Phenotypic and Molecular Characterization of Antimicrobial Resistant Non-pathogenic Staphylococcus Species in Raw Camel Milk From Garissa County, Kenya

Abstract

Staphylococci bacteria are grouped into coagulase-positive Staphylococcus (CoPS) and coagulase-negative Staphylococcus (CoNS) by their ability to produce coagulase. CoNS are traditionally non-pathogenic but recent studies have reported CoNS to cause mastitis and septicaemia in livestock. In humans, CoNS play a role as opportunistic pathogens often involved in catheter-related infections, osteomyelitis, bacteraemia, endocarditis, boils, skin abscesses, cellulitis and urinary tract infections. Both S. aureus and CoNS species harbour resistant genes that confer antimicrobial resistant (AMR) phenotypes which are encoded by a range of genes. Some of these genes are found in mobile genetic elements which facilitate transfer of resistance genes from the non-pathogenic to pathogenic species. As a result of the genetic transfer, both groups can harbour multidrug-resistance (MDR) genes. This study investigated the phenotypic and genotypic AMR profiles of non-pathogenic Staphylococcus species contaminating raw camel milk from Garissa County in Kenya. A total of 231 raw camel milk samples were randomly collected from lactating camels. Smallholder camel farmers were randomly selected from the list of camel farmers provided by the clan heads in each of the sub-Counties. Bacteria were recovered in Buffered Peptone Water (BPW). Staphylococcus isolates were cultured on Mannitol Salt agar (MSA) and Blood Agar (BA). Coagulase and catalase tests were used to characterize the isolates. Thereafter the isolates were confirmed as Staphylococcus species by PCR and sequencing. Antimicrobial resistance profiles of the confirmed isolates were established by Kirby Bauer disk diffusion method. The antimicrobials used included; 10ug ampicillin, 10ug streptomycin, 30ug cephalexin, 15ug erythromycin, 5ug ciprofloxacin, 30ug cefoxitin, 30ug tetracycline and 30ug chloramphenicol. Mueller Hinton Agar (MHA) was streaked with a swab of isolates cultured on Trypticase Soy Agar. Antibiotic disks dispensed on the surface of the MHA using a disk dispenser in duplicates. The plates were incubated at 37°C for 24 hours after which diameters of zones inhibition (mm) were measured using a Vernier calibre and an average obtained. The readings were recorded as either susceptible, intermediate, xii or resistant based on the interpretative breakpoints by the Clinical Laboratory Standards Institute (CLSI) guidelines. Genetic determinants responsible for the resistance phenotypes of Staphylococcus species were determined by Polymerase Chain Reaction (PCR), sequencing, and Blast analysis. Genes encoding aminoglycoside (streptomycin) resistance (aph(6)-Id (strB), beta-lactams (mecA, mecC, blaZ and blaTEM) were analysed. Overall 122 (52.8%) isolates showed yellow mucoid (83.1%) while 83 (68%) were catalase positive. β-haemolysis with clear zones around the yellow colonies was observed in 122 (91.7%) of the isolates cultured in Blood agar (BA) indicating Staphylococcus species. On molecular analysis, 122 (91.7%) isolates were identified as Staphylococcus species at 900bp by amplification of 16S rRNA gene while 102 (83.6%) isolates were confirmed as S. aureus after PCR amplification of nuc gene at corresponding band of 323bp. Highest resistance among the isolates was observed against Cephalexin (81.9%) and Streptomycin (72.1%) while lowest resistance was against Chloramphenicol (1.6%) and Tetracycline (3.3%). High levels of resistance was seen in Methicillin- resistant Coagulase negative Staphylococcus (MRCoNS) (15%) than in Methicillin-resistant S. aureus (MRSA) (9.8%) isolates. MDR was relatively high in all the isolates (43.4%) comprising of 6/20 (30%) MDR-CoNS and 47/102 (46%) MDR S. aureus. Overall, 68 (75.6%) Staphylococci isolates harboured at least one of the antimicrobial resistant genes namely mecA, aph(6)-Id (strB) and blaTEM genes. The aph(6)- Id (strB) gene was detected in 28.3% of the isolates while 2.3% of the isolates harboured mecA gene. Majority of the isolates carried blaZ gene (88.6%) and blaTEM gene (46.6%) while one isolate harboured mecA gene. Both CoPS (4) and CoNS(2) harboured aph(6)-Id (strB) and blaTEM genes. The mecA containing isolate also harboured blaTEM gene. The mecC gene was not detected in all the isolates. The findings showed that CoNS and S. aureus isolates coexist contaminating raw camel milk and carry similar resistance genes that horizontally transfer between the Staphylococcus species. Therefore, continuous monitoring is recommended in order to prevent the spread of AMR.