Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Novel helix loop-mediated isothermal amplification (HAMP) assay for colorimetric detection of Staphylococcus aureus in milk.

Staphylococcus aureus is an important and leading cause of foodborne diseases worldwide. Prompt detection and recall of contaminated foods are crucial to prevent untoward health consequences caused by S. aureus. Helix loop-mediated isothermal amplification (HAMP) is an exciting recent addition to the array of available isothermal-based nucleic acid amplification techniques. This study aimed to develop and evaluate a HAMP assay for detecting S. aureus in milk and milk products. The assay is completed in 75 minutes of isothermal temperature incubation (64 ˚C) and dye-based visual interpretation of results based on colour change. The specificity of the developed assay was ascertained using 27 S. aureus and 17 non S. aureus bacterial strains. The analytical sensitivity of the developed HAMP assay was 9.7 fg/µL of pure S. aureus DNA. The detection limit of the HAMP assay in milk (86 CFU/mL) was 1000x greater than the routinely used endpoint PCR (86 × 103 CFU/mL). The practicality of applying the HAMP assay was also assessed by analysing milk and milk product samples (n = 95) obtained from different dairy farms and retail outlets. The developed test is a more rapid, sensitive, and user-friendly method for the high-throughput screening of S. aureus in food samples and may therefore be suitable for field laboratories. To our knowledge, this is the first study to develop and evaluate the HAMP platform for detecting S. aureus.

Limiting the spillover of zoonotic pathogens from traditional food markets in developing countries and a new market design for risk-proofing.

Traditional food markets are age-old systems that primarily serve the food supply needs of society's less affluent sectors, often operating with minimal infrastructure. These markets are prevalent in low and middle-income countries. However, their hygienic conditions are frequently suboptimal, potentially fostering the emergence and spread of presumptive zoonotic diseases. The recent emergence of zoonotic or potentially zoonotic diseases and their possible links to traditional food markets underscore the need for focused attention on this overlooked issue. The socioeconomic characteristics of traditional food markets reveal that despite the risk of zoonotic pathogen spread, these markets play a crucial role for large segments of the population. These individuals rely on such markets for their livelihood, food, and nutrition. Therefore, a comprehensive set of measures addressing various aspects of traditional food markets is necessary to manage and mitigate the risks of potential zoonotic disease emergence. In this article, we explore various facets of traditional food markets, paying special attention to the risks of zoonotic diseases that urgently require stakeholder attention. We also propose a new market design to prevent the risk of zoonotic spillover and advocate for the development of a Market Hygiene Index for these markets.

Biofilm-forming antimicrobial-resistant pathogenic Escherichia coli: A one health challenge in Northeast India.

This study aimed to investigate the prevalence of Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) in common food animals (cattle, goats, and pigs) reared by tribal communities and smallholder farmers in Northeast India. The isolates were characterized for the presence of virulence genes, extended-spectrum beta-lactamases (ESBL) production, antimicrobial resistance, and biofilm production, and the results were statistically interpreted. In pathotyping 141 E. coli isolates, 10 (7.09%, 95% CI: 3.45%-12.66%) were identified as STEC, 2 (1.42%, 95% CI: 0.17%-5.03%) as atypical-EPEC, and 1 (0.71%, 95% CI: 0.02%-3.89%) as typical-EPEC. None of the isolates were classified as ETEC. Additionally, using the phenotypic combination disc method (ceftazidime with and without clavulanic acid), six isolates (46.1%, 95% CI: 19.22%-74.87%) were determined to be ESBL producers. Among the STEC/EPEC strains, eleven (84.6%, 95% CI: 54.55%-98.08%) and one (7.7%, 95% CI: 0.19%-36.03%) strains were capable of producing strong or moderate biofilms, respectively. PFGE analysis revealed indistinguishable patterns for certain isolates, suggesting clonal relationships. These findings highlight the potential role of food animals reared by tribal communities and smallholder farmers as reservoirs of virulent biofilm-forming E. coli pathotypes, with implications for food contamination and zoonotic infections. Therefore, monitoring these pathogens in food animals is crucial for optimizing public health through one health strategy.

Prevalence and Characterization of Staphylococcus aureus Isolated from Retail Raw Milk Samples in Chennai, India.

