Phenotypic and genotypic characterization of Staphylococcus aureus isolates recovered from bovine milk in central highlands of Ethiopia

1 Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa Ethiopia. 2 Department of Microbiology, Immunology and Parasitology, Black lion school of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia. 3 Central Statistical Agency, Addis Ababa, Ethiopia. 4 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, USA. 5 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, 1920 Coffey Rd. Columbus, OH 43210, USA.


INTRODUCTION
Different studies conducted in Ethiopia have shown that Staphylococcus aureus is implicated in nearly 40% of mastitic cows (Workineh et al., 2002;Dego and Tarke, 2003;Getahun et al., 2008;Abera et al., 2010).Another study in and around Sebeta, Ethiopia, that looked into the prevalence of mastitis in 180 local and crossbreed dairy cows showed that mastitis (majority of which is subclinical in form) can reach a prevalence rate of 52.78% (Hundurra et al., 2005).The prevalence of S. aureus as a cause of mastitis was 44% and that of Staphylococcus epidermis was 14.93% in the study conducted by Hundurra et al. (2005).With such high prevalence of subclinical mastitis in the Ethiopian context, the public health importance of zoonotic pathogens would be of great concern as there is a trend of using raw milk for direct consumption from apparently healthy cows.In addition, most rural Ethiopians have a tradition of consuming milk and milk product as a ready to eat food items in unpasteurized form such as raw milk, Ethiopian cottage cheese (Ayib) and yoghurt (CSA, 2001).
With the ever-increasing situation of foodborne diseases worldwide, the development of antimicrobial resistance in foodborne pathogens has become a very important public health issue.Antimicrobial resistance is becoming an extremely serious global health problem (Carlet et al., 2012).The liberal use of antimicrobials in hospitals and treatment centers as well as the subtherapeutic use in livestock for growth promotion and prophylaxis has greatly contributed to the emergence and persistence of resistant strains of bacteria (Helmuth, 2000;Ray, 2004).
There is a concern that the extensive use of antimicrobials in animal husbandry potentially leads to maintenance of selective pressure and higher frequency of resistance for wide variety of antimicrobial agents (Kumar et al., 2005;Silbergeld et al., 2008).Furthermore, over the last few decades, community-and healthcarerelated infections have become a common phenomenon globally (Carlet et al., 2011;Gagliotti et al., 2011).Specifically, Staphylococcus aureus has been involved in infections associated with in patients in hospitals and community settings (Millar et al., 2007).Livestockassociated methicillin resistant Staphylococcus aureus (LA-MRSA) multilocus sequence type 398 (ST398) has also emerged in the past 10 years.
Initially, it was described in pigs and pig farmers who were carriers, but soon it was also detected in patients in hospitals.Several lineages of this epidemic strain have also been reported to be commonly present in other livestock species (Armand-Lefevre et al., 2005;Voss et al., 2005).The detection of mecA as a confirmatory test for methicillin-resistant S. aureus (MRSA) is widely accepted.However, recent reports indicate that there appears a new mecA homologue, mec ALGA251 that is only 70% nucleotide homology to the conventional mecA gene.This makes the confirmatory test of the conventional mecA gene detection questionable (Stegger et al., 2012).
Although large-scale studies and documentations regarding the problem of antimicrobial resistance (AMR) in Ethiopia do not exist, the available reports indicate that it is already a growing problem and should be considered a public health concern (Gebre-Sealsssie, 2007;DACA, 2009).In the veterinary area, a number of reports indicate that resistance of S. aureus to antimicrobials is a common problem prevailing in different parts of Ethiopia (Legesse et al., 2009;Getahun et al., 2008;Abera et al., 2010;Sori et al., 2011;Daka et al., 2012;Tamiru et al., 2013;Mekuria et al., 2013).A number of studies have been conducted in Ethiopia with a major focus on the prevalence of S. aureus in bovine milk (Getahun et al., 2008;Abera et al., 2010;Workineh et al., 2002;Dego and Tarake, 2003).Most of these researches, however, were concentrated on the importance of this pathogen as a cause to clinical and subclinical mastitis and all of these studies conducted in Ethiopia were done using the conventional phenotypic methods and none of them managed to use a modern molecular technique that involves mecA gene detection.Despite the expense, application of molecular techniques like PCR and DNA fingerprinting renders many advantages over the conventional phenotypic assay in the study of antimicrobial resistance profile of pathogens (Fluit et al., 2001).The current study aim was to determine the prevalence and antimicrobial resistance of S. aureus isolates recovered from raw bovine milk collected in central Ethiopia using both phenotypic and genotypic molecular techniques.

