PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF STAPHYLOCOCCUS AUREUS ISOLATED FROM CATTLE, BUFFALO, SHEEP AND GOAT`S RAWS MILK IN SOHAG GOVERNORATE, EGYPT.

PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF STAPHYLOCOCCUS AUREUS ISOLATED FROM CATTLE, BUFFALO, SHEEP AND GOAT`S RAWS MILK IN SOHAG GOVERNORATE, EGYPT.

ABO-SHAMA, U.H

Microbiology and Immunology Dpt, Fac. Vet. Med., Sohag Univ. Egypt.

Email: usama.shama@yahoo.com

INTRODUCTION

Milk has long been referred to as the most perfect food for human from birth to senility it contains all the nutrients required for rapid growth and healthy development of the body. Milk is an excellent medium for a large number of microorganisms. When the milk is drawn from the udder of a healthy animal, it contains organisms that have entered the teat canal through its opening. They are mechanically flushed out during milking. The number is ranged during milking between several hundreds to several thousand per milliliter (Farzana   et al., 2004).

Staphylococcus aureus infection is estimated to be present in up to 90% of dairy farms and is responsible for 35% of the economic loss in the dairy industry. S. aureus is a facultatively anaerobic Gram-positive bacterium. The majority of S. aureus strains are catalase-positive which constitute the well known pathogenic species S. aureus and forms the basis of traditional identification methodology. The presence of Staphylococcus aureus whichshows up unsanitary conditions in the dairy herd and counts above 10³ UFC in milk increase the risk of staphylococcal toxin production more resistant to the heat processes of pasteurization (Tortora et al., 2005). Normally enterotoxin is produced at temperatures of 40ºC to 45°C, although Smith et al. (1982) detected a production of toxins at temperatures of 10ºC to 46ºC.

Among the bacteria predominantly involved in food-borne poisoning diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food Staphylococcus aureus is one of the most common agents in bacterial food poisoning outbreaks. It is also a major causative pathogen of clinical or subclinical mastitis of dairy domestic ruminants (Le Loir et al., 2003). Staphylococcal food poisoning is due to the absorption of staphylococcal at enterotoxin performed in the food is (Le Loir et al., 2003) characterized by nausea, vomiting, diarrhea, general malaise and weakness. Such symptoms appear within 1-8 hours after consumption of contaminated food. Although the illness is seldom fatal, complications including dehydration and shock, can accompany severe attacks (Balaban and Rasooly, 2001).

Antibiotics are used to treat diseases of cattle, sheep, goat, buffalo and other animals and as well as used as preservatives for milk (Devriese et al., 1997).           S. aureus has been reported to frequently show multiple antimicrobial resistance patterns (Enright, 2003). This may be due to the indiscriminate use of antibiotics has led to the development of multiple antibiotic resistances thereby rendering the antibiotic treatment ineffective. Resistant bacteria occur in soil, water, plants and animals. The resistant bacteria present in environments are in contact with human beings and animals. It has been estimated that nearly equal tonnage of antimicrobial agents are used in man (Farzana et al., 2004). Antimicrobial resistance is a major public health concern in many countries due to the persistent circulation of resistant strains of bacteria in the environment and the possible contamination of water and food (Normanno et al., 2007). S. aureus strains were once nearly uniformly susceptible to semi-synthetic penicillinase-resistant β-lactams (e.g. methicillin, oxacillin), the most commonly used class of antibiotics for skin infection. These strains were termed ‘methicillin resistant Staphylococcus aureus, or MRSA, a term that implied cross-resistance to all β-lactams including all penicillins and cephalosporins.