Milk is an important source of food, and it is also a nutrient-rich medium, which can harbor multiple microorganisms. Staphylococcus aureus is an important foodborne pathogen in food-producing animals, and there have been many reports on its infection and antimicrobial resistance (AMR), which has significant global public health concerns. This study was designed to isolate, characterize, and analyze the AMR pattern of S. aureus from milk samples collected in Chennai, India. A total of 259 raw milk samples from 3 groups: dairy farms, local vendors, and retail outlets were analyzed, and it was found that 34% (89/259) were positive for S. aureus. Positive isolates were further characterized by pulsed-field gel electrophoresis and isolates recovered from different sources, study areas, and locations showed high genetic diversity with no similarity. The presence of AMR has been further assessed by phenotypic methods as per CLSI-M100 performance standards, and all the isolates were susceptible to ampicillin/sulbactam, mupirocin, and tylosin. Additionally, all of the isolates were resistant to ampicillin. There were 28 isolates categorized as multidrug-resistant, which showed resistance to more than 2-3 classes of antimicrobials. This is the first report of inducible clindamycin resistance and mupirocin sensitivity pattern from S. aureus isolates recovered from milk. This study established the occurrence varied with genetic diversity in the isolates prevalent in the study area and divergence pattern of AMR S. aureus. The AMR in these isolates and with methicillin-resistant S. aureus could pose a serious threat to food safety and economic implications.

Livestock, pets and humans as carriers of methicillin-resistant Staphylococcusaureus and comparative evaluation of two PCR protocols for detection.

Staphylococcus aureus are Gram positive bacteria known to acquire antibiotic resistance rapidly and pose a major challenge to clinicians worldwide. Infections by methicillin resistant Staphylococcus aureus (MRSA) are usually associated with increased mortality and prolonging of treatment. Samples (n = 706) from diverse sources (livestock, pets, animal handlers, human hospital) were collected and screened for the presence of MRSA by phenotypic and genotypic methods. The incidence of Staphylococcus aureus was greater in goats (42.00%; 28.20 - 56.80%, confidence interval [CI] 95.00%) followed by cattle (13.50%; 9.20 - 18.80%, CI 95.00%), humans (12.90%; 9.30 - 17.40%, CI 95.00%) and dogs (12.90%; 8.10 - 19.20%, CI 95.00%). Significantly higher incidence of MRSA was observed in dogs (65.00%; 40.80 - 84.60%, CI 95.00%), compared to other hosts namely cattle (48.00%; 26.50 - 64.30%, CI 95.00%), humans (35.00%; 20.20 - 52.50%, CI 95.00%) and goats (10.00%; 1.20 - 30.40%, CI 95.00%). All the S. aureus isolates were further screened for thermostable nuclease (nuc gene) by polymerase chain reaction (PCR). The incidence of nuc gene in cattle, dog, goat and human were found to be 3.30% (1.30 - 6.60%, CI 95.00%), 5.20% (2.30 - 9.90%, CI 95.00%), 28.00% (16.20 - 42.50%, CI 95.00%) and 9.10% (6.00 - 13.00%, CI 95.00%), respectively. Comparative evaluation of two PCR primers (mecA-162 and mecA-310) indicated the former one as more rational choice for detection of MRSA. Overall, the results of our study indicated possible risk of zoonotic transmission of MRSA from canines.

Prevalence of bovine tuberculosis and analysis of risk factors among the dairy farms in and around Guwahati metropolitan city, India.

Bovine tuberculosis (bTB), a neglected zoonotic disease caused by Mycobacterium bovis is being reported worldwide. The present work was carried out from December 2020 to November 2021 to assess the prevalence and risk factors of bTB in peri-urban and urban dairy farms of Guwahati, Assam, India. A questionnaire was used to collect data on knowledge about bTB on 36 farms, and ten animals per farm were screened by single intradermal comparative cervical tuberculin test (SICCT) to determine the prevalence of bTB, giving a total of 360 animals. The demographic data of the farmers revealed that 61.1% respondents were illiterate, 66.7% had no awareness about bovine tuberculosis and 41.7% consumed unpasteurised milk and milk products. SICCT showed that 38 cattle from 18 of the farms were positive reactors for bTB, yielding an overall animal level prevalence of 10.55% (95% confidence interval (CI = 7.58-14.2%) and a 50% herd prevalence (95% CI 32.9-67.1%). Animals 5 years and above were found to be more likely to be positive for bTB (17.18%). The study highlighted the widespread prevalence of bovine tuberculosis in peri-urban and urban dairy farms of Guwahati which gives a picture also about other major cities of India. Hence, it is of utmost importance to undertake a comprehensive epidemiological study in such cities for effective control and prevention of bTB in a one health approach.

