium that is recognized, together with C. perfringens, as the most important etiologic agent of progressive gas gangrene. Clostridium septicum infections are almost always fatal in humans and animals. Despite its clinical and agricultural relevance, there is currently limited knowledge of the diversity and genome structure of C. septicum. This study presents the complete genome sequence of C. septicum DSM 7534T type strain as well as the first comparative analysis of five C. septicum genomes. The taxonomy of C. septicum, as revealed by 16S rRNA analysis as well as by genomic wide indices such as protein-based phylogeny, average nucleotide identity, and digital DNA-DNA hybridization indicates a stable clade. The composition and presence of prophages, CRISPR elements and accessory genetic material was variable in the investigated genomes. This is in contrast to the limited genetic variability described for the phylogenetically and phenotypically related species Clostridium chauvoei. The restriction-modification (RM) systems between two C. septicum genomes were heterogeneous for the RM types they encoded. C. septicum has an open pangenome with 2,311 genes representing the core genes and 1,429 accessory genes. The core genome SNP divergence between genome pairs varied up to 4,886 pairwise SNPs. A vast arsenal of potential virulence genes was detected in the genomes studied. Sequence analysis of these genes revealed that sialidase, hemolysin, and collagenase genes are conserved compared to the α-toxin and hyaluronidase genes. In addition, a conserved gene found in all C. septicum genomes was predicted to encode a leucocidin homolog (beta-channel forming cytolysin) similar (71.10% protein identity) to Clostridium chauvoei toxin A (CctA), which is a potent toxin. In conclusion, our results provide first, valuable insights into strain relatedness and genomic plasticity of C. septicum and contribute to our understanding of the virulence mechanisms of this important human and animal pathogen.