Draft genome sequence of biofilm-forming methicillin-resistant Staphylococcus aureus MTR_V1 strain isolated from a ready-to-eat food in Bangladesh.

This announcement provides the genome sequence of the biofilm-forming methicillin-resistant Staphylococcus aureus MTR_V1 strain isolated from a ready-to-eat food sample in Bangladesh. Our assembled genome had a length of 2.8 Mb, 27 contigs, two CRISPR arrays, 38 predicted antibiotic resistance genes, and 66 predicted virulence factor genes.

Virulence Determinants and Methicillin Resistance in Biofilm-Forming Staphylococcus aureus from Various Food Sources in Bangladesh.

The eradication of staphylococcal infections has become more difficult due to the development of antibiotic resistance and virulence in biofilm-forming Staphylococcus aureus. The presence of the life-threatening zoonotic pathogen, methicillin-resistant S. aureus (MRSA), in foods indicates a public health issue. This study, therefore, aimed to determine virulence factors and methicillin resistance in biofilm-forming S. aureus isolates from different foods and food handlers. A total of 100 PCR-positive S. aureus isolates (97 biofilm formers and three non-biofilm formers) were screened using the disk diffusion method and PCR assay. By PCR, genes encoding virulence factors, e.g., enterotoxin (sea, 30%, 95% CI: 21.90−39.59%), toxic shock syndrome toxin (tst, 20%, 95% CI: 13.34−28.88%), and Panton−Valentine leukocidin toxin (PVL, 15%, 95% CI: 9.31−23.28%), were detected in the S. aureus isolates. By the disk diffusion method, 100% (95% CI: 96.30−100.00%) of S. aureus isolates were phenotypically MRSA in nature, showing 100% resistance to oxacillin and cefoxitin. Moreover, the methicillin-resistant gene mecA was found in 61 (61%, 95% CI: 51.20−69.98%) MRSA isolates. Furthermore, all the S. aureus isolates were phenotypically resistant to ampicillin and penicillin, 30% to erythromycin, and 11% to gentamycin. Among them, 51% (95% CI: 41.35−60.58%) of S. aureus isolates were phenotypically multidrug-resistant in nature, and the multiple antibiotic resistance index varied from 0.33 to 0.55. Genes encoding resistance to beta-lactams (blaZ, 100%, 95% CI: 96.30−100.00%) and tetracyclines (tetA and tetC, 3%, 95% CI: 0.82−8.45%) were found positive in the S. aureus isolates. Genes encoding virulence determinants and MRSA were significantly (p < 0.05) higher in strong biofilm-forming S. aureus than in moderate and non-biofilm-forming isolates. To our knowledge, this is the first study in Bangladesh to incorporate preliminary data on the occurrence of virulence determinants and methicillin resistance, including resistance to clinically important antibiotics, in biofilm-forming S. aureus isolates from different foods and food handlers in Bangladesh, emphasizing a potential threat to human health.

Phenotypic and Genotypic Detection of Biofilm-Forming Staphylococcus aureus from Different Food Sources in Bangladesh.

Staphylococcus aureus is a major foodborne pathogen. The ability of S. aureus to produce biofilm is a significant virulence factor, triggering its persistence in hostile environments. In this study, we screened a total of 420 different food samples and human hand swabs to detect S. aureus and to determine their biofilm formation ability. Samples analyzed were meat, milk, eggs, fish, fast foods, and hand swabs. S. aureus were detected by culturing, staining, biochemical, and PCR. Biofilm formation ability was determined by Congo Red Agar (CRA) plate and Crystal Violet Microtiter Plate (CVMP) tests. The icaA, icaB, icaC, icaD, and bap genes involved in the synthesis of biofilm-forming intracellular adhesion compounds were detected by PCR. About 23.81% (100/420; 95% CI: 14.17−29.98%) of the samples harbored S. aureus, as revealed by detection of the nuc gene. The CRA plate test revealed 20% of S. aureus isolates as strong biofilm producers and 69% and 11% as intermediate and non-biofilm producers, respectively. By the CVMP staining method, 20%, 77%, and 3% of the isolates were found to be strong, intermediate, and non-biofilm producers. Furthermore, 21% of S. aureus isolates carried at least one biofilm-forming gene, where icaA, icaB, icaC, icaD, and bap genes were detected in 15%, 20%, 7%, 20%, and 10% of the S. aureus isolates, respectively. Bivariate analysis showed highly significant correlations (p < 0.001) between any of the two adhesion genes of S. aureus isolates. To the best of our knowledge, this is the first study in Bangladesh describing the detection of biofilm-forming S. aureus from foods and hand swabs using molecular-based evidence. Our findings suggest that food samples should be deemed a potential reservoir of biofilm-forming S. aureus, which indicates a potential public health significance.

