Ready-to-eat foods: A potential vehicle for spread of coagulase-positive staphylococci and antibiotic-resistant Staphylococcus aureus in Buea municipality, South West Cameroon

Background The consumption of ready-to-eat (RTE) foods contaminated with coagulase-positive staphylococci (CoPS) and especially Staphylococcus aureus puts consumers at potential risk of foodborne disease or colonization and subsequent infection. This cross-sectional study determined the levels of CoPS and the presence of S. aureus in RTE foods sold in Buea municipality. Method A total of 420 RTE food samples comprising 70 each of cake, bread, fruit salad, meat-hot-pot, suya and boiled rice were randomly purchased from February to August 2020. The CoPS counts were determined by culturing on Baird-Parker agar and S. aureus identified by amplification of the nuc gene using polymerase chain reaction. All S. aureus isolates were screened for the presence of classical staphylococcal enterotoxin genes and each isolate challenged with 11 antibiotics to determine their antibiotic susceptibility profiles. Oxacillin-resistant S. aureus were analyzed for the presence of mecA gene. Result Overall, 161 (38.3%) samples had detectable levels of coagulase-positive staphylococci ranging from 2.0-5.81 log10CFU/g. Based on CoPS levels, 37 (8.81%) of the 420 RTE food samples, only fruit salad and meat-hot-pot, had unsatisfactory microbiological quality. A total of 72 S. aureus isolates, comprising 52.78% from fruit salad, 16.67% from meat-hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread and 4.17% from cake were recovered. None of the S. aureus isolates possessed any of the classical enterotoxin genes. All the isolates were sensitive to vancomycin and ofloxacin while 68 (94.44%) and 66 (91.67%) were sensitive to oxacillin and ciprofloxacin, respectively. Resistance to penicillin (93.06%) was highest followed by amoxicillin (91.67%) and erythromycin (79.17%). Four isolates were identified as methicillin-resistant S. aureus all of which carried the mecA gene. A total of 24 antibiotypes were identified. Conclusion Our findings showed that RTE foods sold in the Buea municipality are likely vehicles for transmission of CoPS and antibiotic-resistant S. aureus.

This study was carried out on RTE foods from vendors in Buea municipality; the administrative  If negative after 4 h, the tube was re-examined at 24 h.

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The number of coagulase positive staphylococci per g of sample was calculated as follows: suspended in the PBS. The tubes were vortexed at 14000 rpm for 10 sec. The bacterial solution was 198 heated in a water bath at 100℃ for 15min after which it was removed and immediately chilled in ice for 199 another 15min. The bacterial solution was allowed to thaw at 37 ℃ in the water bath and again chilled in 200 ice for another 15min. The tubes were centrifuged at full speed (14000rpm) for 5 min and the 201 supernatant was pipetted into another labeled tube and used as template DNA in PCR assay.

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Presumptive S. aureus isolates were confirmed by amplification of a 280 bp fragment of the nuc gene of 203 S. aureus using primer sequences previously described (Table 2). Each PCR reaction in this study comprised 2X BioMix Red master-mix (10μL), 1μL of each primer 207 from a 10μM working stock (final concentration, 0.5μM), 1μL DNA template and nuclease free water to 208 make a final volume of 20µL. Each PCR run had a negative control which was nuclease-free water and a 209 positive control which was a previously characterized S. aureus isolate stored in the laboratory.

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The PCR cycling conditions were optimized with initial denaturation at 95°C for 5min followed by 35 211 cycles of denaturation at 94°C / 60sec, annealing at 54°C / 40sec and extension at 72°C / 60sec. Final extension was at 72°C for 7min and the reaction was stopped by holding tubes at 4°C until removed 213 from the thermal cycler (MyCycler™ Thermal Cycler BIORAD, USA). The PCR products were 214 electrophoresed in a 1.5% agarose gel stained with SYBR Safe DNA Gel stain (Invitrogen) for 1 hour at 215 90V in 1X Tris borate EDTA, visualized and photographed with a Molecular Imager Gel Doc XR   (Table 1).

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The multiplex PCR run was optimized at initial denaturation for 5 min at 94 °C followed by 30 cycles of 222 denaturation at 94°C for 60sec, annealing at 50°C for 60sec and extension at 72°C for 60sec. A final 223 extension at 72°C for 5min was done at the end of the cycles. A singleplex PCR run was performed for 224 detection of enterotoxin E using the same cycling conditions except for the annealing temperature that 225 was set at 48°C. All PCR products were electrophoretically separated in 1.5% agarose gel as described 226 above.  counts were transformed to log 10 CFU/mL prior to statistical analysis. Data on the prevalence of S. aureus and multidrug resistant isolates were analysed using a chi-squared test to determine whether 259 there were significant differences in their prevalence between sample types. The confidence level was 260 held at 95% and p-value at less than 5% for all analysis.      the most contaminated sample with S. aureus followed by boiled rice and meat-hot-pot (Table 5).  The mecA gene (Fig 1) was detected in 5.56 % (4/72) of the S. aureus isolates from three RTE food 300 types while none of the S. aureus isolates possessed any of the classical enterotoxin genes (Table 5).

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A total of 24 antibiotypes were identified in this study (Table 7). The most prevalent antibiotype was P-

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AML-E-AZM-C that was detected in 9 (12.5%) of the 72 S. aureus isolates. More antibiotypes were 316 identified in fruit salad followed by meat-hot-pot.

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The high rate of consumption of RTE foods in Cameroon and especially in the Buea municipality 319 coupled with the scarcity of data on the microbiological quality of these foods is a call for concern. 320 Elsewhere, there are several reports that highlight the role of RTE foods in the emergence or   CoPS and it ranged from 2 -2.91 log 10 CFU/g (Table 3). While these 12 samples had borderline quality, 364 the remaining 58 (82.86%) samples had satisfactory quality (Table 4) In this study, majority of the samples of boiled rice (75.71%), bread (61.43%) and cake (70.0%) (