Antibiotic Susceptibility Profile of Methicillin Resistant Staphylococci aureus in Poultry Farm, in Zaria, Nigeria

Methicillin resistant S. aureus is a notorious pathogenic microorganism. Though, antimicrobials such as Penicillin and other β-lactams are commonly used in livestock production for the treatment of disease associated with S. aureus, and to improve production; the development of resistance through the production of β-lactamase enzyme against β-lactam antibiotics by S. aureus creates a huge some of economic burden [1]. This limitation had prompted the development and use of methicillin antibiotics; a new synthetic β-lactam drug that resisted β-lactamase producing S. aureus [2]. The use of methicillin was stopped due to its toxicity, development of MecA resistance gene and wide spread of resistance after a few years of production [3]. These accruing problems call for concern as resistance to methicillin is not mediated through the production of β-lactamase, but rather acquisition of mobile genetic element known as staphylococcal cassette chromosome mec (SCCmec) [4]. This staphylococcal cassette chromosome is majorly possessed by coagulase-negative staphylococci (CNS) that carries MecA gene, which encodes for an altered penicillin-binding protein (PBP2a or PBP2’) [5]. The PBP2a according to Sarah and Robert, [6] has a lower affinity for β-lactam antimicrobials than the normal PBP such that these antimicrobials are deactivated. The wide spread of this gene might be attributed to the ability of some of the organisms to acquire a competitive advantage property that could aid inherent resistance, leading to proliferation in bacteria population [7]. Community acquisition of MecA gene as found in dairy, pig, cat, poultry, cattle and poultry farm worker with phenotypical and genotypical indistinguishable MecA gene, suggests a cross-species transmission (livestock-associated MRSA (LA-MRSA)) either by contact or indirectly via the food chain, water, air, manure and sludge-fertilized soils [1,5,8,9], which could be endemic in rural areas with low medical facilities in zoonotic disease outbreak [10]. Furthermore, the staphylococcal cassette chromosome contains additional insertional DNA sequences that allow for incorporation of additional antimicrobial resistance markers [11]. These insertional sequences explain why many methicillin-resistant staphylococci are resistant to non-β-lactam antimicrobials that act through mechanisms other than interference with bacterial cell wall synthesis (e.g., macrolides, fluoroquinolones) and thus why methicillin-resistant strains can be multi-drug resistant. In both humans and animals, in-apparent colonization is far more common than outright infection, and colonization is more often transient than chronic [12].


Introduction
Methicillin resistant S. aureus is a notorious pathogenic microorganism. Though, antimicrobials such as Penicillin and other β-lactams are commonly used in livestock production for the treatment of disease associated with S. aureus, and to improve production; the development of resistance through the production of β-lactamase enzyme against β-lactam antibiotics by S. aureus creates a huge some of economic burden [1]. This limitation had prompted the development and use of methicillin antibiotics; a new synthetic β-lactam drug that resisted β-lactamase producing S. aureus [2]. The use of methicillin was stopped due to its toxicity, development of MecA resistance gene and wide spread of resistance after a few years of production [3]. These accruing problems call for concern as resistance to methicillin is not mediated through the production of β-lactamase, but rather acquisition of mobile genetic element known as staphylococcal cassette chromosome mec (SCCmec) [4]. This staphylococcal cassette chromosome is majorly possessed by coagulase-negative staphylococci (CNS) that carries MecA gene, which encodes for an altered penicillin-binding protein (PBP2a or PBP2') [5]. The PBP2a according to Sarah and Robert, [6] has a lower affinity for β-lactam antimicrobials than the normal PBP such that these antimicrobials are deactivated. The wide spread of this gene might be attributed to the ability of some of the organisms to acquire a competitive advantage property that could aid inherent resistance, leading to proliferation in bacteria population [7]. Community acquisition of MecA gene as found in dairy, pig, cat, poultry, cattle and poultry farm worker with phenotypical and genotypical indistinguishable MecA gene, suggests a cross-species transmission (livestock-associated MRSA (LA-MRSA)) either by contact or indirectly via the food chain, water, air, manure and sludge-fertilized soils [1,5,8,9], which could be endemic in rural areas with low medical facilities in zoonotic disease outbreak [10]. Furthermore, the staphylococcal cassette chromosome contains additional insertional DNA sequences that allow for incorporation of additional antimicrobial resistance markers [11]. These insertional sequences explain why many methicillin-resistant staphylococci are resistant to non-β-lactam antimicrobials that act through mechanisms other than interference with bacterial cell wall synthesis (e.g., macrolides, fluoroquinolones) and thus why methicillin-resistant strains can be multi-drug resistant. In both humans and animals, in-apparent colonization is far more common than outright infection, and colonization is more often transient than chronic [12].
Till date, there are no comprehensive data on the situation of LA-MRSA in Zaria, Nigeria and although infections with MRSA are much less reported than carriage. Hence the need to evaluate the occurrence and antibiotics susceptibility profile of MRSA in poultry farms in Zaria, Nigeria in other to investigate into the pathogenicity potential of MRSA in our environment and to curb resistance spread through the provision of information for surveillance purpose becomes imperative.