Genomic characteristics, virulence, and antimicrobial resistance in avian pathogenic Escherichia coli MTR_BAU02 strain isolated from layer farm in Bangladesh.

BACKGROUND: Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is one of the most significant infectious diseases affecting poultry worldwide. OBJECTIVES: This study aimed to determine the genomic diversity, virulence factor genes (VFGs), and antimicrobial resistance genes (ARGs) in the APEC MTR_BAU02 strain isolated from a layer chicken using whole-genome sequencing (WGS). METHODS: Paired-end (2 × 250) WGS was performed using Illumina MiSeq sequencer (Illumina, San Diego, CA) and de novo assembly was performed using SPAdes. Core genome multilocus sequence typing (cgMLST) analysis between APEC MTR_BAU02 and all of the ST1196 E. coli strains retrieved from the National Center for Biotechnology Information (NCBI) GenBank database was performed using the BacWGSTdb 2.0 server. We utilized different databases to detect ARGs, VFGs, and genomic functional features of the APEC MTR_BAU02 strain. RESULTS: The complete genome of APEC MTR_BAU02 consists of 94 contigs comprising 4,924,680 bp (51.1% guanine-cytosine [GC] content), including 4681 protein-coding sequences, one chromosome, and one plasmid, and was assigned to ST1196. The closest relatives of APEC MTR_BAU02 were four isolates originating from human clinical specimens (diarrhetic stool) in Bangladesh and two clinical isolates originating from chicken in India, which differed by 694 core genome multilocus sequence typing (cgMLST) alleles. One hundred and twenty-two ARGs and 92 VFGs were identified in the APEC MTR_BAU02 genome. Metabolic functional annotations detected 380 SEED subsystems including genes coding for carbohydrate metabolism, protein metabolism, cofactors, vitamins, prosthetic groups and pigments, respiration, membrane transport, stress response, motility and chemotaxis, and virulence, disease, and defense. CONCLUSION: This study reports the genome sequence of a multidrug-resistant APEC strain isolated from layer birds in Bangladesh. The ARGs and VFGs, widespread in APEC MTR_BAU02, are similar to those found in human isolates, and highlight the growing threat of antimicrobial resistance in both poultry and humans.

Detection of blaTEM, blaCTX-M, blaCMY, and blaSHV Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolated from Migratory Birds Travelling to Bangladesh.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli cause severe health hazards. Migratory birds are reservoirs and transmitters of many pathogens including ESBL-producing E. coli. To examine migratory birds as potential carriers of ESBL-producing E. coli and E. coli-carrying antibiotic resistance genes, 55 PCR-positive E. coli isolates were screened using the disk diffusion method, double-disk synergy test, and further polymerase chain reaction (PCR) tests. Genes encoding resistance to tetracycline [tetA, 100% (35/35); tetB, 31.43% (11/35)], fluoroquinolone [qnrA, 35.71% (10/28); qnrB, 25% (7/28)], and streptomycin [aadA1, 90.24% (37/41)] were detected in the isolated E. coli. Of the 55 E. coli isolates, 21 (38.18%) were ESBL producers, and all of them were multidrug resistant. All the ESBL-producing E. coli isolates harbored at least two or more beta-lactamase genes, of which blaTEM, blaCMY, blaCTX-M, and blaSHV were detected in 95.24%, 90.48%, 85.71%, and 42.86% of isolates, respectively. All the beta-lactamase genes were present in four of the ESBL-producing E. coli isolates. Furthermore, 95.24% of ESBL-producing E. coli isolates were positive for one or more antibiotic resistance genes. To the best of our knowledge, this is the first study to detect E. coli-carrying antibiotic resistance genes including beta-lactamase blaCMY and blaSHV originating from migratory birds in Bangladesh. These results suggest that migratory birds are potential carriers of ESBL-producing E. coli along with other clinically important antibiotic resistance genes which may have detrimental impacts on human health.