Antibiotic-resistant S. aureus isolates pose a severe challenge to both veterinary and health professions and dairy cattle producers because they have a negative impact on therapy (Brouillette and Malouin, 2005). The usage of antibiotics correlates with the emergence and maintenance of antibiotic-resistant traits within pathogenic strains (Shitandi and Sternesjö, 2004). These traits are coded for by particular genes that may be carried on the bacterial chromosome, plasmids, transposons or on gene cassettes that are incorporated into integrons (Rychlik et al., 2006), thus are easily transferred among isolates. Multiple antibiotic resistant S. aureus strains have been isolated from milk obtained from cattle, beef and human samples in many parts of the world (Petinaki et al., 2001; Pesavento et al., 2007). The prevalence of antibiotic resistance usually varies between isolates from the different sampled stations and even between isolates from different herds on the same farm (Waage et al., 2002).

Determination of levels of S. aureus and an evaluation of the antibiotic-resistant phenotypes of the isolates could serve as a tool for determining the hygiene standards implemented during milking. Data on antibiotic resistance could also be used to characterize these opportunistic pathogens, which may further limit the risks associated with the consumption of contaminated milk and its products (Evenson et al., 1988).

Therefore, the aims of this study were determine the prevalence rate of S. aureus in cow, buffalo, sheep and goat raw milk in Sohag Governorate, Egypt by using conventional methods and molecular technique and further characterization of their antimicrobial resistance.

MATERIALS and METHODS

Sample collection: A total of 340 of fresh raw milk sample (120 cow`s samples, 140 buffalo`s samples, 40 sheep`s and 40 goat`s samples) were randomly collected from various places in dairy farms and dairy shops for cow’s and buffalo’s milk samples and from dairy farms for sheep and goat’s milk samples, all samples were collected from Sohag Governorate, Egypt. Properly packed samples were placed on ice or frozen refrigerant packs in an insulated box, and then stored in laboratory at 4°C until all the microbial examination was performed, which was within 2 h. Aseptic techniques were applied, wherein all the equipments were pre-sterilized prior to analysis. The samples collected from dairy shops were tested for heat treatment testig (Lampert; 1975).

Isolation and Identification of Staphylococcus aureus:

The samples were processed immediately upon arrival using aseptic techniques. To detect S. aureus, 1mL of each milk sample was inoculated on Baird-Parker agar (Baird-Parker, 1962) (Difco, USA) and Mannitol Salt Agar (Cruickshank et al., 1973; F.D.A., 2001; Bendahou et al., 2008) (Oxoid, USA). After 24 - 48 h of incubation at 37°C, suspected colonies were sub-cultured on blood agar plate (Difco, USA) and incubated for 24 h at 37°C.

Identification of bacterial cultures under microscope:

The purified cultures of bacteria isolates obtained on Mannitol salt agar media plates were further examined under the microscope for their morphological characters(convex elevation and smooth margins colonies and by gram staining the typical colonies were showed gram-positive cocci occurring in bunched, grapelike irregular clusters were taken as presumptive staphylococcus species.

Biochemical Identification: Biochemical tests were performed to confirm S. aureus using Catalase test, Coagulase test, Oxidase test, Acetoin production, Voges-Proskauer reactions, and D-mannitol fermentation tests were conducted on suspected colonies All S. aureus strains were positive for all tests except for oxidase test negative. (CLSI, 2007; Huong et al., 2010). For sugar fermentation S. aureus strains ferment glucose, lactose, sucrose, maltose and manitol and produces acid (CLSI, 2007).

Molecular biology technique (PCR): (Riffon et al., 2001)

In this study PCR technique used for confirmation presence of S. aureus in 10 raw milk samples from cow, buffalo, sheep and goat which selected by detection of clfA and nuc genes from culture samples through the following steps:

DNA Isolation: Isolation of genomic DNA was carried out by picking three to five colonies from freshly subcultured S. aureus. Colonies were homogenized in 50 μl TE buffer [10 mm of Tris HCl, 1 mm of ethylenediaminetetraacetic acid (EDTA), pH 8.0], followed by the addition of 1 μl lysostaphin (1.8 U/μl; Sigma, USA). After incubation of 1 h at 37°C, 1 μl proteinase K was added and the suspension was reincubated for 2 h at 56 °C. Proteinase K was finally inactivated through boiling of the mixture for 10 min. After centrifugation at 10.000 r.p.m for 5 min the supernatant was cooled on ice before use.