Sampling areas and sample types
Small-scale bovine milk producing co-operatives were conveniently selected from urban and per-urban regions of central Ethiopia [Asela, Debre-Zeit, Addis Ababa (Akaki/Kaleti) and Selale, the areas surrounding and supplying milk to the capital city, Addis Ababa, with a population of 2.7 million people (CSA, 2007).A total of 577 samples were conveniently selected from milk collection centers and milk processing plants in the aforementioned regions and were analyzed.The samples included raw on-farm pooled milk (n=433) and combined bulk tank milk (n=44), pasteurized milk (n=65), yogurt (n=20) and cheese (n=15) samples.

Culture and identification of S. aureus
Raw/pasteurized milk samples (25 ml) and 25 g of milk products (cheese and yoghurt) were analyzed following methods as recommended by the U.S. Food and Drug Administration: Bacteriological Analytical Manual (BAM: http://www.cfsan.fda.gov/_ebam/bam-5.html).Briefly, 25 ml of milk were pre-enriched in 225 ml of Mueller-Hinton broth (Difco, Detroit, MI, USA) with 6.5% NaCl.After incubation at 37C for 24 h aerobically, aliquots were plated onto Mannitol Salt agar plates (Difco).Presumptive positive colonies were confirmed biochemically using Gram stain, catalase, and coagulase tests.Confirmation of S. aureus isolates was done by nuc gene detection using PCR.

Genomic DNA extraction
DNA was extracted directly from all the 109 S. aureus isolates.Genomic DNA extraction was performed using DNeasy blood and tissue kit (QIAGEN, Hilden, Germany).Bacterial DNA was extracted according to the protocol provided by the manufacture.

Species identification
PCR was done for the detection of the nuc genes as previously described (Table 1) (Brakstad et al., 1992).This gene is a marker gene used to confirm isolates as S. aureus.

Screening for methicillin resistance genes
The primers used in the case of mecA gene detection were according to methods described elsewhere (Table 1) (Oliveira and Lencastre, 2002).The thermocycling conditions used for mecA gene detection were: Initial denaturing at 94°C for 2 min followed by 30 cycles of 94°C for 2 min, 55°C for 1 min, 72°C for 2 min and a final extension step of 72°C for 2 min.10 μl of the PCR product of each isolate tested were electrophoresed on 1% agarose gel containing 5 μl of 10 mg/ml ethidium bromide for 1 h at 120 v using 0.5X Tris-borate EDTA (TBE) as running buffer.PCR product size estimation was done using Gene ruler 100-bp DNA ladder (exACTGene 100 bp DNA ladder, Fisher Scientific, Fairlawn, NJ).For PCR amplification of mecA gene, the MRSA ATCC 43300 strain was used as a positive control.

PFGE genotyping
Thirty nine (39) S. aureus isolates were randomly selected from four geographic locations to determine the clonal relatedness of isolates.DNA fingerprinting of the selected isolates was performed using pulsed-field gel electrophoresis (PFGE).
PFGE for Staphylococcus aureus was done as previously described (McDougal et al., 2003).Briefly, isolates were grown on Trypticase Soy Agar (TSA) at 37°C for 16-18 h.Cell suspension buffer (10 mM Tris-HCl pH 7.2, 20 mM Nacl, 50 mM EDTA) was used to suspend and adjust the bacterial concentration to an optical density (OD) of 1.35.TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA,) was used to prepare agarose embedded cells.After the bacterial cells were lysed (2 µl of 2 mg/ml lysostaphin was added for each sample), intact genomic DNA was digested with 20 l of SmaI restriction enzyme (New England Biolabs, Ipswich, MA, USA) incubated at 25C for 4 h.The Pulsenet universal strain Salmonella enterica serovar Braenderup H9812 was used as a molecular standard marker.The DNA fragments were separated by CHEF-DR  III Pulsed-Field Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA).PFGE running conditions were as follows: initial switch 5.2s, final switch 43.9 s, voltage 6 V/cm include angle 120, run time 19 h and pump 50-60.Cluster analysis was performed using the unweighted pair group method with arithmetic averages with 2.0% band position tolerances and 1.5% optimization values using Bionumerics software, version 5.1 (Applied Math inc., Belgium).Similarity coefficients were calculating using Dice Coefficient Similarity Index.PFGE banding patterns with a similarity index >70% were grouped within the same genotypic cluster.