Development of novel isothermal-based DNA amplification assay for detection of pig tissues in adulterated meat.

For the first time, we describe an innovative polymerase spiral reaction (PSR) assay for the rapid, simple, and accurate detection of pig tissues or pork in adulterated meat including heat-treated and processed ones. The PSR assay specifically targeting the mitochondrial cytochrome b (cyt-b) gene of the pig was successfully optimized permitting assay results in 65 min time. The developed detection method was 100% specific amplifying only the cyt-b gene and displaying negative results with all the tested non-pork meats. The sensitivity of the developed PSR (760 fg porcine DNA) was tenfold better than the end-point PCR and able to detect heat-treated (121 °C) and adulterated (0.5% pork in beef) meat and processed pork products such as sausages, salami, meatball, soup, curry, etc. The developed PSR-based method can be used for point-of-care detection with minimum instrumentation and technical expertise to guarantee instant clearance of exported and imported meat products. This is the first time that PSR has been adapted for food authenticity purposes.

Microbiome analysis reveals potential for modulation of gut microbiota through polysaccharide-based prebiotic feeding in Oreochromis niloticus (Linnaeus, 1758).

Characterization and functional profiling of the gut microbiota are essential for guiding nutritional interventions in fish and achieving favorable host-microbe interactions. Thus, we conducted a 30 days study to explore and document the gut microbial community of O. niloticus, as well as to evaluate the effects of a polysaccharide-based prebiotics with 0.5% and 0.75% Aloe vera extract on the gut microbiome through genomic analysis. The V3-V4 region of 16S rRNA was amplified and sequenced using Illumina HiSeq 2500, resulting in 1,000,199 reads for operational taxonomic unit (OTU) identification. Out of 8,894 OTUs, 1,181 were selected for further analysis. Our results revealed that Planctomycetes, Firmicutes, Proteobacteria, Verrucomicrobia, Actinobacteria, and Fusobacteria were the dominant phyla in both control and treatment samples. Higher doses of prebiotics were found to improve Planctomycetes and Firmicutes while decreasing Proteobacteria and Verrucomicrobia. We observed increasing trends in the abundance of Bacilli, Bacillaceae, and Bacillus bacteria at the class, family, and genus levels, respectively, in a dose-dependent manner. These findings were consistent with the conventional colony count data, which showed a higher prevalence of Bacillus in prebiotic-supplemented groups. Moreover, predicted functional analysis using PICRUSt indicated a dose-dependent upregulation in glycolysis V, superpathway of glycol metabolism and degradation, glucose and xylose degradation, glycolysis II, and sulfoglycolysis pathways. Most of the energy, protein, and amino acid synthesis pathways were upregulated only at lower doses of prebiotic treatment. Our findings suggest that the gut microbiome of O. niloticus can be optimized through nutritional interventions with plant-based polysaccharides for improved growth performance in commercial fish.

Isolation, susceptibility profiles and genomic analysis of a colistin-resistant Salmonella enterica serovar Kentucky strain COL-R.

Salmonella enterica serovar Kentucky is a frequent cause for clinical infections in human patients. They are isolated and reported with multidrug resistance from the foods of animal origin from various countries. However, studies inferring the colistin resistance are limited. Hence, the current study reports the genetic factors and genomic analysis of the colistin-resistant Salmonella enterica serovar Kentucky strain COL-R for better understanding of its pathogenic potential and phylogenetic relatedness. The S. Kentucky strain COL-R was successfully isolated from chicken meat during ongoing surveillance of food of animal origin. Antimicrobial susceptibility testing revealed resistance to cefoxitin, erythromycin, gentamicin, tetracycline, and most disturbingly to ciprofloxacin and colistin (broth microdilution method). Whole-genome sequence of the COL-R strain was subjected to various in silico analysis to identify the virulence factors, antimicrobial resistance genes, pathogenicity islands and sequence type. The S. Kentucky COL-R strain belonged to sequence type (ST) 198 with a high probability (0.943) of being a human pathogen. Besides presence of integrated phage in the S. Kentucky COL-R genome, 38 genes conferring resistance to various antimicrobials and disinfectants were also identified. Nucleotide Polymorphism analysis indicated triple mutations in gyrA and parC genes conferring fluoroquinolone resistance. Phylogenomic analysis with 31 other S. Kentucky genomes revealed discernible clusters with S. Kentucky COL-R strain latching onto a cluster of high diversity (geographic location and isolation sources). Taken together, our results document the first occurrence of colistin resistance in a fluoroquinolone resistant S. Kentucky COL-R strain isolated from retail chicken and provide crucial information on the genomic features of the strain. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03559-2.

