Characterization and control of multi-drug resistant enterotoxigenic Staphylococcus aureus and Escherichia coli isolated from Arabic shawarma kiosks in the Klang Valley, Malaysia

Despite several efforts being made to solve the problem arising from antimicrobial resistance remains one of the major health issues globally with significant impact on health worldwide. Survival of multidrug resistant enterotoxigenic S. aureus and E. coli with biofilm forming ability in ready to eat food is a possible danger to the health of consumers especially in societies where most of the population depend on public food for their daily meals. These organisms can serve as sources of outbreak leading to morbidity and mortality particularly in persons with compromised immunity. However, there is a need in the area of food safety, quality and preservation to counter the rising of resistant pathogens and the limitation of synthetic chemical additives in the food system. This study aimed to detect the presence of S. aureus and E. coli in chicken shawarma sandwiches, presence of enterotoxins, investigate their resistance to commonly used antibiotics, their biofilm formation potential and to evaluate the antimicrobial activity of white vinegar against them. Sixty Shawarma samples were purchased from 20 selected kiosks within Klang valley; Kajang, South City Serdang, One South Serdang and Kuala Lumpur. The samples were purchased in three visits to each restaurant and subjected to microbiological analysis for the detection of S. aureus and E. coli. Following the analysis, the isolates were identified using biochemical tests and PCR. The identified isolates were then screened for the presence of enterotoxins using PCR and tested for antimicrobial resistance using Clinical and Laboratory Standard Institute (CLSI) guidelines to detect the multidrug resistant (MDR) strains. The MDR S. aureus and E. coli strains were also subjected to biofilm formation. The MDR enterotoxigenic S. aureus isolates with strong biofilm formation potential were challenged with white vinegar using disc diffusion technique, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Sessile minimum inhibitory concentration (SMIC) and minimum biofilm eradication concentration (MBEC) were used to assess the activity of the white vinegar against the biofilms of the multidrug resistant enterotoxigenic strains of S. aureus. Finally, chicken breast meats were contaminated with the MDR biofilm forming enterotoxigenic S. aureus isolates and treated with various concentrations of white vinegar (0.50, 1.00, 1.50 and 2.00%) at various exposure times (10, 20 and 30 minutes) to assess the white vinegar for its potency to serve as food sanitizer. Out of the 60 shawarma samples, 60% (n=36) were positive for S. aureus while 52% (n=31) were positive for E. coli. Of the 36 S. aureus, 19.7% (n=7) were found to harbour staphylococcal enterotoxin A (sea) and none harboured staphylococcal enterotoxin D (sed). Heat-labile (lt) and heat-stable (st) gene of enterotoxigenic E. coli (ETEC) was not detected in all E. coli strains. Moreover, out of the 36 S. aureus, 33% (n=12) were MDR and 54.8% (n=17) of the 31 E. coli isolates were MDR. Out of the 12 MDR S. aureus only 33% (4/12) were enterotoxigenic with strong BFP. The strain S3.2 was the most resistant to white vinegar 5.00% concentration with 15.33± 0.58 mm inhibition zone while S4.2 was the most susceptible with 17.33± 0.58 mm inhibition zone. The MIC and MBC results showed that S3.2 was inhibited at 0.31% concentration while the remaining strains S4.2, S8.2 and S10.1 were inhibited at 0.62% MIC. Meanwhile, S3.2 and S10.1 were totally killed at 0.62% MBC while S4.2 and S8.2 were totally killed at 1.25% MBC. However, the SMIC was lower for S3.2 at 0.62% and higher for S4.2, S8.2 and S10.1 at 1.250%. The MBEC result showed that the biofilm of S3.2 was eradicated at 1.25% while that of S10.1 at 2.50%. The S.42 and S8.2 were found to be resistant to the highest MBEC (2.50%) prepared in this study. On the contaminated chicken breast, 0.50% did not record significant log reduction while 1.00% and 1.50% concentrations exhibited an effect on the bacteria especially at 30 minutes of exposure. Lastly, the 2.00% concentration of white vinegar recorded a significant activity especially at 30-minute time of exposure with more than 3 Log reductions in all the four isolates of S. aureus. The results obtained in this study indicated the promising applications of white vinegar as natural sanitizer agent for raw chicken meat shawama. Moreover, white vinegar has proved to be a potent anti-biofilm agent that is safe to be used in foods such as Shawarma. However, the effects of white vinegar mechanisms of action on food pathogens should be determined in future studies. In addition, it is highly recommended that white vinegar is used on raw chicken meat to inhibit the growth of multidrug resistant organisms.

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