Data analysis
Descriptive statistics were used for the frequency of isolation of S.

Isolation of S. aureus isolates from raw bovine milk and milk products
Overall, 120 Staphylococcus aureus isolates were obtained from the milk and milk product samples with a prevalence of 21% (120/577).In addition, Coagulase Negative staphylococcus (CoNS) species were also isolated from 361/577 (63%) milk and milk product samples analyzed.Considering the raw bovine milk samples only, the overall prevalence of S. aureus was 24% (103/433) for individual on-farm pooled milk and 39% (17/44) for combined bulk tank milk samples.All of the milk product samples tested were negative for S. aureus.However, 17/65 (26%), 2/20 (10%) and 8/15 (53%) of pasteurized milk, yoghurt and cheese samples were contaminated with coagulase negative Staphylococcus (CoNS) isolates, respectively (Table 2).Of the 120 S. aureus isolates, only 110 were recovered after shipping to USA for molecular analysis.Further analysis of 110 of the S. aureus isolates using the nuc gene PCR assay revealed that, 109 of the isolates were confirmed to be S. aureus that made the overall PCR confirmed prevalence to be 109/577(19%).

Phenotypic and genotypic antibiotic resistance profile of S. aureus
Of the total 109 S. aureus isolates tested for susceptibility to 12 antimicrobials, all were found to be susceptible to vancomycin.The highest rate of resistance was observed for penicillin (83%) and the lowest rate was noted for amoxicillin/clavulanic acid (amc) (3%) and gentamicin (gen) (3%) (Table 3).Fourteen isolates (13%) recovered from raw milk sample were found to be susceptible to more than one of the antimicrobials tested while 57% of the isolates were multidrug resistant.
Isolates resistant to more than one antimicrobial were detected from all the four geographic locations (Addis Ababa, Debre-Zeit, Selale and Asela) (Table 3).However, the proportions of resistance to each antimicrobial varied by location.For example, penicillin (pen) resistance was higher for isolates from Addis Ababa (92%) as compared to isolates from Selale (88%), Debre-Zeit (75%) and Asela (69%).Resistance to cefoxitin (Fox) was relatively higher in Asela isolates (62%) as compared to 48% for Selale and 58.3% for both Addis Ababa and Debre-Zeit isolates.Resistance to ciprofloxacin (cip) was only found in the isolates from the Selale region (7%).In addition, resistance to erythromycin was only evident in the isolates from the Asela and Selale areas (peri-urban areas) with 8 and 7% respectively.When stratifying the study sites into urban and peri-urban settings, resistance to erythromycin and ciprofloxacin was only evident in the peri-urban areas.Overall, the proportion of S. aureus isolates with resistance to more than one antimicrobial from the different regions was as follows: Selale (53%), Asela (69%), Addis Ababa (58%), and Debre-Zeit (58%).These differences were not statistically significant (p>0.05).When stratifying the study sites into peri-urban and urban, the resistance proportions were not statistically significant between the two regions.
In addition to the phenotypic characterization, all the 109 S. aureus isolates were screened for the presence of the mecA gene using PCR.None of the isolates were found to carry the mecA gene, indicating the absence of MRSA in the samples.Phenotypically, as shown in Table 3, there were 58 isolates that were resistant to cefoxitin (Fox) and yet were mecA negative.
isolates (Figure 1).Cluster-A contains eight isolates that are 74% clonally related based on banding pattern identity.A total of 7/8 isolates in this cluster share the penicillin resistance phenotype and one isolate was susceptible to all the antimicrobials tested.This cluster also has two sub-clusters; sub-cluster-IA and IIA.
Isolates in sub-cluster-IA share banding similarity at 79% and contains 5 isolates: 3 isolates from Selale (collection centre 6-farmer 1 and 8; collection centre 5-farmer 2); 1 isolate from Asela (collection centre 3-farmer 17) and 1 isolate from Debre-Zeit (collection centre 4-farmer 5).Isolates from Selale region under sub-cluster-IA (collection centre 5-farmer 2 and collection centre 6-farmer 1) had shown a 93% identity based on PFGE banding pattern and phenotypically they share resistance to penicillin.In addition, the third isolate recovered from Asela (collection centre 3farmer 17) also share banding pattern identity at 92% with the above isolates within the same subcluster-IA.This isolate also shares phenotypically Pen-Fox resistance pattern with isolate from Selale collection centre 5-farmer 2 and from Debre-Zeit (collection centre 4-farmer 5).
A 94% banding pattern identity was observed for isolates in sub-cluster-II A. This sub-cluster contains three isolates from three different geographical locations, Selale, Addis Ababa and Debre-Zeit.An isolate from Debre-Zeit collection centre 2-bulk tank 1 and an isolate from Selale collection centre 9-farmer 15 showed a 96.3% banding pattern identity within the sub-cluster.The isolates from Debre-Zeit and Addis Ababa shared the same penicillin resistance profile while the isolate from Selale was susceptible to all the antimicrobials tested.
Cluster-B isolates are 74% clonally related based on banding pattern identity and contains two isolates-one from Asela (collection center 3 farmer-12) and one isolate from Addis Ababa (collection centre 2-farmer 15).These clonal isolates share phenotypically Pen-Fox resistance pattern.
Cluster C contains 2 isolates that were found to be 87% clonally.These isolates were from two geographically distinct regions.One was from Debre-Zeit (collection centre 3-farmer 17) and the other was from Addis Ababa (collection centre 1farmer 3).These isolate had different resistance patterns.There are also several sporadic isolates representing the different geographical areas that include: four isolates from Addis Ababa, ten isolates from Selale, four isolates from Asela and nine isolates from Debre-Zeit.