A study on the occurrence of Burkholderia cepacia complex in ultrasound gels used in different veterinary clinical settings in India.

Burkholderia cepacia complex (Bcc) organisms are emerging multidrug-resistant pathogens. They are opportunistic and cause severe diseases in humans that may result in fatal outcomes. They are mainly reported as nosocomial pathogens, and transmission often occurs through contaminated pharmaceutical products. From 1993 to 2019, 14 Bcc outbreaks caused by contaminated ultrasound gels (USGs) have been reported in several countries, including India. We screened a total of 63 samples of USGs from various veterinary and human clinical care centers across 17 states of India and isolated 32 Bcc strains of Burkholderia cenocepacia (46.8%), B. cepacia (31.3%), B. pseudomultivorans (18.8%) and B. contaminans (3.1%) species. Some isolates were co-existent in a single ultrasound gel sample. The isolation from unopened gel bottles revealed the intrinsic contamination from manufacturing sites. The MALDI-TOF analysis to identify the Bcc at the species level was supported by the partial sequencing of the recA gene for accurate species identification. The phylogenetic analysis revealed that isolates shared clades with human clinical isolates, which is an important situation because of the possible infections of Bcc by USGs both in humans and animals. The pulsed field gel electrophoresis (PFGE) typing identified the genetic variation among the Bcc isolates present in the USGs. The findings indicated USGs as the potential source of Bcc species.

Development of a novel visual isothermal amplification assay for rapid detection of Brucella spp.

Brucellosis is an economically important livestock disease worldwide besides having a noteworthy impact on human health. In this study, a rapid, simple, and ultra-sensitive nuclei-acid diagnostic technique was developed for the detection of brucellosis harnessing saltatory rolling circle amplification (SRCA). The diagnostic method was developed using World Organization for Animal Health (WOAH) approved primers targeting the bcsp31 gene of the Brucella genome. The assay can be accomplished within 90 min at a temperature of 65 °C without the requirement of sophisticated instrumentation. The result interpretation can be done with the naked eye with the aid of SYBR green dye. The developed technique displayed 100% specificity by amplifying only 10 reference and field strains of Brucella spp. and there was no cross-reactivity with the other tested pathogens. The lower limit of detections of SRCA and end-point PCR assays were 9.7 fg/µL (2.7 genome copies of Brucella) and 970 fg/µL, respectively. Thus, the developed SRCA assay was found to be 100× more sensitive than the end-point PCR assay. To the best of our knowledge, our study is the first one to develop an SRCA-based assay for the detection of brucellosis and it can be a diagnostic tool for resource-constrained laboratories and veterinary hospitals.

Novel sensitive isothermal-based diagnostic technique for the detection of African swine fever virus.

A rapid, simple, and sensitive diagnostic technique for the detection of African swine fever virus (ASFV) nucleic acid was developed for testing clinical samples in the field or resource-constrained settings. In the current study, the saltatory rolling-circle amplification (SRCA) technique was used for the first time to detect ASFV. The technique was developed using World Organization for Animal Health (WOAH)-approved primers targeting the p72 gene of the ASFV genome. The assay can be performed within 90 minutes at an isothermal temperature of 58°C without a requirement for sophisticated instrumentation. The results can be interpreted by examination with the naked eye with the aid of SYBR Green dye. This assay exhibited 100% specificity, producing amplicons only from ASFV-positive samples, and there was no cross-reactivity with other pathogenic viruses and bacteria of pigs that were tested. The lower limits of detection of SRCA, endpoint PCR, and real-time PCR assays were 48.4 copies/µL, 4.84 × 103 copies/µL, and 4.84 × 103 copies/µL, respectively. Thus, the newly developed SRCA assay was found to be 100 times more sensitive than endpoint and real-time PCR assays. Clinical tissue samples obtained from ASFV-infected domestic pigs and other clinical samples collected during 2020-22 from animals with suspected ASFV infection were tested using the SRCA assay, and a 100% accuracy rate, negative predictive value, and positive predictive value were demonstrated. The results indicate that the SRCA assay is a simple yet sensitive method for the detection of ASFV that may improve the diagnostic capacity of field laboratories, especially during outbreaks. This novel diagnostic technique is completely compliant with the World Health Organization's "ASSURED" criteria advocated for disease diagnosis, as it is affordable, specific, sensitive, user-friendly, rapid and robust, equipment-free, and deliverable. Therefore, this SRCA assay may be preferable to other complex molecular techniques for diagnosing African swine fever.

