Molecular characterization of selected pathogens in milk

Abstract

Milk is a nutrient-rich liquid produced by the mammary glands of mammals including cow. It is an excellent source of vitamins, minerals and nutrients and it thus consumed by humans. The composition of milk makes it an excellent growth medium for microorganisms and thus very prone to microbial and chemical contamination. Contamination of affects its quality and this frequently occurs when standard operating procedures and farm management techniques during the production and processing procedures are not strictly enforced. The consumption of contaminated milk has been associated with a variety of health complications. The aim of this study was to isolate and characterize selected pathogens (S. aureus, S. agalactiae, and E. coli) in milk using phenotypic and molecular methods (PCR). One hundred and two milk samples comprising 24 raw, 37 bulk tank and 41 pasteurized milk were randomly collected from supermarkets, shops as well as both commercial and communal farms in the North-West province of South Africa in winter and spring, 2018. Target pathogens S. aureus, S. agalactiae, and E. coli were isolated using selective media Mannitol Salt agar (MSA) plates, Columbia agar supplemented with 5% (v/v) sheep blood and Eosin Methylene Blue agar (EMBA), respectively. Based on colonial morphologies, 62 presumptive isolates comprising 21 S. aureus (yellow colonies), 20 S. agalactiae (golden yellow) and 21 E. coli (metallic sheen colonies) were obtained. The presumptive isolates were subjected preliminary identification test for E. coli (oxidase and the triple sugar iron agar test), S. agalactiae and S. aureus serological assays, respectively. The haemolytic activity of presumptive S. aureus and S. agalactiae was determined using ox-blood agar supplemented with 5% (w/v) bovine blood. S. aureus (ATCC® 29213), E. coli (ATCC® 25922) and S. agalactiae (ATCC® 13813) were used as positive control strains while K. pneumonia (ATCC® 13883) was used as a negative control strain. Genomic DNA was

extracted from 62 presumptive isolates and bacterial 16S rRNA gene was amplified as an internal control. The identities of potential S. aureus, S. agalactiae and E. coli was confirmed through PCR amplification of the nuc, VI and C1 and the uidA gene fragments respectively. The antimicrobial resistance profiles of all confirmed S. aureus, S. agalactiae and E. coli isolates was determined using the disc diffusion technique. PCR targeting classical S. aureus virulence genes: sea, seb, sec, sta and Etb genes was used to determine the clinical significance of the isolates. All E. coli isolates were screened for the presence virulence genes (stx1, stx2 and hlyA) using specific primers. In addition, all the 9 confirmed S. agalactiae isolates were screened by multiplex PCR analysis for the presence of the Cluster PI-1, hylB, fbsA, cps C, D, E, cpsJ, K, eneuB virulence genes. All the potential S. aureus, S. agalactiae and E. coli isolates were Gram-positive cocci appearing in clusters, Gram-positive cocci appearing in chains (presumptively) and Gram-negative rod-shaped cells, respectively. Large proportions (76.2%) of the presumptive E. coli isolates were oxidase positive. All the 62 presumptive isolates harboured the bacterial 16S rRNA PCR gene fragments. Despite the fact that only nine (45%) of the presumptive S. agalactiae were positive for the C1 gene, large proportions (18/21; 85.7%) and (14/21; 66.7%) of the presumptive S. aureus and E. coli isolates harboured the nuc and uidA genes respectively. Virulence gene analysis of the 18 S. aureus isolates indicated that 6 (33.3%) were positive for seb virulence gene while only 3 (16.7%) of these isolates harboured the sec gene. The exfoliative toxin b (etb) was detected in 5 (27.8%) of the S. aureus isolates. PCR analysis revealed that S. agalactiae isolates only 2 (22.2%) were positive for cpsJ, K and eneuB virulence genes. A large proportion (12; 86%) of E. coli isolates harboured the three STEC virulence genes assessed in this study. Generally, the majority (10; 71%) possessing the hlyA gene. The stx2 gene was also

detected in 8 (57.1%) of the isolates. On the contrary, only four E. coli isolates were positive for the stx1 gene. Phenotypic determination of the virulence potentials (haemolytic patterns on blood agar) of S. aureus and S. agalactiae isolates revealed that 7 (38.89%) and 6 (66.6%) isolates respectively displayed beta (ᵦ) haemolytic patterns. All the 41 PCR confirmed isolates comprising 18 S. aureus, 9 S. agalactiae and 14 E. coli isolates were subjected to the disc diffusion antibiotic susceptibility test in order to determine their drug resistant profiles against a panel of 8 antimicrobial agents. Results indicated that all (100%) of the S. aureus isolates from milk samples were resistant to Cefotaxime. Large proportions of the S. aureus isolates from tank milk in Mafikeng were resistant to Ertapenem (80.95%), Piperacillin (76.19%) and Ticarcillin (47.62). In addition, small proportions of isolates from pasteurized milk in Zeerust were resistant to Cefepime (33.33%) and Amoxicillin (19.05%). On the contrary, none of the S. aureus isolates was resistant to Aztreonam and Ceftazidime. Similarly, all (100%) the nine PCR confirmed S. agalactiae isolates were resistant to multiple antibiotics. Moreover, drug resistance was frequently observed against Cefotaxime, Ceftazidime, Amoxicillin, Cefepime when compared to Ertapenem and Ticarcillin. A large proportion (100%) of the isolates from tank milk in Klerksdorp were resistant to Cefotaxime. Large proportions of S. agalactiae from pasteurized milk from Coligny were resistant to Ceftazidime (80%), Amoxicillin (60%) and of Cefepime (55%). On the contrary, small proportion of isolates of tank milk in Rustenburg were resistant to [of Ertapenem (45%) and Ticarcillin (35%). Despite this, none of these isolates was resistant to Piperacillin and Aztreonam. Large proportions of E. coli isolates of raw milk from Rooigrond were resistant toof Piperacillin (71.43%), Ticarcillin (61.90%) and Amoxicillin (57.14%). On the contrary, small

proportions 23.81%) of the isolates from raw milk from Lichtenburg were resistant to Cefepime and Ertapenem respectively. All the E. coli isolates in this study were susceptible to Aztreonam and Ceftazidime. From the findings of this study, it is evident that these multi-drug resistant and virulent isolates may cause infections in humans when consumed in contaminated milk. Moreover, infections caused by the isolates may be difficult to treat using these antibiotics and thus amplified the need to constantly search for alternative antibacterial agents.