Sample collection
Fifty (50) samples of fresh chicken droplets were collected aseptically in a clean sterile universal bottle from five poultry farms (Hanwa new extension, Kongo, Zangon, A.B.U staff quarters Samaru, Dakace quarters) located in Zaria metropolis and were transported on an Ice pack to the laboratory for bacteriological examination.
Five milli litre (5 ml) of the stock solution (4 µg/ml) were aseptically mixed with sterilized mannitol salt agar and distribute into petri-dish and allowed to solidify. The dried agar surface was inoculated with the standard inoculum of the test isolates by streaking and incubated at 37°C for 24-48 hrs. This was repeated for all the isolates. Brain heart infusion agar (BHI) was mixed with 6 µg/ml of Vancomycin and distributed into petri-dishes and allowed to solidify. Overnight culture of the test isolates were standardized to an inoculum size of 106 cfu/ml. The plates were allowed to dry at room temperature and then incubated at 37°C for 24-48 hrs. This was repeated for all the resistant isolates.

Result Sample collection and identification of S. aureus isolates
Out of the 250 chicken droplets collected, isolates that produced round smooth, glistening deep golden to white colonies on nutrient agar were selected for microscopy. The microscopy result showed that 157 isolates were Gram positive Staphylococcus spp. with uniform sized cocci appearance, occurring predominantly in characteristic clusters or bunches and retained the purple colour of crystal violet. On mannitol salt agar, 98 isolates of Staphylococcus spp. fermented mannitol to acid and produced golden yellow colouration within 24 hrs of incubation.
The biochemical characterization of the isolates showed that all the 98 S. aureus were catalase positive, 88 (89.8%) were coagulase positive, 10(10.2%) were coagulase negative while 87 and 88 of the isolates were deoxyribonuclease and β-lactamase producing S. aureus respectively ( Table 1).

Antibiotic susceptibility profile of S. aureus from poultry farms in Zaria, Nigeria
The antibiotic susceptibility profile of the 88 isolates of S. aureus that were β-lactamase producers were evaluated against the 8 antibiotics commonly used in poultry farm in Zaria metropolis. Figure 1 depicts the susceptibility and the resistance of the isolates to the 8 antibiotics. The result showed that the isolates were 90.8% susceptible to Ciprofloxacin, 76.2% to Vancomycin, 72.2% to Pefloxacin, 65.6% to Gentamicin, 58.8% to Methicillin, 57.6% to Oxacillin, 49.6% to Ampicillin and 25.3% to Tetracycline. Their percentage resistance varied from 9.2, 23.9, 27.8, 34.4 and 42.4 for Ciprofloxacin, Vancomycin, Pefloxacin, Gentamycin and Oxacillin, respectively ( Figure 1). The results showed that most of the S. aureus from poultry farms in Zaria, Nigeria are resistant to β-lactam (Methicillin, Ampicillin, Oxacillin) and tetracyclines, and the pattern of antibiotic resistance also varies from one isolate to another. The isolates were also found to be 44.3% (39) multidrug resistant (44.3%), 40.9% XDR (40.9%) while 14.8% were neither MDR nor XDR. The multiple antibiotic resistance index (MARI) at ≥0.4 was observed to be high (60%), indicating an environment with pre-exposure to the

Staph. species identification, isolation and microscopy
Gram staining and microscopy was also carried out to identify Gram positive organisms while further morphological characterization of the colonies isolated from concentrated Mannitol salt agar 3 (17.5%) of 12.5 g NaCl to 1 L of Mannitol salt agar in other to inhibit the growth of other organism was carried out using the method described by Onaolapo" Split the sentence in two: "Gram staining and microscopy was also carried out to identify Gram positive organisms. " Followed by "Further morphological characterization of the colonies isolated from concentrated Mannitol salt agar (17.5%; 12.5 g NaCl to 1 L of agar in order to inhibit the growth of other organism) was carried out using the method described by Onaolapo [12].