Lifestyle, Epstein-Barr virus infection, and other factors could impede nasopharyngeal cancer survivorship: a five-year cross-sectional study in North Eastern India.

Nasopharyngeal Carcinoma (NPC) is one of the leading cancers in India's north-eastern (NE) region affecting a section of the population each year. A proportion of the NPC cases are observed to recur even after therapy, indicating the involvement of other factors. We aimed to explore the NPC and Epstein-Barr virus (EBV) burden in the NE region and investigate the prognostic factors for the NPC patients' poor survival and recurrence. NPC patients' information was obtained from different state hospitals between 2014 and 2019. PCR and Sanger sequencing were performed to detect EBV types. Statistical analysis, including forest plot analysis, Kaplan-Mayer survival plot, Log-rank test, cox hazard regression, and Aalen's additive regression model, were performed to determine prognostic factors for the NPC patients' lower survival and recurrence. We observed an increased incidence of NPC and EBV infection in the past five years. Step-wise statistical analyses pointed out that variable such as non-professionals (B = 1.02, HR = 2.8, 95%CI = 1.5,4.9) workers (B = 0.92, HR = 2.5, 95%CI = 1.4,4.4), kitchen cum bedroom (B = 0.61, HR = 1.8, 95%CI = 1.2,2.8), mosquito repellent (B = 0.60, HR = 1.7, 95%CI = 1.1,2.7), nasal congestion (B = 0.60, HR = 1.8, 95%CI = 1.2,2.8), lower haemoglobin level (B = 0.92, HR = 2.5, 95%CI = 1.3,4.9), tumor stage IV (B = 2.8, HR = 1.8, 95%CI = 1.6,14.3), N2 (B = 1.4, HR = 4.0, 95%CI = 1.8,9.1), N3 (B = 1.9, HR = 6.4, 95%CI = 2.8,15.3), and M+ (B = 2.02, HR = 7.5, 95%CI = 4.1,13.7) revealed significant correlation with NPC patients' poor prognosis (p < 0.05). The presence of viral factors also showed a significant association with NPC patients' decreased survival. We concluded that factors related to day-to-day life with EBV infection could be the individual predictor for NPC incidence, lower survival, and disease recurrence. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00789-5.

Prevalence, toxinotyping, antimicrobial susceptibility and biofilm-forming ability of Clostridium perfringens isolated from free-living rodents and shrews.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Development and evaluation of a novel polymerase spiral reaction based testing technique for same-day visual detection of Campylobacter coli in pork.

The developed polymerase spiral reaction-based technique specifically amplified the ceuE gene of C. coli and involved a three-step centrifugation method for DNA extraction. PSR, real-time and end-point PCR were able to detect 62 fg, 620 fg and 6.2 pg C. coli DNA/tube, respectively. PSR detection limits for artificially contaminated pork samples without enrichment, with 12 h enrichment and after 24 h enrichment were 1000 CFU/g, 100 CFU/g, and 10 CFU/g samples, respectively which were ten times better than real-time PCR. The detection performance of PSR (with 12 h enrichment) was also compared to culture (ISO10272-1:2017) method using 75 naturally-contaminated samples, which revealed the sensitivity, specificity, PPV, NPV and accuracy of 100% (95%CI, 73.2%-100%), 98.4% (95%CI, 90%-99.9%), 93.3% (95%CI, 66%-99.6%), 100% (95%CI, 92.5%-100%) and 98.7% (95%CI, 92.8%-99.9%), respectively. The advantage and novelty of this assay are its equipment-free nature, dye-based interpretation by the naked eye, and the requirement of one enzyme and one primer pair. This assay could be a better alternative to other molecular methods and may help in reducing the possible troubles (e.g., gastroenteritis, hospitalization, or death) of belated detection of C. coli in food products. This is the primary report applying the PSR for C. coli detection.

Genomic insights of beta-lactamase producing Klebsiella quasipneumoniae subsp. similipneumoniae belonging to sequence type 1699 from retail market fish, India.