PCR Amplification: For PCR amplification, a reaction mixture of (20 μl) contained 0.7 μl of primer 1 (10 pm/ μl), 0.7 μl of primer 2 (10 pm/ μl), 0.4 μl of deoxynucleoside triphosphate, 2.0 μl of 10X thermophilic 2 buffer (Promega), 1.2 μl of MgCl (25 mm; Promega), 0.1 μl of Taq DNA polymerase (5 U/ μl; Promega), and 12.9 μl of distilled water. Finally, 2.0 μl of the DNA preparation was added to each 0.2-ml reaction tube. A negative control (with no template DNA) as well as a positive control “Diagnostic PCR product of S. aureus DNA isolated from reference strain” were included in each PCR. The tubes were subjected to thermal cycling (Progene, Ouxford, Cambridge, U.K) with the same programme described previously by Nashev et al. (2004). The PCR products (10 μl) were electrophoresed on 2% agarose gels with 1X TAE buffer (40 mm Tris- HCl, 1 mm EDTA, 1.14 ml glacial acetic acid, pH 7.8) at 70–100 V. Characterization of the strains was performed by PCR amplification of virulence and regulatory genes. This included the genes encoding clumping factor (clfA) and thermonuclease (nuc). For both genes, reaction mixtures (25 μl) included 2 μl template DNA, 10 × PCR buffer (Sigma, USA), 25 mM  MgCl2 , 200 μM of the four dNTPs, 10 pmol of each of the 2 primers (Integrated DNA Technologies, Inc., Coralville, Iowa), and 1U Taq DNA polymerase (Sigma, USA). Amplification parameters and primer sequences were used as described by Straub et al. (1999) (Table 1). Amplified products were separated by agarose gel electrophoresis (1%) agarose containing 0.5 mg ethidium bromide per ml (all reagent from Promega Corp., Madison). Visualized and photographed under UV illumination. The sizes of the amplication products were estimated by comparison with a 100 bp DNA ladder (Promega Corp., Madison). 

Table 1: Primers for amplification of the Staphylococcal genes.

Antibiotic susceptibility tests: Antimicrobial susceptibility tests by disc diffusion method has been used with antibiotic discs (Oxoid, USA). Antibiotic susceptibility tests were performed on all S. aureus isolates to determine their antibiotic-resistance profiles (Kirby et al., 1966). Fresh overnight cultures were prepared and used for antibiotic sensitivity tests. An aliquot (100μL) from each isolate suspension was spread plated on Mueller Hinton agar (Oxoid, USA). Susceptibilities of the isolates to a panel of eleven different antibiotic discs (Table, 2) were determined. Antibiotic discs were gently pressed on to the inoculated Mueller Hinton agar to ensure intimate contact with the surface and the plates were incubated aerobically at 37 °C for 18 h – 24 h. S. aureus isolates were then classified as resistant, intermediate resistant or susceptible according to the inhibition zone diameter for individual antimicrobial agents according to the interpretation table (supplied by the Oxoid, USA).Multiple antibiotic resistant (MAR) phenotypes were recorded for isolates showing resistance to three and more antibiotics (Rota et al., 1996).

Table 2: List of antimicrobial agents tested and their symbol and volumes.

RESULTS

In this study the prevalence rate of S. aureus isolated from cow, buffalo, sheep and goat`s raw milk obtained from Sohag Governorate, Egypt were made. One hundred and thirty six of 340 samples (40%) were positive forS.aureus. Forty four raw cow`s milk (36.7%), 65 raw buffalo`s milk (46.4%), 12 raw sheep`s milk (30%) and 15 raw goat`s milk (37.5%) samples were contaminated with S. aureus (Table, 3).

Table 3: Prevalence of Staphylococcus aureus in raw milk isolated from cow, buffalo, sheep and goat raw milk originating from Sohag Governorate.