Molecular detection and antibiotyping of multi-drug resistant Enterococcus faecium from healthy broiler chickens in Bangladesh.

BACKGROUND: Enterococcus faecium is a ubiquitously distributed member of the intestinal microbiota of both humans and animals. Antibiotic resistant E. faecium are a major public health concern. OBJECTIVES: This study aimed to detect multi-drug resistant (MDR) E. faecium and their antibiotic resistance genes from broiler chickens in Bangladesh. METHODS: A total of 100 faecal samples of healthy broilers were screened by conventional methods and polymerase chain reaction (PCR) to detect E. faecium and their resistance genes. Disk diffusion test was employed to determine antibiotic profiles. RESULTS: By PCR, among 100 samples, 45% [95% confidence interval (CI): 35.62%-54.76%] were positive for E. faecium. Based on antibiogram, all the E. faecium isolates were found resistant to ampicillin, and frequently (93.33%-55.56%) resistant to ceftriaxone, cefotaxime, streptomycin, erythromycin, and imipenem; moderate to lower (26.67%-4.44%) resistance to tetracycline, ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and vancomycin. Interestingly, 80% (95% CI: 66.18%-89.10%) E. faecium isolates were MDR in nature. In addition, the indices of multiple antibiotic resistance (MAR) ranged from 0.08 to 0.83. By bivariate analysis, high positive significant correlations were observed between resistance profiles of erythromycin and imipenem, ciprofloxacin and norfloxacin, erythromycin and streptomycin, ceftriaxone and cefotaxime, tetracycline and chloramphenicol, and streptomycin and imipenem. Furthermore, the prevalence of resistance genes of E. faecium was 58.33% (tetA), 33.33% (tetB), 35.56% (blaTEM ), 60% (CITM), 13.33% (aadA1), and 12% (SHV). CONCLUSIONS: To the best of our knowledge, this is the first study in Bangladesh to detect MDR and MAR E. faecium and their associated resistance genes. The detection of MDR and MAR E. faecium and their corresponding resistance genes from healthy broilers is of public health concern because of their potential to enter into the food chain.

Migratory birds travelling to Bangladesh are potential carriers of multi-drug resistant Enterococcus spp., Salmonella spp., and Vibrio spp.

