Prevalence and Susceptibility Patterns Associated with Staphylococcus Aureus Presence in Marketed Milk and Milk Products.

Introduction: Staphylococcus aureus is a major foodborne pathogen that poses a serious threat to public health. Indiscriminate use of antibiotics increases evolution of antibiotic resistant strains. This study aimed to determine the presence of Staphylococcus aureus in milk and milk products and their antimicrobial susceptibility patterns. Methods: A total of 334 samples were collected for analysis in the laboratories. To determine antimicrobial susceptibility patterns, selected antibiotics from different classes were used: Penicillin G, Erythromycin, Vancomycin, Chloramphenicol, Tetracycline, Gentamycin, Methicillin, and Ciprooxacin. Results: The presence of S. aureus in milk and milk products was found occurring in 21.56% of all the samples. In raw milk analyzed, 64.81% of samples were contaminated by Staphylococcus aureus, 20.54% in pasteurized milk, 10.71% in yogurt, and 3.57% in ice cream. All isolates were found to be 100 % sensitive to Tetracycline, Ciprooxacin, Erythromycin, and Methicillin. Infrequent sensitivity was found in Gentamicin and Vancomycin. Resistance to Penicillin G was occasionally observed across the different sources of milk and milk products. Resistance to Gentamicin (42 %) and Vancomycin (11 %) was seldom observed in isolates, hence occurring in yogurt samples only. Conclusion and Recommendation: The research hypothesis was rejected based on the presence of pathogenic Staphylococcus aureus across the different samples analyzed. It is recommended that Tetracycline, Ciprooxacin, Methicillin, and Erythromycin antibiotics should be used for the treatment of Staphylococcus aureus infections based on the susceptibility test outcome.


Introduction
Staphylococcus aureus (S. aureus) is a Gram-positive and spherical coccus of about (0.8-1.0 micron) in diameter appearing in grape-like clusters. S. aureus are aerobes and facultative anaerobes that thrive at an optimum temperature of 37º C while the temperature at which they are active ranges between 12-44º C. They have an optimum pH of 7.5. They have been found to grow well on ordinary lab media forming a golden yellow color in agar [1]. Pathogenic microorganisms are introduced in milk products when raw milk used to process them is contaminated or by cross-contamination [2].
In Kenya, 86% of milk is marketed raw and only 14% is processed [3]. Raw milk can be 20-50% cheaper than the formal and pasteurized milk on the market [3]. Unprocessed milk is also sold in desired quantities which give the low-income earners access since they can buy as little as they can afford [3].
Milk handling equipment is one of the most signi cant sources of microbial contamination in milk [2]. S. aureus produces enterotoxins that are virulent factors responsible for food poisoning in humans. In essence, enterotoxins are basic proteins that are resistant to heat, acid, and digestive enzymes.
Enterotoxins produced cannot be destroyed by exposure to 100˚C for 30 minutes, therefore posing a serious challenge to their elimination from the contaminated food and other edibles [1]. Enterotoxins consumed incubate under favorable conditions and after 4-6 hours, symptoms of infection appear such as nausea, dizziness, severe abdominal pains and cramps, loss of appetite, diarrhea, and vomiting up to 24 hours. This period of incubation is of great signi cance since it differentiates food poisoning caused by S. aureus to that caused by Salmonella infection which appears 24-48 hours after eating contaminated food.
The genetic plasticity of S. aureus has facilitated the emergence of persistent and multidrug-resistant strains that have a major impact on human and animal health [4]. Staphylococcus aureus can grow at low temperatures and therefore posing a serious threat. In Kenya, there exists limited information on the prevalence of S. aureus in raw milk while none has been documented on milk products. Susceptibility patterns of S. aureus isolated from milk and milk products have also not been well documented.

Sampling design
Purposive sampling was employed in choosing supermarkets. Four supermarkets were selected in the Nairobi central business district based on the availability of all the required target samples in their storage facilities. Milk products from the supermarkets were randomly picked from the storage facilities taking into consideration the expiry date as indicated in the packet while applying the concept of Hazard Analysis and Critical Control Point (HACCP). Those with a shelf life of three days and below to expiry were excluded. Raw milk was randomly obtained from shops in the selected points of distribution between 6.00 am and 7.00 am to acquire milk directly from farmers. Blinding of the samples and concealment of supermarkets and shop outlets was done to avert bias and con ict of interest.

Microbiological isolation in Blood agar and MacConkey agar
MacConkey agar without crystal violet was preferred since it permits the growth of Gram-positive bacteria. Gram staining was done and microscopy was done on the selected colonies. The presence of S. aureus was determined from milk and milk product samples using standard con rmatory methods for the identi cation of S. aureus.

Subculture of suspected isolates in Mannitol salt agar
Mannitol salt agar was used as an indicator media for S. aureus. Isolates of Staphylococcus origin were sub-cultured in Mannitol Salt Agar (MSA) using the standard protocol for bacterial culture. After incubation at 37° C, golden yellow halo colonies that were big, spherical, raised, and smooth appearing in clusters were observed to grow fermenting Mannitol salt agar, and hence the characteristic yellow color caused by S. aureus.