Fig. 1: Typical amplicons of the clumping factor (clfA) gene of some S. aureus strains. Lane M: 100-bp-molecular-size DNA ladder, Lane 1 – 3: strains of S. aureus detected from cow’s raw milk, Lane 4 – 6: strains of S. aureus detected from buffalo’s raw milk, Lane 7 - 8:  strain of S. aureus detected from sheep raw milk, Lane 9 - 10:  strain of S. aureus detected from goat raw milk, Lane 11: Negative control “unamplified PCR product”  and  Lane 12: Positive control “ amplification of the 985 bp fragment of  S. aureus from the extracted  DNA of S. aureus reference strain”.

Fig. 2: Typical amplicons of the thermonuclease (nuc) gene of some S. aureus strains. Lane M: 100-bp-molecular-size DNA ladder, Lane 1 – 3: strains of S. aureus detected from cow’s raw milk, Lane 4 – 6: strains of S. aureus detected from buffalo’s raw milk, Lanes 7 - 8:  strains of S. aureus detected from sheep raw milk, Lane 9 - 10:  strains of S. aureus detected from goat raw milk, Lane 11: Negative control “unamplified PCR product”and Lane 12: Positive control “amplification of the 270 bp fragment of  S. aureus from the extracted  DNA of S. aureus reference strain”.

The antibiotic susceptibility tests for S. aureus isolates were subjected to eleven antimicrobial agents, from different antibiotic classes were used. Antibiotics of veterinary and human health relevance were also considered. A summary of the percentage of S. aureus that were resistant to these antibiotics is provided in table (4).

Table 4: Antibiotic susceptibility profiles of Staphylococcus aureus isolated from cow, buffalo, sheep and goat raw milk originating from Sohag Governorate, Egypt.  

Result of antimicrobial susceptibility are shown in Figure 3. In total S. aureus originating from cow’s and buffalo’s milk were more resistant than those of goat and sheep origin. The resistance pattern of         S. aureus isolates originated from raw milk from cow’s and buffalo’s of this study area revealed that most of the isolates (83.7%) were resistant to one or more antimicrobial agent. Eighty isolates (13.2%) were resistant to single antibiotic and 35 isolates (25.7%) showed resistance to 2 antimicrobial agents. Multiresistance was found in 44.8% of S. aureus isolates. Resistance to Penicillin G was the most common finding (54.5%, 44.6%), followed by Ampicillin (45.5%,43.1%), Oxacillin (43.1%,40%), Amoxicilin/clavulanic acid (29.5%,35.4%) Erythromycin (20.5%,19.2%) and Chloramphenicol (6.7%,4.6%) for cow and buffalo milk respectively. On the other hand several isolates were found susceptible to the Ciprofloxacin,Cefoxitin, Tetracyclin, Rifampicin and Vancomycin antibiotics.

In general observation the large percentage of Penicillin G (10μg), Ampicillin (10μg), Oxacillin (1μg) and Amoxicilin/clavulanic acid (30 μg) resistant S. aureus were isolated from the study area. These were also resistant to several other antibiotics. Therefore, the one can easily conclude that these are Methicillin resistant S. aureus (MRSA).

In sheep and goat raw milk, a resistant to Erythromycin (16.7%, 26.7%) followed by resistance to Amoxicillin-Clavulanic Acid, Ampicillin, Oxacillin and Penicillin G (8.7%, 6.7%) for sheep and goat milk respectively.

Antimicrobial susceptibility test showed similar results between cow and buffalo as well as between sheep and goat this may be due to the same rearing, breeding and handling condition to these animals and their milk production in Egypt.

Fig. 3: Comparison of sensitivity to antibiotics in S. aureus from raw cow, buffalo, sheep and goat milk.

