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.

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.

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.

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.