Screening of methicillin resistant Staphylococcus aureus from dogs and cats in Hyderabad Pakistan

Methicillin-resistant Staphylococcus aureus (MRSA) is a “superberg”, an important cause of human nosocomial and as a universal community-acquired infection cause resistant to β-lactam antibiotics. Till to date no detailed and authentic report on MRSA in nasal swabs of diseased dogs and cats reported. This study was planned to check the presence of MRSA in nasal swabs of dogs and cats. Total 100 samples were collected, and 37 samples were positive with S. aureus in both diseased dogs and cats which were confirmed by their colony characteristic, morphology, gram stain reaction and biochemical properties. These isolates were subjected to minimum inhibitory concentration (MIC) through Micro broth dilution method by using methicillin in 96 well plates. Among them, 8/37 (21.62%) were found methicillin resistant. In diseased dog, 5/21 (23.80%) and cat 3/16 (18.75%) isolated were found moderately resistant to methicillin as they grew at 32μg/μl concentration of methicillin. mec-A gene in methicillin resistant samples were detected through polymerase chain reaction (PCR). This resistance was due to phenotypically and moderate level of the resistance against methicillin with absence of mec-A gene shows that the examined pets were not the reservoir of mec-A gene. However, more studies are recommended to further confirm that, pets are reservoir of mec-A gene or not or this may be due to involvement of sharing mechanism of resistance to penicillin group. So far, this is first study on MRSA in diseased dog and cats, which revealed that, there is no MRSA in this area of Pakistan.


Introduction
Antibiotics are generally used to treat for variety of bacterial infection in veterinary and human practice and these antibiotics produced their action by either stopping the bacterial growth or directly killing microbes. The broad spectrum antibiotics have killed all type of bacteria including gram positive and gram negative whereas narrow spectrum antibiotics are effected on either gram positive or gram negative bacteria [1]. Antibiotic agent of β-lactam antibiotics contains a β-lactam ring in their molecular structures. The β-lactam resistant penicillin consist of methicillin, oxacillin and cloxacillin were used in first line of treatment as penicillin-resistant S. aureus and the β-lactam antibiotics are frequently used for Staphylococcal infections as a result antibiotic acquired resistance has developed [2]. The different derivates of Penicillin including Methicillin has been used for the infection occurred through S. aureus [3]. The most penicillin derivatives are resistant to 90% of S. aureus strains However, many species of S. aureus have developed resistance recognized as MRSA [4]. The first antibiotic was Methicillin which introduced in human medicine used as methicillin resistant Staphylococcal infection in 1950s and more than 80% of S. aureus have produced Penicillinase enzyme, caused resistant to β-lactam antibiotics which commonly used to treat Staphylococcal infections in dogs and cats [5]. Antimicrobial resistance is a major threat in veterinary and human medicine and the most important antimicrobial resistance has developed due to use and misuse of antibiotics in veterinary field and human medicine [6]. MRSA known as an important cause of human nosocomial and as a universal community-acquired infection cause resistant to β--lactam antibiotics was facilitated by the mec-A gene, present in bacterial cell wall which encodes to penicillin-binding protein (PBP2a) for reducing the affinity of β-lactam antibiotics [7]. MRSA kills more people than the acquired immunodeficiency syndrome (AIDS) because of it known as the "superbug" [8]. The mobile genetic element (mec-A) gene is fragment of a 21kb to 60-kb Staphylococcal chromosome cassette mec (SCCmec). The mec-A gene is characterized by the combination of ccr and mec which encode resistance to β-lactam antibiotics [9]. Pets contain emerging and prevalent pathogenic species of Staphylococcus aureus [10]. Dogs and cats can be a potential reservoir of MRSA infection for human and contact with large part of human population so there is the potential for transfer of MRSA or mec-A gene among human and cats and dogs. The risk factor for MRSA infections were intravenous catheter and drugs used in veterinary medicine in cats and dogs and MRSA infection is becoming a public health concern because dogs and cats are in close connection with their owners, risk them for transmission of highly pathogenic strain of MRSA [11]. Considering this screening present study was designed to check the presence of highly pathogenic and zoonotic organism in pets (cats and dogs) from in order to analyses the possible pathogenic thread of spread of MRSA from cats and dogs to human. Results of this study will provide information to the veterinary field as well as human clinicians about the prevalence of Methicillin resistant strains of S. aureus in pets.

