Beef Hygiene and the Prevalence of Antimicrobial Resistant Shiga Toxin-producing Escherichia Coli in Kajiado Slaughterhouses, Kenya

Abstract

Local slaughterhouses in Kenya have the responsibility of availing to the public wholesome beef and meat of other animal species. These slaughter facilities have great variability in human, technical and infrastructural capacity, and ultimately in slaughter hygiene and safety (SHMS) levels. The aim of this study was to determine the prevalence and antimicrobial resistance of STEC and address the underlying factors to its occurrence and reduction in beef. The study had six objectives, i) to review the literature associating slaughter practices with meat hazards prioritized in Kenya, ii) to determine the level of knowledge, attitudes and practices (KAPs) of slaughterhouse workers on SHMS across 7 small and medium slaughterhouses (SMS) in Kajiado County, iii) to determine bacteriological and chemical quality of slaughterhouse tap water and efficacy of hot water in carcass washing, iv) to determine the effect of 8 beef cooking methods on the survival of Pediococcus surrogates of E. coli O157:H7, v) to determine the effect of delayed beef refrigeration on growth of spoilage bacteria and vi) to determine the prevalence of antimicrobial resistant Shiga-toxin producing E. coli (STEC) on carcasses, hides and recto-anal junction mucosa (RAJ) of cattle across 5 slaughterhouses in Kajiado County. A Google Scholar search guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) was run to identify relevant articles for review. Semi-structured questionnaires were administered to 78 slaughterhouses workers to assess their knowledge, attitudes and practices on slaughter hygiene and meat safety. Slaughterhouse tap water from 5 facilities along the Namanga-Bissil-Kajiado-Isinya-Kiserian trade route were sampled and analyzed for total and faecal coliform, E. coli, Salmonella spp., Shigella spp., water pH and temperature, electrical conductivity (EC), total dissolved solids (TDS), turbidity, iron (Fe), aluminium (Al), fluoride, nitrate, nitrite and ammoniacal nitrogen. Beef rib surfaces inoculated with standard E. coli ATCC 25922 were washed with water heated to 70, 75, 80, 85, 90 and 95 °C and control (20.7 °C) for 10 sec to determined log reduction and spreading of inoculum, and effect of treatments on carcass appearance. The effect of beef cooking methods; boiling (95 °C/1 hr), simmering (85 °C/1 hr), roasting (163 °C/1 hr), pressure cooking (85kPa/ 80 ° C / 30 min), sous vide cooking to three degrees of doneness, that is rare (60 ° C ), medium (71 ° C ) and very well done (82 ° C ) all for 1 hr and pan-broiling (163 °C/ 6 min per side) of burger patties were assessed for their lethality against Bactoferm® xxv LHP-DRY, a Pediococcus surrogate of E. coli O157:H7 and endpoint core temperatures of cooked beef. Procured beef steaks were refrigerated promptly (0 hr), and after four (4 hr) and eight (8 hr) hours of delay and stored for 12 days. Change in pH and beef was assessed every 4th day. Microbial growth was analysed culturally on day 4, 8 and 12 on Plate Count Agar (PCA), de Man-Rogosa Sharpe (MRS) agar, Streptomycin-thallous acetate-actidione agar (STAA), Pseudomonas agar, Violet-Red Bile Glucose (VRBG) agar, and MacConkey agar, and non-culturally by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Two hundred (200) beef carcasses, 196 hides, 205 recto-anal junction mucosa samples were sampled. A Total Viable Count (TVC) was done on carcasses on PCA. All samples were enriched in Tryptone Soy Broth (TSB), enumerated on MacConkey Sorbitol agar (SMAC) and confirmed on Eosin Methylene Blue (EMB). Isolates with colourless colony morphology on SMAC and subsequently greenish metallic sheen on EMB were suspected to be E. coli O157:H7. Confirmation was analysed by Multiplex PCR characterization of Stx1, Stx2 and eae virulence genes. Isolates positive for at least 1 virulence gene were tested for antimicrobial susceptibility against , tetracycline, streptomycin, gentamycin, sulfamethazole, erythromycin, ceftiofur, cephalexin, cefuroxime, nalidixic acid and chloramphenicol. Objective one revealed that fascioliosis and cysticercosis remain the most prevalent parasitic meat hazards in cattle and pig carcasses, respectively. Zoonotic E. coli had been identified as a leading bacterial hazard, however, the lack of routine microbial examination renders its surveillance inadequate. The second objective on KAPs revealed that out of 78 respondents, 80.8 % had not received any kind of slaughter-related training, majority had moderate to low knowledge, neutral attitudes towards SHMS, while practices in carcass decontamination, sanitizing of facility and tools and medical testing were poor. Experiment 1 of objective three revealed that 80 %, 60 % and 20 % of water samples failed to meet the WHO guidelines for total coliform, E. coli, and Salmonella, and all water sample temperatures ranged from 23.4 to 26.1 ° C exceeding the WHO guidelines of 15 ° C . Experiment 2 reported increase in carcass microbial log reductions with increasing wash water temperatures. Water at 95 to 75 ° C yielded carcasses with unacceptable appearance. Objective 4 reported that pressure-cooking, very well done sous-vide, oven roasting, medium done sous-vide, boiling, and rare done sous-vide, simmering, and pan-broiling achieved 5.23, 5.13, 4.83, 4.13, 4.0, 3.83, 3.26, and 2.83 log reductions CFU/g, respectively. Kenyan beef cooking methods; boiling, simmering, and roasting attained endpoint core temperatures >71.1 ° C guaranteeing the instant 5-log reduction of STEC. xxvi Objective 5 examined the effect of delaying beef refrigeration by 0, 4 and 8 hours on population diversity of spoilage microorganisms. The quantitatively dominant microflora were Enterobactereacea and Pseudomonas spp. with mean microbial counts and pH increments being highest in the 0 hr promptly refrigerated treatment. Objective 6 characterized 135 out of 601 (22.46 %) bovine samples by Multiplex PCR and isolates positive for virulence genes were reported in 14.00 % (28/ 200) of carcasses, 28.06 % (55/ 196) of hides and 25.37 % (52/ 205) of RAJ. Of the 135 STEC isolates, 95 (70.35 %) were viable for AMR analysis. High resistance was observed against cefalexin (85.26 %) and erythromycin (83.16 %) while 21 (22.1 %) isolates had multi-drug resistance to three to five classes of antibiotics. The study concluded that low to medium levels of knowledge of SHMS, minimal training, inadequate PPEs and sanitation facilities, and faecally contaminated piped water, impacts negatively on the attitude and practices of slaughterhouse workers as well as on beef hygiene and safety. Furthermore, hot water carcass washing at 70 °C for 10 seconds is an effective carcass decontaminating intervention that can be adopted in local SMS while high-temperature long-time beef cooking methods are lethal against STEC. The current practice in local butcheries of hanging beef carcasses at ambient temperatures during the day before refrigeration in the evenings, leads to dehydration of the carcasses, and thereby slows down microbial growth and ultimately slowing down spoilage. The study proposes the review of the current Meat Control Act and standards of meat inspection, by clearly outlining good hygienic practices (GHPs) and HACCP in slaughter operations. The Kenyan Standard on bovine meat specifications should broaden the microbiological criteria of bovine carcasses by adopting a zero tolerance policy for O157 and non-O157 STEC. Recommendation for practice include capacity building of slaughterhouse workers, meat retailers and consumers, establishment of water treatment and monitoring plans, upgrading and availing sanitation facilities, and strict supervision of worker practices. Future studies on meat spoilage are also recommended.