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.

Fish has always been an integral part of Bengali cuisine and economy. Fish could also be a potential reservoir of pathogens. This study aimed to inquisite the distribution of virulence, biofilm formation, and antimicrobial resistance of Enterococcus faecalis isolated from wild and cultivated fish in Bangladesh. A total of 132 koi fish (Anabas scandens) and catfish (Heteropneustes fossilis) were collected from different markets in the Mymensingh district and analyzed to detect E. faecalis. E. faecalis was detected by conventional culture and polymerase chain reaction (PCR), followed by the detection of virulence genes by PCR. Antibiotic susceptibility was determined using the disk diffusion method, and biofilm-forming ability was investigated by crystal violet microtiter plate (CVMP) methods. A total of 47 wild and 40 cultured fish samples were confirmed positive for E. faecalis by PCR. The CVMP method revealed four per cent of isolates from cultured fish as strong biofilm formers, but no strong producers were found from the wild fish. In the PCR test, 45% of the isolates from the wild and cultivated fish samples were found to be positive for at least one biofilm-producing virulence gene, where agg, ace, gelE, pil, and fsrC genes were detected in 80, 95, 100, 93, and 100% of the isolates, respectively. Many of the isolates from both types of samples were multidrug resistant (MDR) (73% in local fish and 100% in cultured fish), with 100% resistance to erythromycin, linezolid, penicillin, and rifampicin in E. faecalis from cultured fish and 73.08, 69.23, 69.23, and 76.92%, respectively, in E. faecalis from wild fish. This study shows that E. faecalis from wild fish have a higher frequency of virulence genes and biofilm-forming genes than cultivated fish. However, compared to wild fish, cultured fish were found to carry E. faecalis that was more highly multidrug resistant. Present findings suggest that both wild and cultured fish could be potential sources for MDR E. faecalis, having potential public health implications.

Enterococci are commensal bacteria that inhabit the digestive tracts of animals and humans. The transmission of antibiotic-resistant genes through human–animal contact poses a potential public health risk worldwide, as zoonoses from wildlife reservoirs can occur on every continent. The purpose of this study was to detect Enterococcus spp. in rhesus macaques (Macaca mulatta) and to investigate their resistance patterns, virulence profiles, and biofilm-forming ability. Conventional screening of rectal swabs (n = 67) from macaques was followed by polymerase chain reaction (PCR). The biofilm-forming enterococci were determined using the Congo red agar plate assay. Using the disk diffusion test (DDT), antibiogram profiles were determined, followed by resistance and virulence genes identification by PCR. PCR for bacterial species confirmation revealed that 65.7% (44/67) and 22.4% (15/67) of the samples tested positive for E. faecalis and E. faecium, respectively. All the isolated enterococci were biofilm formers. In the DDT, enterococcal isolates exhibited high to moderate resistance to penicillin, rifampin, ampicillin, erythromycin, vancomycin, and linezolid. In the PCR assays, the resistance gene bla_TEM was detected in 61.4% (27/44) of E. faecalis and 60% (9/15) of E. faecium isolates. Interestingly, 88.63 % (39/44) of E. faecalis and 100% (15/15) of E. faecium isolates were phenotypically multidrug-resistant. Virulence genes (agg, fsrA, fsrB, fsrC, gelE, sprE, pil, and ace) were more frequent in E. faecalis compared to E. faecium; however, isolates of both Enterococcus spp. were found negative for the cyl gene. As far as we know, the present study has detected, for the first time in Bangladesh, the presence of virulence genes in MDR biofilm-forming enterococci isolated from rhesus macaques. The findings of this study suggest employing epidemiological surveillance along with the one-health approach to monitor these pathogens in wild animals in Bangladesh, which will aid in preventing their potential transmission to humans.

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.