                %

DISCUSSION

In this study the prevalence rate of S. aureus isolated from cow, buffalo, sheep and goat`s raw milk obtained from Sohag Governorate, Egypt and further characterization of their antimicrobial resistance were made. One hundred and thirty six of 340 samples (40%) were positive forS. aureus. Forty four raw cow`s milk (36.7%), 65 raw buffalo`s milk (46.4%), 12 raw sheep`s milk (30%) and 15 raw goat`s milk (37.5%) samples were contaminated with S. aureus.

This contamination rate is similar that observed in the surveys previously conducted in other countries on several kinds of raw milk, the prevalence of S. aureus in  the present study was 40% such as isolated by Mekonnen et al. (2011) (39.5%) and Zakary et al. (2011) (40%). On other hands, higher incidence was reported by Haggag, 2006 (56%), Ozkan et al. (2007) (98.7%), Ateba et al. (2010) (100%) and Lingathurai and Vellathurai, (2011) (61.70%). While lower prevalence was reported by Shah et al. (1985), Ekici et al. (2004), Fagundes et al. (2010), Kumar and Prasad, (2010), Malahat et al. (2010), Tambekar and Bhutda, (2010) and Santana et al. (2010) who found 8.3 %, 18.18%, 7.3%, 6.6%, 14% 17.39% and 18.80% prevalence respectively.

Although the prevalence of S. aureus has been reported to vary with the size and geographic region of the area sampled, a high proportion of these bacteria in milk relates to poor hygiene practices. Based on observations made during the collection of samples, the improper hygiene and poor farm management practices contributed to the presence of S. aureus in the milk. S. aureus is normally resident in humans; therefore the S. aureus present in cow’s milk may have resulted from transmission from humans, which raises questions regarding the hygiene practices followed. In this study area milk was obtained from animals by washing hands of person deal with animal and/or the utensils and containers used. In certain cases, untreated groundwater was used to wash the containers that were used for milking. This may have contributed to the high level of S. aureus isolated. Improving the hygienic conditions of the milking environment and/or utensils may reduce the prevalence of S. aureus in milk and prevent its transmission to humans.

S. aureus is the most pathogenic among the mastitis causing agents in cattle, globally (Grabber et al., 2009; Vyletlova et al., 2010; Persson et al., 2011; Arga et al., 2012). S. aureus is the most frequently isolated from the clinical cases of mastitis in small ruminants (Bergonier et al., 2003; Vyletlova, 2009; Arga et al., 2012). Da Silva et al. (2004) detected that S. aureus represented 37% of the isolates from subclinical mastitis in goats. Contreras et al. (2007) identified also Staphylococcus spp. as the most prevalent pathogens in small ruminants and refer to necessity to eliminate this pathogen because of risk of milk contamination by thermostable toxins.

There are several disadvantages associated with microbiological culture which are as follows: it is limited by the dynamic nature of infections of the milk sample, milk culture may yield no bacteria from truly infected milks due to the presence of very low numbers of bacteria in samples and negative cultures may also be due to the presence of residual therapeutic antibiotics that may inhibit bacterial growth in vitro. The presence of leucocytes in milk samples may also inhibit growth of bacteria. Moreover, microbiological culture of milk is time consuming and species identification by standard biochemical methods requires more than 48 hours for completing (Riffon et al., 2001).

Due to limitation of cultural methods, PCR has been developed to identify various bacteria in milk samples. The development of PCR- based methods provides a promising option for the rapid identification of bacteria. With this method, identification of bacterial pathogens can be made in hours, rather than days, as conventional cultural methods require. PCR can also improve the level of detection due to its high sensitivity. Theoretically, only a few cell of pathogen are necessary to yield a positive diagnosis (phueketes et al., 2001). Rapid identification methods, in particular nucleic acid based tests, have the potential to be extremely specific and can also discriminate between closely related organisms such as other species of the staphylococci (Riffon et al., 2001).

The ability of S. aureus to adhere to extracellular matrix proteins is thought to be essential for the colonization and the establishment of infections (Salasia et al., 2004). S. aureus possesses various adhesion genes, including clfA, fnbA, and can (El-Sayed et al., 2005). Amplification of gene sequences by PCR such as nuc and clfA is used for identification of Staph. aureus (Akineden et al., 2001 and Nashev et al., 2004).