Biochemical test and β-lactamase production test
The following conventional biochemical tests; catalase, coagulase and deoxyribonuclease (DNase) tests as described by Cheesbrough [13] were also adopted to distinguish S. aureus from other forms of Staph. spp. The test tube method according to Lennette et al., [14] and Plateacidimetric method according to Cheesbrough [13] were also used to determine the ability of the identified S. aureus to produce β-lactamase.

Antibiotic susceptibility test and multiple antibiotic resistance index (MARI) evaluation
The susceptibility profiles of the identified S. aureus was tested against eight selected antibiotics (ampicillin, ciprofloxacillin, methicillin, tetracycline, Vancomycin, gentamicin, pefloxacin and oxacillin) using disc diffusion method as described by Cheesbrough [13] and the corresponding results interpreted using CLSI [15]. The multiple antibiotic resistant (MAR) index was determined for each isolate. This is defined as the number of antibiotics to which the organism is resistant to, divided by the total number of antibiotics tested [16,17].

Minimum inhibitory concentration (MIC) to oxacillin
Resistance to methicillin was confirmed by the determination of the MIC of Oxacillin to the isolates. A working stock solution of 128 µg/ ml was prepared. This working solution (2 ml) was then serially diluted in nutrient broth (2 ml) up to the last tube. Eighteen hours cultures of the isolates were standardized to contain about 106 cfu/ml inoculum size. The diluted antibiotic was aseptically inoculated with 1-2 drops of the standardized inoculum. The test tubes were inoculated at 35°C for 18 hrs and this was repeated for all the resistant isolates.

Determination of vancomycin resistance
Isolates that were resistant to oxacillin from the minimum inhibitory concentration results were picked for this test. Fresh stock solution of 4 µg/ml and 6 µg/ml of Vancomycin were prepared. The result showed the biochemical characteristics of the identified S. aureus from different farm sources. N=number of farms, n=number of S. aureus from various farms in Zaria, metropolis.  [17] is shown in Table 2.

Minimum inhibitory concentration (MIC) to oxacillin
The result of the MIC of oxacillin against the 35 isolates that were was resistant to methicillin showed that 74.3% of the isolates had high MIC ≥64 μg/ml and the remaining 25.7% had MIC of 2 μg/ml (Table  3). This is as shown in Table 3. The MIC break points for oxacillin are MIC of ≤2 µg/ml is susceptible while that of ≥4 µg/ml is resistant [18]. Table 4 shows that the 74.3% (26) isolates that showed high MIC value against Oxacillin were tested against Vancomycin. The result showed that 80.8% (21) of the isolates were resistant to Vancomycin while 19.2% (5) were sensitive even after 48 hrs incubation on mannitol salt agar impregnated with 4 μg/ml Vancomycin. The isolates were also grown on Brain heart infusion agar impregnated with 6 μg/ml Vancomycin. The result showed that 88.5% (23) of the isolates were resistant while 21.5% (3) were sensitive. This is shown in Table 4.

