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Clostridium perfringens (C. perfringens), is a spore-forming and toxin-producing pathogenic Gram-positive rod-shaped bacterium with immense public health/zoonotic concern. Rodents are well-known reservoirs and vectors for a large number of zoonoses and strong links have been recognized between synanthropic rodents and foodborne disease outbreaks throughout the world. To date, no study has been conducted for studying the prevalence of C. perfringens in rodents and shrews. In this study, we investigated faecal samples from free-living rodents and shrews trapped in Meghalaya, a North-eastern hill state of India for the presence of virulent and antimicrobial-resistant C. perfringens. A total of 122 animals comprising six species of rodents and one species of shrews were trapped: Mus musculus (n = 15), Mus booduga (n = 7), Rattus rattus (n = 9), Rattus norvegicus (n = 3), Bandicota indica (n = 30), Bandicota bengalensis (n = 32) and Suncus murinus (n = 26). The faecal swabs were collected and processed for the isolation of C. perfringens. Toxinotyping was done using PCR. Antimicrobial susceptibility testing and biofilm forming ability testing were done using Kirby Bauer disc diffusion method and crystal violet assay. C. perfringens was isolated from 27 of the 122 faecal swabs (22.1%), from six species of rodents and shrews. Five of the host species were rodents, Bandicota bengalensis (25%), Bandicota indica (16.7%), Rattus norvegicus (33.3%), Mus musculus (13.3%), Mus booduga (42.8%) and Suncus murinus (shrew) (29.6%). The common toxinotype was type A (59.2%) followed by Type A with beta2 toxin (33.3%), Type C (3.7%) and Type C with beta2 toxin (3.7%). None of the isolates harboured cpe, etx, iap, and NetB genes and therefore none was typed as either B, D, E, F, or G. Nine isolates (33.3%) turned out to be multi-drug resistant (MDR), displaying resistance to three or more categories of antibiotics tested. Twenty-three out of twenty-seven isolates (85.2%) were forming biofilms. Globally, this is the first study to report the prevalence of C. perfringens and its virulence profile and antimicrobial resistance in free-living rodents and shrews. The rodents and shrews can potentially contaminate the food and environment and can infect humans and livestock with multi-drug resistant/virulent Type A and Type C C. perfringens. [Display omitted] • Globally, first study to describe the faecal prevalence of Clostridium perfringens in free-living rodents and shrews. • C. perfringens is 22.1% prevalent in free-living rodents and shrews. • C. perfringens type A, type A cpb2 +, type C, and type C cpb2 + strains were detected. • Multi-drug resistance was displayed by 33.3% of isolates. • Biofilm formation was identified in 85.2% of isolates. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • PSR assay was developed to detect Campylobacter coli for the first time. • The assay involved simple 3-step centrifugation based DNA extraction method. • The assay involved pre-addition of HNB dye for result interpretation. • Analytical sensitivity and detection limit was 62 fg DNA/tube and 100 CFU/g pork. • The assay is equipment-free and requires single enzyme and a primer pair. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is a widespread zoonotic foodborne pathogen and one of the major concerns in the poultry-based food industry. There is a need for a rapid, robust and equipment free detection system for C. jejuni as the conventional culture method is labour and resource-intensive. In this study, we have developed a simple saltatory rolling circle amplification (SRCA) assay integrating a cost-effective three-step centrifugation-based DNA extraction method for rapid and visual detection of C. jejuni in chicken meat. The specificity of the SRCA assay was established by using 17 C. jejuni and 18 non -C. jejuni strains. SRCA, end-point and real-time PCR were able to detect 50 fg, 5 pg and 500 fg C. jejuni DNA/tube, respectively. In the artificial-spiking study conducted in C. jejuni -free chicken meat, a limit of detection (LoD) of 3 log CFU/g and 2 log CFU/g were attained in 120 min (without enrichment) and 14 h (with 12 h enrichment), respectively. The LoD of the SRCA assay was 10- and 100-fold better than the end-point and real-time PCR, respectively. Therefore, keeping the end-user of resource-limited food analysis laboratories in mind, we have successfully developed a SRCA assay for rapid and accurate detection of C. jejuni in chicken meat. - Developed a novel SRCA assay to detect Campylobacter jejuni in chicken meat. - LoD of 3 log CFU/g and 2 log CFU/g were attained in 120 min and 14 h, respectively. - The method was validated in artificially spiked and naturally contaminated chicken meat samples. - A simple method that enables sophisticated-equipment free operation and visual interpretation of results. [ABSTRACT FROM AUTHOR]