Amplification of genes encoding clumping factor (clfA) and thermonuclease (nuc) gene by Polymerase Chain Reaction was used for the genotypic characterization of isolated S. aureus strains. Amplification of the clumping factor (clfA) gene resulted in a single amplicon with a size of approximately 985 bp for all 10 tested S. aureus strains isolated from raw milk samples indicating no size polymorphisms of this gene (Figure1). This agreed with (Marrack and Kappler, 1990). While, Amplification of the nuc gene produced an amplicon of 270 bp in all 10 examined S. aureus isolated from raw milk samples (Figure 2). This result as that recorded by Rosenstraus et al. (1998) and Kim et al. (2001). Specificity of the PCR products was demonstrated with 100% of the tested isolates. This specificity of S. aureus was agreed to the results recorded by Ozkan et al. (2007) and Karahan et al. (2011). Lower results were detected by Karahan and Cetinkaya., 2007 (80.5%), El-Jakee et al., 2010 (65.9%) and Malahat et al. 2010 (21%).

The accuracy of PCR in the detection of S. aureus in raw milk samples was more than the cultural methods. No culture positive samples were negative in PCR..` PCR amplification of the clfA and nuc genes could be used as a powerful tool for the identification of S. aureus in milk samples.

The antibiotic susceptibility tests for S. aureus isolates were subjected to eleven antimicrobial agents, from different antibiotic classes were used. Antibiotics of veterinary and human health relevance were also considered. The evolution of antibiotic resistance in S. aureus strains is a serious cause of concern in dairy animals (Wang et al. 2008). The results presented here are similar to previous studies in which Gram-positive bacteria were generally susceptible to Vancomycin, Chloramphenicol, Cefoxitin and Ciprofloxacin (Gündan et al., 2006; Pereira et al., 2009). These drugs are no longer used in veterinary medicine in many countries (Pace and Yang, 2006), which may account for the results reported here.

As shown in Table. 4, a large percentage of S. aureus isolates isolated from cow and buffalo’s raw milk were Methicillin resistant S. aureus (MRSA). These strains in intra-mammary dissemination often produce incurable severe intra-herd infections (Moon et al., 2007; Kumar et al., 2010). MRSA strains have been observed to be multi-drug resistant, such as Aminoglycosides, Macrolides, Lincosamides, Streptogramins, Tetracyclines, etc., which are often used in the treatment of mastitis (Wang et al., 2008; Kumar et al., 2010). Lee (2003) reported that there are only a few reports on MRSA associated with mastitis although the transmission of bovine MRSA to humans is possible and may contribute to outbreaks in animal and human populations. Hence, it is necessary to know which endemic strains of S. aureus in dairy cattle populations are highly pathogenic and methicillin-resistant.

High levels of MRSA have been identified in patients in the United States and some European countries (Mark et al., 2003). In these countries, 44.4%, 34.7%, 41.8% and 32.4% of isolates from patients in the United States, France, Italy and Spain, respectively, were resistant to methicillin. Since antibiotic-resistant isolates might be transmitted to humans by the consumption of food products containing such resistant bacteria, the use of antibiotics as growth promoters in animal husbandry, especially of those commonly used for both human and animal care should be avoided (Wise, 2007).

A further objective of the study was to characterize and compare the antibiotic-resistance profiles of S. aureus isolated from the study area. The motivation for this was the fact that there were no clear studies conducted in this area. Furthermore, the presence of antibiotic resistant S. aureus has been reported to negatively affect the treatment of its associated infections in humans and animals (Petinaki et al., 2001, Brouillette and Malouin, 2005; Moneoang and Bezuidenhout, 2009). Investigation of the antibiotic-resistance profiles of these isolates may serve as a tool in assessing both the sanitary conditions employed during milking and the health risks that humans may encounter when infected by antibiotic-resistant strains.

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