Discussion
Previous studies on poultry farms have recognized S. aureus as an important pathogenic organism [19], and based on geographical locations and site of sample collection, different percentages of S. aureus from poultry farms have been reported. In Nigeria, Suleiman et al., [20] had reported 84% (84) S. aureus from tracheal swabs in Maiduguri, Adeyeye and Adewale [21] in Ogbomoso reported 100% (30) from nasal swabs while in Zaria, from chicken droplets, 39.2% (98) of the total samples (250) evaluated in this study were observed to be S. aureus. This showed that the percentages of S. aureus from different samples sources varies. The most commonly isolated of this specie is coagulase positive (89.8% (88)) while coagulase negative were 10.2% (10) ( Table 1). The report of Roda and Fatema [22] concurred with the high percentages of coagulase positive S. aureus (42.4% (14)) in poultry farms compared with the coagulase negative (30.3%) S. aureus. The susceptibility profile of the S. aureus to commonly used antibiotics in the hospital and poultry management showed that the isolates were highly susceptible to Ciprofloxacin, Vancomycin, Pefloxacin and Gentamycin. The isolates showed high resistance (74.7% and 50.4%) to Tetracycline and Ampicillin respectively while 41.2% of the isolates were resistant to Methicillin and produced β-lactamase enzyme ( Figure  1). The observed variation in the percentage susceptibility profile expressed by S. aureus in this study also concurred with the report of Roda and Fatema [22] who observed a variation of 3 to 66.66%. The study of Roda and Fatema [22] and the work of Yaqub et al., [23,24] also observed high resistance to penicillin and tetracycline. This could be associated with the vast used of these antibiotics for the treatment of animal diseases [25]. Among these, 44.3% were found to be multidrug resistant, 40.9% XDR while 14.8% were neither MDR nor XDR ( Table  2). This high occurrence of multidrug resistance in this study might be due to the production of beta-lactamase enzyme and horizontal gene transfer of antibiotic resistant gene [5,9]. The MAR index result showed that 40% of the isolates had MAR index of ≤0.3 while 60% had MARI of ≥0.4; indicating that the S. aureus tested were pre-exposed to the antibiotics used in this study. The result of the MIC of oxacillin against the 35 isolates that were resistant to Methicillin showed that 74.3% of the isolates had high MIC value of ≥64 µg/ml and the remaining 25.7% had MIC of 2 µg/ml (Table 3). This form of high resistance according to Cohn and Middleton,[3] and Sarah and Robert, [6] could be linked with an altered penicillin-binding protein, PBP2a, which is encoded by the mecA gene. The 74.3% (26) isolates that showed high MIC value against Oxacillin were tested against Vancomycin. The result showed that 80.8% (21) of the isolates were resistant to Vancomycin while 19.2% (5) were sensitive even after 48 hrs incubation on mannitol salt agar impregnated with 4 µg/ml Vancomycin. The isolates were also grown on Brain heart infusion agar impregnated with 6 µg/ml Vancomycin. The result showed that 88.5% (23) of the isolates were still resistant while 21.5% (3) were sensitive (Table 4). Though the mechanism of Vancomycin resistance in S. aureus still remain unclear, the report of Natasha et al., [25], had suggested that there is a likelihood of clinical treatment failure when Vancomycin is used to treat infections where the MIC is above 2 μg/ml. Howden et al., [26] suggested that resistance to Vancomycin (the principal drug used to treat MRSA infections) might have emerged as a result of point mutation (six nucleotide substitutions), which occurred in graRS, encoding a putative twocomponent regulatory sensor, leading to a change from a polar to a nonpolar amino acid (T136I) in the conserved histidine region of  the predicted protein, while the work of Hiramatsu et al., [27] had suggested that this persistent resistance may be due to the acquisition of the vanA resistance determinant from enterococci which is plasmid mediated. Other inferences like the work of Cui et al., [28], and Sieradzki and Tomasz [29] ascribed the thickened cell wall containing dipeptides capable of binding Vancomycin as the major cause of the resistance to Vancomycin. This thickened cell wall according to Susana and Alexander [30] is caused by transposon Tn1546, acquired from Vancomycin-resistant Enterococcus faecalis, which is known to alter cell wall structure and metabolism. An observation of this high resistance in our environment should been indeed a major concern to clinicians, veterinarians, pharmaceutical companies, and consumers worldwide due to the ability of acquired Methicillin and Vancomycin resistant gene to render diseases associated with S. aureus such as dermatitis, pneumonia, septicaemia to osteomyelitis and meningitis in humans and swine as well as bovine mastitis in cattle and bumblefoot disease in poultry untreatable [31]. Although no clinical trials suggest superiority of Vancomycin over any comparator antibiotics, some studies have provided evidence of its inferiority [32]. Therefore strong consideration like the use of Ciprofloxacin as observed in this study, or a combination of Vancomycin and Piperacillin-Tazobactam or Oxacillin or Cefepime [33] should be used as alternative agents in the treatment of serious S. aureus infections from poultry farms in Zaria, Nigeria.

Conclusion and Recommendations
Methicillin-resistant S. aureus (MRSA), once restricted to hospitals is spreading rapidly in poultry farms in Zaria, Nigeria and this could play a potential role in disseminating pathogens between animal and human    resulting into community acquired MRSA. This study established the first complete S. aureus isolates to be Vancomycin resistanct with an elevated Vancomycin MIC within the susceptible range in Zaria, Nigeria among poultry farms. It also showed that MRSA is able to develop Vancomycin resistance, in which the spread of this resistant trait might influence untreatable diseases in zoonotic outbreak. To improve the efficacy of Vancomycin therapy we suggest a further study on the combination of Vancomycin with Ciprofloxacin or Gentamicin or Pefloxacin to infections associated with highly resistant MRSA. Also antibiotic surveillance and control on the use of beta-lactam antibiotics including other classes of antibiotics in our community should be emphasized.