Pathogenic, antibiotic-resistant, and biofilm-forming bacteria can be transferred to humans through the consumption of contaminated seafood. The present study was carried out to determine antibiotic resistance profiles and virulence determinants in biofilm-forming Enterococcus faecium isolated from seafood in Bangladesh. A total of 150 seafood samples, including shrimp (n = 50), crabs (n = 25), and marine fish (n = 75), were screened using cultural, staining, biochemical, polymerase chain reaction (PCR), Congo red (CR), and disk diffusion (DD) assays. In PCR, E. faecium was detected in 27.3% (41/150; CI_95% 20.8; 34.9) of samples, where marine fish (34.7%, CI_95% 24.9; 45.9) had the highest prevalence (p < 0.05) compared to crabs (32%, CI_95% 17.2; 51.6) and shrimp (14%, CI_95% 7.0; 26.1). Thirty-two (78.1%, CI_95% 63.3; 88.0) of the E. faecium isolates were determined to be biofilm formers in the CR test, where 43.9% (18/41, CI_95% 29.9; 59.0) and 34.2% (14/41, CI_95% 21.6; 49.5) of the isolates were strong and intermediate biofilm formers, respectively. In PCR, virulence genes, i.e., pil (100%), ace (92.7%), agg (68.3%), fsrA (65.9%), gelE (63.4%), sprE (53.7%), fsrB (51.2%), and fsrC (43.9%), were detected in E. faecium isolates. All the E. faecium isolates were phenotypically resistant to ≥3 antimicrobial categories and ≥3 antibiotics, including WHO-classified reserve antibiotics linezolid (70.7%) and fosfomycin (19.5%). Moreover, the multiple antibiotic resistance index ranged up to 0.8, showing resistance to ten antibiotics and eight antibiotic classes. In this study, the prevalence of virulence genes and antibiotic resistance was significantly greater (p < 0.05) in strong biofilm-forming E. faecium strains as compared to strains with intermediate and non-biofilm-forming abilities. As far as we know, this study, for the first time in Bangladesh, determined antibiotic resistance and detected virulence genes in biofilm-forming E. faecium isolated from seafood samples. The data from this study could play a significant role in evaluating potential health hazards linked to the ingestion of uncooked or minimally processed seafood.

Objective: This study focused on the antibiogram profiling of Staphylococcus aureus and coagu¬lase-negative Staphylococcus spp. (CoNS) and the detection of icaA and blaZ genes from bovine raw milk samples. Materials and Methods: Bovine milk samples were collected from dairy farms, and Staphylococcus spp. were isolated and identified via conventional and molecular screening. Disk diffusion test (DDT) was implemented to determine the resistance pattern. Biofilm and β-lactamase-produc¬ing Staphylococcus spp. were identified via amplification of the icaA and blaZ genes. Methicillin-resistant Staphylococcus aureus and CoNS were identified by DDT and PCR of the mecA gene. Results: From 63 samples, 35 were confirmed as Staphylococcus spp., of which 16 (25.39%) S. aureus isolates were coagulase-positive, while 19 (30.16%) were negative. PCR confirmed that 50% (8/16) of S. aureus and 36.84% (7/19) of CoNS possessed the icaA gene. All S. aureus iso¬lates were found resistant to penicillin-G (P) both phenotypically and genotypically. The isolates were also resistant to erythromycin (ERY) and oxytetracycline (TET). While CoNS showed high to reduced resistance against P, TET, ERY, and azithromycin, no S. aureus isolates were resistant to sulfamethoxazole, while 10.53% of CoNS isolates were. All S. aureus and CoNS isolates were sus¬ceptible to vancomycin and gentamicin. MR was exhibited by 37.5% of S. aureus and 42.10% of CoNS isolates. Moreover, S. aureus and CoNS had 56.25% and 52.63% multidrug-resistant (MDR) isolates, respectively. Conclusion: The present study revealed the presence of a biofilm-producing, MDR staphylococcal strain in milk that might endanger consumers. Routine surveillance and monitoring, along with antimicrobial resistance learning, can reduce risks.