Antimicrobial resistance (AMR) is a major health crisis globally. Migratory birds could be a potential source for antibiotic resistant (ABR) bacteria. Not much is known about their role in the transmission of ABR in Bangladesh. In this study, a total of 66 freshly dropped fecal materials of migratory birds were analyzed. Bacterial isolation and identification were based on cultural properties, biochemical tests, and polymerase chain reaction (PCR). The disk diffusion method was employed to evaluate antibiogram profiles. By PCR, out of 66 samples, the detection rate of Enterococcus spp. (60.61%; 95% confidence interval: 48.55-71.50%) was found significantly higher than Salmonella spp. (21.21%; 95% CI: 13.08-32.51%) and Vibrio spp. (39.40%; 95% CI: 28.50-51.45%). Enterococcus isolates were frequently found resistant (100-40%) to ampicillin, streptomycin, meropenem, erythromycin, and gentamicin; Salmonella isolates were frequently resistant (72-43%) to chloramphenicol, tetracycline, ampicillin, streptomycin, and erythromycin; and Vibrio spp. isolates were frequently resistant (77-31%) to vancomycin, ampicillin, erythromycin, tetracycline, and streptomycin. In addition, 60% (95% CI: 44.60-73.65%) Enterococcus spp., 85.71% (95% CI: 60.06-97.46%) Salmonella spp., and 76.92% (95% CI: 57.95-88.97%) Vibrio spp. isolates were multi-drug resistant (MDR) in nature. Three isolates (one from each bacterium) were found resistant against six classes of antibiotics. The bivariate analysis revealed strong associations (both positive and negative) between several antibiotic pairs which were resistant to isolated organisms. To the best of our knowledge, this is the first study in detecting MDR Enterococcus spp., Salmonella spp., and Vibrio spp. from migratory birds travelling to Bangladesh. Frequent detection of MDR bacteria from migratory birds travelling to Bangladesh suggests that these birds have the potential to carry and spread ABR bacteria and could implicate potential risks to public health. We recommend that these birds should be kept under an AMR surveillance program to minimize the potential risk of contamination of the environment with ABR as well as to reduce their hazardous impacts on health.

Virulence Determinants and Multidrug Resistance of Escherichia coli Isolated from Migratory Birds.

Migratory birds are carriers of multidrug resistant pathogenic Escherichia coli. However, their roles in the dissemination of these resistant pathogens are still being neglected in Bangladesh. The present study was therefore carried out to detect multidrug resistant E. coli. In addition, these isolates were also screened for the presence of avian pathogenic E. coli (APEC)-associated virulence genes. A total of 66 fecal matter samples of migratory birds were screened. E. coli were isolated and identified by culturing and biochemical tests followed by polymerase chain reaction (PCR). APEC-associated virulence genes were detected by PCR. Disk diffusion assays were employed to investigate antibiogram profiles. Bivariate analysis was performed to assess correlations in resistance patterns between antimicrobials and to assess associations between virulence genes of E. coli. Among the 66 samples assessed by PCR, 55 (83.33%) were found positive for E. coli. Of these 55 isolates, the APEC-associated virulence gene fimC was detected in 67.27% of the isolates, which was significantly higher than in the cases of iucD (29.09%) and papC (5.45%) genes. In addition, three isolates were found positive for all three virulence genes, while 23 and 12 isolates were positive for one and two virulence genes respectively. In the bivariate analysis, significant associations were detected between fimC and iucD virulence genes. Using the antibiogram, all E. coli isolates were found to be multidrug resistant (MDR). The isolates exhibited 100% resistance against ampicillin and erythromycin in addition to varying percentages of resistance against streptomycin, tetracycline, ciprofloxacin, and chloramphenicol. Highly positive correlations between tetracycline and ciprofloxacin, chloramphenicol and ciprofloxacin, chloramphenicol and tetracycline were observed by bivariate analysis. To the best of our knowledge, this is the first study that reports APEC-associated virulence genes of MDR E. coli from migratory birds in Bangladesh. Results indicate that migratory birds are reservoirs of MDR E. coli isolates carrying APEC-associated virulence genes, which can seriously contribute to the development of human and animal diseases.

Zoonotic Diseases: Etiology, Impact, and Control.

Most humans are in contact with animals in a way or another. A zoonotic disease is a disease or infection that can be transmitted naturally from vertebrate animals to humans or from humans to vertebrate animals. More than 60% of human pathogens are zoonotic in origin. This includes a wide variety of bacteria, viruses, fungi, protozoa, parasites, and other pathogens. Factors such as climate change, urbanization, animal migration and trade, travel and tourism, vector biology, anthropogenic factors, and natural factors have greatly influenced the emergence, re-emergence, distribution, and patterns of zoonoses. As time goes on, there are more emerging and re-emerging zoonotic diseases. In this review, we reviewed the etiology of major zoonotic diseases, their impact on human health, and control measures for better management. We also highlighted COVID-19, a newly emerging zoonotic disease of likely bat origin that has affected millions of humans along with devastating global consequences. The implementation of One Health measures is highly recommended for the effective prevention and control of possible zoonosis.

