Pathotyping and antimicrobial resistance-characterization of Staphylococcus aureus in milk for human consumption in Marsabit and Isiolo Counties, Kenya

Abstract

Staphylococcus aureus (S. aureus) is a ubiquitous Gram-positive bacterium commonly encountered in the environment as well as mucus membranes of animals and humans. S. aureus food poisoning results from consumption of preformed S. aureus enterotoxins in food. The risk posed by contamination of milk intended for human consumption by pathogenic S. aureus in pastoral areas in Kenya is still not well documented, yet this information is critical for ensuring safety to consumers who sometimes may take unpasteurized milk. The prevalence of antimicrobial resistant (AMR)-S. aureus including methicillin-resistant S. aureus (MRSA) in livestock raw milk consumed by pastoralists in Kenya remains unclear. This study therefore determined the phenotypes, genetic determinants for virulence associated with S. aureus enterotoxins, the phenotypic resistance profiles and the genetic determinants responsible for antimicrobial resistance phenotypes of milk borne S. aureus. In addition, the relationship between antimicrobial usage (AMU) and emergence of multidrug-resistant (MDR) S. aureus, including MRSA in raw milk of livestock was determined by correlation analysis. A cross-sectional study design involving 188 households in the two counties was conducted. In total, 603 milk samples from 57 zebu cattle, 346 Galla goats, 8 red Maasai and dorper sheep, 4 one-humped camels (Camelus dromedaries) and 188 pooled from all animals were collected from Isiolo and Marsabit counties of Kenya. S. aureus isolates were cultured from milk samples using a selective media, mannitol salt agar (MSA). Suspect colonies of S. aureus were further analysed using biochemical tests including Gram staining, catalase activity, mannitol fermentation, coagulase activity and β-hemolysis. The isolates were confirmed by amplification of S. aureus specific staphylococcal terminase gene (satm) and BLAST analysis. xviii The isolates confirmed to be potentially pathogenic S. aureus were further evaluated to determine whether they harbour enterotoxin genes responsible for milk-borne food poisoning. Oligonucleotide primers were designed and used for the amplification of sea, seb, sec, sed and see enterotoxin encoding genes. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method according to Clinical Laboratory Standards Institute Guidelines. Moreover, genetic determinants responsible for the resistance phenotypes of S. aureus were analyzed by PCR, sequencing, and Blast analysis. Genes encoding for resistance to oxacillin (mecA), tetracyclines (tetK, tetM), other beta-lactams (blaZ), aminoglysosides [aac (6’)/aph (2”), aph (3’)-IIIa], and macrolides (msrA/ermA) were used for the analysis. Data on the commonly used and/or sold antimicrobials in the study area were collected from wholesalers and veterinary pharmacies for a period of one year. Correlation between AMU and occurrence of resistance was determined by Pearson’s correlation coefficient (r) method. Overall, potentially pathogenic S. aureus harboring enterotoxic genes were detected in 85 (14.09%, 95% CI: 11.55–17.1%) of the total milk samples. Genes encoding enterotoxins were detected in the S. aureus bacteria isolated from the milk samples. At least one type of S. aureus enterotoxin gene (SE) was detected in 74.11% (95% CI: 63.91–82.24%) of the 85 isolates. The most frequently encountered gene in the two counties was see (51; 60%, 95% CI: 49.73–69.76%) followed by sea (22; 25.88%, 95% CI: 17.76–36.09%) and sec (19; 22.35%, 95% CI: 14.8–32.29%). None of the isolates tested positive for sed. Overall, 21 of the 85 (24.7%, 95% CI: 16.76–34.83%) strains harbored more than one enterotoxin gene. More than half of the S. aureus isolates harbored at least one of the enterotoxin coding genes, indicating milk samples contaminated by S. aureus could have a high chance of causing staphylococcal food intoxication. xix The S. aureus isolates were mainly resistant to tetracycline (79%), ampicillin (58%), and oxacillin (33%), respectively. A few isolates (5–18%) were resistant to clindamycin, cephalexin, erythromycin, kanamycin, and ciprofloxacin. Most of the MDR-S. aureus isolates were MRSA (94%). The genetic determinants found in the AMR isolates included tetK/tetM (96.5%/19%) for tetracycline, blaZ (79%) for penicillin, aac (6’)/aph (2”)/aph (3’)-IIIa (53%) for aminoglycosides, mecA (41%) for oxacillin, and msrA/ermA (24%/7%) for macrolides. The antimicrobial classes used in the study site were as follows; 4,168 kg of oxytetracycline, 70 kg of sulfonamides, 49.7 kg of aminoglycosides, 46 kg of beta-lactams, 39.4 kg of macrolides, and 0.52 kg for trimethoprim. Oxytetracycline usage was correlated to tetK/tetM (r = 0.62/1) detection, penicillins to mecA/blaZ (r = 0.86/0.98), aminoglycoside to aac (6’)/aph (2”)/aph (3’)-IIIa (r = 0.76/-13), and macrolide usages for detection of ermA/msrA (r = 0.94/0.77). There was association between AMU and the occurrence of MDRSA and the tetM detection. In conclusion, milk consumed in Marsabit and Isiolo counties is contaminated with multidrug resistant S. aureus that harbour enterotoxigenic and antimicrobial resistance genes hence its consumption could increase the risk of staphylococcal food poisoning. AMU appeared to be associated with occurrence of MDRSA and the tetM detection. Judicious use of antimicrobials is recommended in the study areas to mitigate against development of AMR Key Words: Staphylococcus aureus: raw milk: enterotoxins genes; Antimicrobial usage; Multidrug resistant-MRSA; Kenya