Isolation of Candida in Sabouraud`s dextrose agar
The germ tube test was done as a con rmatory test for Candida albicans. A wet mount was examined microscopically for germ tubes, following the standard procedure for fungal isolation and staining technique. A positive result was indicated by a short hyphal extension arising laterally from a yeast cell with no constriction at the point of origin. A negative result had no hyphal extension arising from a yeast cell.

Biochemical tests
Identi cation of S. aureus by catalase test For the Gram-positive cocci, the standard protocol for the catalase test was performed to distinguish catalase-negative Streptococcus sp from catalase-positive Staphylococcus sp. Isolates identi ed as catalase-positive were further identi ed by the coagulase test. Pathogenic S. aureus is both catalase and coagulase positive.

Identi cation of S. aureus by coagulase test
The standard protocol for evaluation of coagulation was done and appropriate observations made in line with the protocol. Clotting was evaluated at 30 min intervals for the rst 4 hours of the test and then after 24 hours of incubation. The capacity of S. aureus to coagulate plasma is the principal characteristic of pathogenic S. aureus and is highly correlated to the capacity to produce enterotoxins harmful to the tissues of the infected host [5].

Differentiation of Staphylococcus epidermidis from Staphylococcus saprophyticus
Staphylococcus epidermidis was differentiated from Staphylococcus saprophyticus by novobiocin susceptibility test. Staphylococcus epidermidis is sensitive while Staphylococcus saprophyticus is resistant to novobiocin antibiotic. The zone diameter of inhibition was measured. Zone of inhibition < 12 mm was interpreted as resistant while ≥ 16 mm was interpreted as sensitive.

Microbiological identi cation of Escherichia coli by IMViC tests
The indole test was performed by growing pure cultures on sterile Tryptophan broth for 24-48 hours per the identi cation protocol. A positive result was indicated by a red layer at the top of the tube. Methyl red test and Voges-Proskauer test were both done in Methyl red-Voges-Proskauer broth. A positive Methyl red test was identi ed by the development of a red color after the addition of methyl red reagent. In the Voges-Proskauer test, a positive result was indicated by the development of a red-brown color after the addition of Barritt s B reagent. A citrate utilization test was performed on Simmons citrate agar. A positive citrate result was indicated by growth and a blue color change.

Antimicrobial sensitivity test
To establish antimicrobial sensitivity testing, the correctly identi ed S. aureus was thawed at room temperature, and the sample used for subculture. Panels of selected antibiotics commonly used in the s A a n d B a r r i empirical treatment of S aureus infections informed the choice of antibiotics used as follows: Penicillin G (10 µg) a penicillin, Erythromycin (15 µg) a macrolide, Vancomycin (5 µg) a glycopeptide, Chloramphenicol (30 µg), Tetracycline (30 µg) a tetracycline, Gentamycin (30 µg) an (aminoglycoside), Methicillin (10 µg) a penicillin and Cipro oxacin (5 µg) a uoroquinolone. Their effects on the growth of S. aureus were evaluated using the standard protocol for sensitivity testing.

Results
Subculture of suspected isolates in Blood agar and Mannitol salt agar Pathogenic S. aureus showed β-hemolysis in blood agar after incubation while S. epidermidis exhibited γhemolysis. In Mannitol salt agar, the presence of growth and change of pH in the media (red to yellow) was regarded as con rmative identi cation of the salt-tolerant Staphylococci.

Microbial contamination of milk and milk products
Besides the targeted S. aureus, other bacteria and a fungus were isolated from milk and milk products in this study. These were Staphylococcus epidermidis, Staphylococcus saprophyticus, Bacillus species (sp), and Escherichia coli bacteria while the fungus was Candida as shown in (Table 1), P = 0.00001. There was a signi cant difference in contamination set at P < 0.05.
a Other bacterial contaminants were also isolated from raw milk and milk products in varying levels.

Biochemical tests on Staphylococcus species isolated
Biochemical tests of microorganisms found to be of Staphylococcus origin were done and the results showed that Staphylococcus aureus coagulated rabbit plasma forming sticky clots in the test tubes. They were also catalase-positive in 3% hydrogen peroxide as opposed to the other Staphylococcus sp.  Sensitivity pro les obtained from samples of milk and milk products.
Staphylococcus aureus isolated from pasteurized milk in packets Zones of inhibition of the growth of S. aureus in pasteurized milk when exposed to 8 different types of antibiotics showed that there was signi cantly higher inhibition by Gentamycin (mean inhibition zone 26.9 mm), Erythromycin (Ery) (mean 26.7 mm) and Penicillin (mean 26.4 mm) than the other antibiotics. Out of the 23 S. aureus isolates, 5 isolates were sensitive to Gentamycin (Gen) and 1 isolate was sensitive to Penicillin (Pen). All the 19 isolates were sensitive to Tetracycline (Ten), Cipro oxacin (Chl), Erythromycin, and Methicillin (Met). 13 of the S. aureus isolates were resistant to Penicillin and none of the isolates was resistant to the other seven antibiotics, P = 0.0001 (Table 3).