Klebsiella quasipneumoniae is a recently described species and often misidentified as Klebsiella pneumoniae. Here, we report the genomic characterization of Klebsiella quasipneumoniae subsp. similipneumoniae (India238 strain) isolated from fish. The annotated genome acknowledged the presence of blaCTX-M-15, blaOKP-B-1, fosA5, oqxAB and virulence genes. The strain with ST1699 and serotypes KL52 and OL103 also harboured insertion sequences (ISs): ISKpn26 and ISEc9. Three complete phage genomes were identified in contigs 1 and 6 of the bacterial genome, enhancing the prospects of genome manipulation. The study highlights the pitfall of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae. This is the first Indian study documenting the incidence of extended-spectrum beta-lactamase (ESBL)-producing K. quasipneumoniae subsp. similipneumoniae from a non-clinical environment, equipped with virulomes and associated mobile genetic elements. Given that fish can act as a potential vector for transmission of antimicrobial resistance genes, our findings have paramount importance on human health.

Pulsed-field gel electrophoresis fingerprinting of Listeria monocytogenes isolates recovered from foods of animal origin and fishes in North-Eastern India.

L isteria monocytogenes is a pathogen of great concern to the food industry. The present study was aimed to explore the clonal relationships amongst L. monocytogenes strains isolated from foods of animal origin (milk, beef, chevon (goat meat), pork and chicken) and fish. Forty-seven L. monocytogenes strains were characterized by pulsed-field gel electrophoresis (PFGE). The PFGE analysis using ApaI and AscI enzymes revealed 37 pulsotypes, with Simpson's discriminatory index of 0.987. This study demonstrated the presence of a few similar L. monocytogenes pulsotypes in different foods of animal origin in different places and years of isolation and this indicates that some L. monocytogenes subtypes may be ubiquitous which are acclimatizing and persisting in different foods of animal origin. This also emphasizes the importance of cross-contamination in local wet markets. Thus, the understanding of genetic diversity will contribute to the development of rational and workable strategies to control this important zoonotic infection.

Avian strains of emerging pathogen Escherichia fergusonii are phylogenetically diverse and harbor the greatest AMR dissemination potential among different sources: Comparative genomic evidence.

Introduction: Escherichia fergusonii is regarded as an emerging pathogen with zoonotic potential. In the current study, we undertook source-wise comparative genomic analyses (resistome, virulome, mobilome and pangenome) to understand the antimicrobial resistance, virulence, mobile genetic elements and phylogenetic diversity of E. fergusonii. Methods: Six E. fergusonii strains (5 multidrug resistant strains and 1 biofilm former) were isolated from poultry (duck faeces and retail chicken samples). Following confirmation by phenotypic and molecular methods, the isolates were further characterized and their genomes were sequenced. Comparative resisto-virulo-mobilome analyses and pangenomics were performed for E. fergusonii genomes, while including 125 other E. fergusonii genomes available from NCBI database. Results and discussion: Avian and porcine strains of E. fergusonii were found to carry significantly higher number of antimicrobial resistance genes (p < 0.05) and mobile genetic elements (plasmids, transposons and integrons) (p < 0.05), while the pathogenic potential of bovine strains was significantly higher compared to other strains (p < 0.05). Pan-genome development trends indicated open pan-genome for all strains (0 < γ < 1). Genomic diversity of avian strains was found to be greater than that from other sources. Phylogenetic analysis revealed close clustering among isolates of similar isolation source and geographical location. Indian isolates of E. fergusonii clustered closely with those from Chinese and a singleton Australian isolate. Overall, being the first pangenomic study on E. fergusonii, our analysis provided important cues on genomic features of the emerging pathogen E. fergusonii while highlighting the potential role of avian strains in dissemination of AMR.

Isolation and sero-genomo-epidemiological studies on Brucella infection in dairy cattle in Meghalaya, India.

In this study, we report the serological, bacteriological and whole genome sequencing data of a 6 years study of Brucella abortus in Meghalaya, India. Investigation of 3060 sera samples indicated overall prevalence of 6.4% by Rose Bengal Plate Test and 10.7% by ELISA. Considerably higher prevalence was observed among milk samples (17.5%, n = 362) and in blood samples (37.7%, n = 262) by direct PCR. Clinical samples (n = 94) from late abortion cases yielded 11 B. abortus isolates. Multi-locus sequence typing indicated circulation of single sequence type, ST1. Whole genome sequencing (n = 8) and phylogenomic analysis revealed close clustering of majority of isolates in two clusters alongwith genomes from other countries, indicating global relatedness among B. abortus. Taken together, the results of our study revealed the putative hotspot of infection in the dairy-dominant districts of the state and also calls for concerted One Health based action for prevention and control of this zoonotic disease.