DISCUSSION
A number of studies have been conducted in Ethiopia on the prevalence of S. aureus in bovine milk (Workineh et al., 2002;Dego and Tarake, 2003;Getahun et al., 2008;Abera et al., 2010).Most of these researches; however, were concentrated on the importance of this pathogen as a cause to clinical and subclinical mastitis and yet its public health importance has not been well addressed.The prevalence of S. aureus in the current study is lower than what was reported previously.A study in Ethiopia (Abera et al., 2010), for example, reported the prevalence of S. aureus in mastitic cows to be 42%.The prevalence of S. aureus as a cause of mastitis was also reported to be 44% by a different study in Ethiopia (Hundurra et al., 2005).Another study done in Ethiopia also showed a higher prevalence of S. aureus (52%) in bovine milk in Jimma (Sori et al., 2011).The discrepancy in prevalence of S. aureus among the present study and the above studies can partially be explained by the very type of milk samples analyzed in the respective studies.In the previous studies, mastitic bovine milk was the focus and cultured for S. aureus, while milk samples for the current study was picked regardless of their mastitic status.In addition, the fact that the milk specimen for the current study has been obtained from on-farm pooled and bulk tank, rather than the cow specifically, would also explain the discrepancy in the prevalence rate.
Prevalence of S. aureus is reported to vary greatly among different countries and production systems.The prevalence of S. aureus reported here is lower than what has been reported elsewhere.Some workers from Brazil reported a 70% prevalence of S. aureus from unpasteurized bovine milk samples (Rall et al., 2008) and a study in Zimbabwe also reported a prevalence of 58% for S. aureus recovered from raw bovine milk (Gran et al., 2003).Despite a lower prevalence of S. aureus in the current study, 21% prevalence is of paramount risk for the consumer as there is a lack of refrigeration facilities and the consumption of raw milk is a common feeding tradition in Ethiopia.
Regarding the antimicrobial resistance profile of S. aureus, all the isolates were susceptible to vancomycin.This is in contrast with some reports in Ethiopia which showed 3 to 38% vancomycin resistance rate in S. aureus isolates from different parts of Ethiopia (Sori et al., 2011;Daka et al., 2012;Mekuria et al., 2013).The highest rate of resistance was observed for penicillin (83%) and the lowest rate was noted for amoxicillin/ clavulanic acid (3%) and genamicin (3%).The penicillin resistance proportion observed in the present study is lower than what has been reported from mastitis cow in Ethiopia.A study conducted in Adama town in Ethiopia that looked at the antibiotic resistance profile of S. aureus from mastitic cows reported a 94% resistance to penicillin (Abera et al., 2010).These workers also reported that resistance to trimethoprim-sulfamethoxazole (SXT), gentamicin (gen) and streptomycin (str) was 58, 0, and 6% respectively.The current study, however, showed a much lower resistance proportion for SXT (5%) and a higher rate of resistance for str (8%).The discrepancy observed here might be due to methods of antimicrobial resistance determination variability based on location, or type of sample collected.The current study revealed that 14 isolate (13%) recovered from raw milk sample were found to be susceptible to the tested antimicrobials while 57% of the isolates were resistant to more than one of the antimicrobials tested.Multidrug resistance reported here is higher than what was reported elsewhere (Adwan, 2006).
A recent study in Ethiopia (Mekuria et al., 2013) reported a 45% prevalence of multidrug resistant S. aureus isolates most of which were originating from milk.A study conducted in Palestine looked into the antimicrobial resistance profiles of S. aureus isolated from sub-clinical mastitis cases and revealed that 53% of the isolates were resistant to at least three antimicrobials (Adwan, 2006).Higher multidrug resistance (79%) rate were also reported for S. aureus isolates recovered from human clinical samples in Gondar University Hospital, located in Northwest Ethiopia (Belay et al., 2013).Further studies focusing on the possible epidemiological link between the human multiple drug resistant S. aureus isolates and those of animal/food origin would help in understanding the dynamics of transmission of resistance traits and the public health risk.