Molecular Detection of Avian Pathogenic Escherichia coli (APEC) for the First Time in Layer Farms in Bangladesh and Their Antibiotic Resistance Patterns.

Avian pathogenic Escherichia coli (APEC) causes significant economic losses in poultry industries. Here, we determined for the first time in Bangladesh, the prevalence of APEC-associated virulence genes in E. coli isolated from layer farms and their antibiotic resistance patterns. A total of 99 samples comprising internal organs, feces, and air were collected from 32 layer farms. Isolation was performed by culturing samples on eosin-methylene blue agar plates, while the molecular detection of APEC was performed by PCR, and antibiograms were performed by disk diffusion. Among the samples, 36 were positive for the APEC-associated virulence genes fimC, iucD, and papC. Out of 36 isolates, 7, 18, and 11 were positive, respectively, for three virulence genes (papC, fimC, and iucD), two virulence genes, and a single virulence gene. Although the detection of virulence genes was significantly higher in the internal organs, the air and feces were also positive. The antibiograms revealed that all the isolates (100%) were resistant to ampicillin and tetracycline; 97.2%, to chloramphenicol and erythromycin; 55.5%, to enrofloxacin; 50.0%, to norfloxacin and ciprofloxacin; 19.4%, to streptomycin; 11.1%, to colistin; and 8.33%, to gentamicin. Interestingly, all the isolates were multidrug-resistant (MDR). Spearman's rank correlation coefficient analysis revealed the strongest significant correlation between norfloxacin and ciprofloxacin resistance. This is the first study in Bangladesh describing the molecular detection of APEC in layer farms. Isolated APEC can now be used for detailed genetic characterization and assessing the impact on public health.

Emergence of colistin-resistant Escherichia coli in poultry, house flies, and pond water in Mymensingh, Bangladesh.

OBJECTIVE: Emergence of colistin-resistant Escherichia coli (CREC) has generated a sense of public alarm. The objective of this study was to detect the CREC and identification of the gene responsible for such resistance. MATERIALS AND METHODS: A total of 150 samples comprising poultry cloacal swab, house flies (Musca domestica), and pond water were collected randomly from Mymensingh, Bangladesh and analyzed. Isolation and identification of E. coli were done based on culture and E. coli 16S rRNA gene-specific polymerase chain reaction (PCR). Phenotypic detection of CREC was done by disk diffusion method. Finally, colistin resistance genes were detected by PCR by using colistin resistant gene mcr3 specific primers. RESULTS: Among the 150 samples, phenotypically 18.00% (n = 27/150) isolates were found as colistin resistant. By PCR, 8.00% of the E. coli isolates were found positive for the presence of mcr3 gene. CONCLUSIONS: Colistin resistant E. coli carrying mcr3 are detected in poultry, house flies and water that are of great public health concern.

Molecular detection of multidrug and colistin-resistant Escherichia coli isolated from house flies in various environmental settings.

Aim: To detect antibiotic-resistant pathogenic Escherichia coli in house flies captured from various environmental settings. Materials & methods: Isolation and identification of E. coli was performed based on culture and PCR. Antimicrobial susceptibility testing was conducted using disk diffusion test. Detection of virulence and antimicrobial resistance genes was carried out using PCR methods. Results: The prevalence of E. coli in flies was 61% with highest in dairy farms (70.67%) followed by food centers (65.33%), dustbins (64%) and area close to hospital (44%). The prevalence rates of tetA, tetB, ereA, CITM, SHV, mcr-3 and catA1 resistance determinants were 88.75, 48.52, 41.67, 37.17, 27.77, 18.37 and 14.81%, respectively. Conclusion: Multidrug-resistant pathogenic E. coli are widely distributed in house flies, which requires a one-health approach to delineate their control.