Staphylococcus aureus isolated from fermented milk
Zones of inhibition of S. aureus isolated from fermented milk when exposed to 8 different antibiotics showed that there was signi cantly higher inhibition by Cipro oxacin (mean inhibition zone 27.5 mm), Tetracycline (mean 26.3 mm), Erythromycin (mean 26.3 mm) and Chloramphenicol (mean 26.0 mm) than the other antibiotics ( Table 4). Out of the 12 S. aureus isolates, 5 isolates were sensitive to Gentamycin. All 12 isolates were sensitive to Tetracycline, Cipro oxacin, Erythromycin, and Methicillin. There was no resistant isolate of S. aureus from fermented milk to any of the antimicrobial agents they were exposed to, P = 0.0001.    (Table 6).    Table 9 Resistant isolates expressed in percentage (%). i This study revealed a higher prevalence rate of S. aureus in raw milk 64.81% compared to the other milk products investigated. Microbial contamination in milk could be associated with unhygienic milking and poor handling practices that could be promoting poor milk. In Pasteurized milk, 20.54% of S. aureus isolates were detected and 10.71% and 3.57% in yogurt and ice-cream respectively. Most of the bacterial contaminants must have been signi cantly eliminated during the process of pasteurization. However, the presence of S. aureus in pasteurized milk in other cases indicates the process was not satisfactorily done. Its presence could also be due to exogenous contamination after pasteurization [7]. Staphylococcus aureus could also nd access to pasteurized milk during the cooling and packaging of the products into their various packets for branding and distribution to the outlets [8].
Fermented milk is characterized by acid production, avor additives, and cultured bacteria. This environment could have therefore been competitively harsh for S. aureus to survive and thrive well and hence the possible cause of few isolates isolated compared to raw milk and pasteurized milk. Low temperatures below 5˚C inhibit growth and multiplication of S. aureus according to [9], and this could be the reason why its presence in ice-cream was notably very low compared to all other ndings of this study. Contamination of the various milk and milk products as was found in this study revealed that the levels of contamination were signi cantly different as well as the number of samples contaminated. The microbial counts were signi cantly lower in farmers` raw milk and highest in fermented milk. An implication that farmers milk was of better quality but the quality deteriorated along the supply chain due to the proliferation of the microorganisms initially present in milk or/and due to cross-contamination. It is demonstrated ( Table 2) that the results on the level of contamination increased after a send-off from the farm level by farmers. The quality of milk signi cantly decreases after send-off by farmers [10].
From the ndings of this study, S. aureus investigated were largely sensitive to the antimicrobial agents used. There was, however, occasional resistance to penicillin G. All S. aureus isolates investigated from the different sources (Pasteurized milk, yogurt, Ice cream, and raw milk) were found to be 100% sensitive to Tetracycline, Cipro oxacin, Erythromycin, and Methicillin. None of the isolates was found sensitive to Chloramphenicol while in Vancomycin 11.00% of the isolates were found sensitive in raw milk (Table 7).
Resistance to Vancomycin was found in isolates obtained from yogurt (11%). This difference in activity by Vancomycin was thought to be brought about by the ability of S. aureus to acquire more resistance due to selective pressure.
Resistance to Penicillin G was variant across the four different sources; Pasteurized milk, yogurt, icecream, and raw milk as 57%, 37%, 50%, and 4% respectively (Table 9). Resistance to Penicillin G antibiotics as presented in the current study could have been caused by the bacterial enzymes which destroy the antibiotic before it can act on the pathogen [11]. This mechanism is mostly used by microorganisms for defense against antimicrobial agents. Besides, frequent use of Penicillin G in the treatment of both herds of cattle and humans is also among the possible cause of the emergence of more resistant strains of S. aureus and hence posing a serious challenge to public health. Resistance to Penicillin G (100.00%) and Gentamicin (10.00%) was observed by [12)] while [13] observed that all S. aureus isolates were susceptible to Cipro oxacin, Gentamicin, and Vancomycin. Isolates were also resistant to Penicillin (56%) and Tetracycline (22%).
Resistance to gentamicin was found occurring in isolates obtained from yogurt (42%). Resistance to gentamicin (aminoglycosides) is due to the evolving mechanism of S. aureus strains to inhibit the aminoglycoside action which occurs via protonated amine and/or hydroxyl interactions with the ribosomal RNA of the bacterial 30 s ribosomal subunit. These mechanisms of aminoglycoside resistance and genetic disorder exhibited by strains are either: aminoglycoside modifying enzymes, ribosomal mutations, or active e ux of the drug out of the bacteria [14]. Occasional resistance by S. aureus to antibiotics could be associated with earlier exposure of these drugs to isolates which may have enhanced the development of resistance. Likewise, failure to follow physicians` instructions resulting in the frequent use of antibiotics can result in the emergence of multi-drug resistant strains. Also, the irresponsible use of antibiotics in animal husbandry could bring about increased antibiotic resistance by S. aureus. These wrong practices can result in the mutation of the organismal genes and therefore becoming more resistant to drugs commonly used in empirical treatment.