Genotypic investigation of all the PCR confirmed S. aureus (n=109) isolates showed that none of the isolates were mecA positive.However, phenotypically, there were 58 (53%) isolates that were resistance to cefoxitin.In previous studies, isolates that were phenotypically resistant to β-lactams and yet genotypically lacked the mecA gene were identified (Stegger et al., 2012;García-Álvarez et al., 2011).A possible explanation for the finding in the present study could be-resistant to cefoxitin, without the mecA gene, may be due to either the over-production or over-expression of penicillinase or alteration of other penicillin-binding proteins (Caierao et al., 2004).
PFGE analysis of 39 S. aureus isolates identified three separate clonal clusters and also several sporadic isolates.This is the first study of its kind in Ethiopia.The result from molecular characterization (PFGE) indicated that the isolates contained in each cluster and subclusters represent two up to four different geographical areas.For example, cluster-A contained isolates from Addis Ababa, Asela, Selale and Debre-Zeit that are hundreds of kilometers apart.The presence of limited diversity among S. aureus isolates of bovine origin with a broad geographic distribution is possibly related to the large number of mastitis cases in this region (Fitzgerald et al., 1997;Sabour et al., 2004).However, further studies using genotypic characterization of S. aureus isolates collected at the level of the cow are warranted, as other factors may be involved in the origin of isolates in this study since samples were collected from pooled milk.
Most of the isolates in each cluster and sub-clusters phenotypically share antimicrobial resistance pattern indicating the presence of a common resistance gene pool within a broad geographic area.Based on the result, it is also possible that cross contamination between different milk collection centers within the same geographical area may occur.For example, in Selale, a number of milk collection centers that are several kilometers apart share the same bulk tank containers and milk field technician who tests the standard of milk at milk collection centers and this may also be possible source of cross contamination.Study that involves tracing down this line of contamination by milk container, however, is needed to verify this notion.

Conclusion
The result from this study reveals that milk in the central high lands of Ethiopia was contaminated with S. aureus which may imply public health significance.The overall prevalence of S. aureus was found to be 21%.PFGE analysis of 39 S. aureus isolates identified 3 separate clusters and also several sporadic isolates.Most of S. aureus isolates in this study showed resistance to a range of antimicrobials tested.Overall, 13% of all the isolates were found to be susceptible to all the antimicrobials while 57% of them were resistant to more than one of the antimicrobials tested.In addition, S. aureus strains isolated from infections in humans or from human nasal colonization should be investigated and compared to those of animal origin in this geographic location to generate a comprehensive picture of the current situation regarding antimicrobial resistance and the public health concern with regards to S. aureus in Ethiopia.In summary, S. aureus isolates recovered from raw bovine milk in this study were found to be resistant to multiply antimicrobials.This warrants further investigation of antimicrobial usage in both the human and veterinary medical sectors.

Table 1 .
Primer-pairs used for the detection of different genes of S. aureus using PCR.

Table 2 .
Prevalence of Staphylococci spp. in the different sample types.

Table 3 .
Proportion of antimicrobial resistance among S. aureus isolates recovered from different locations.