Study area
All samples were collected from nasal swabs of diseased dogs and cats from the Hyderabad and its vicinity. The clinically ill dogs and cats were selected in this study and samples were collected from different Pets clinics of Hyderabad and its vicinity. Collection of sample A total of 100 samples were collected, 50 samples were collected from diseased dogs and 50 samples were collected from the diseased cats and brought to the veterinary clinics for collection of nasal swabs. Samples were collected in Tran swab tube containing Amies medium. All samples were brought to the bacteriology section of Central Veterinary Diagnostic Laboratory (CVDL) Tandojam in a portable cooler containing an ice pack and then swabs were refrigerated at 4 °C until being processed within 48 hr of collection for isolation and identification of S. aureus by chain of laboratory technique including colony characteristics, gram staining, and growth on blood agar. The isolates of S. aureus were cultured on Tryptic Soy Broth (TSB) at 37 °C for further experimental use. Antimicrobial susceptibility test The S. aureus isolates were determined through Minimum inhibitory concentration test by using (Micro Broth 2-fold dilution method) on Muller Hinton (MH) agar (Oxide, UK) and 1:1000 dilution was prepared for MIC test for that 6μl of bacterial culture of S. aureus in Nutrient broth were added into 6ml of Muller Hinton broth. 96 well plates of micro titer tray were used for MIC test and a concentration 1280μl/ml of Methicillin (Sigma, USA) was used for minimum inhibitory concentration against S. aureus. S. aureus isolates in nutrient broth culture were added in each well of micro titer tray. In first well 180μl of culture were added and in remaining all wells 100μl of bacterial culture were added than 20μl of Methicillin (Sigma, USA) were added in first well and mixed by push up of Eppendorf tube then 100μl of culture from first well were introduced into next well and repeat till last well. The final sub cultures of bacterial isolates were discarding. Then MIC plates were placed in incubator at 37 °C  After DNA extraction, PCR was performed in Applied Bio system 2720 thermal cycler (Eppendorf). Total of 50μlof PCR reaction mixture was prepared for each supernatant DNA for that 25μl PCR master mixture was added into Eppendorf tube then 17μl of RNase free water was added into same Eppendorf tube then added 2μl of forward primer followed by 2μl of reverse prime were mixed with 4μl of supernatant DNA Gently, mixed by vortex and then kept tubes in thermal cycler. A thermal cycler was used for amplification of DNA then cycling condition of PCR was set such as initial denaturation at 95 °C for 05 minutes, followed by 35 cycles of denaturation at 95 °C for 01minute annealing at 55 °C for 45 seconds, extension at 72 °C for 45 seconds and final cycling of amplification was done at 72 °C for 5minutes. The amplified product was analyzed through gel electrophoresis processes by using 3μl ethidium bromide and 2% agarose gel and DNA band was visualized by ultraviolet light.

Results Percentage prevalence of S. aureus in diseased dogs and cats
To isolates the S. aureus, one hundred samples were collected from nasal swabs of diseased dogs and cats. All samples were cultured on different media to isolates pure colony of S. aureus then pure cultures of organism were confirmed through grams staining. Further pure cultures were confirmed by the biochemical properties. From all samples only 37 were found positive with S. aureus colony in both diseased dogs and cats (Table 1).

Percentage prevalence of S. aureus isolated from nasal swabs of diseased dogs and cats.
A total of 50/50 samples were collected from nasal swabs of diseased dogs and cats, only 21 and 16 samples were found positive with S. aureus isolates shown in (Table 2).

Colony characteristic of Staphylococcus aureus isolated from diseased Dogs and Cats
The colonies of Staphylococcus aureus produced yellow or white colony and typical characteristic of βhemolysis of red blood cell was recorded on blood agar.

Minimum inhibitory concentration of S. aureus isolates against methicillin from nasal swabs of diseased cats
Total of three samples were shown their resistant at the concentration of 16μg/ml in diseased cats while remaining shown no resistant at any concentration of methicillin. The positive sign indicates the bacterial resistant against methicillin and negative sign shows no growth of S. aureus against methicillin ( During present study, no positive strain of Methicillin resistant S. aureus from nasal swabs of diseased dogs and cats but, 8% of pet samples were infected with MRSA and in addition 5 (10%) samples of dog were positive with MRSA and 3 (6%) cat samples were positive with the MRSA which is in disagreement with our study as we found no positive samples for MRSA.
The bacterial isolates of this study were confirmed by their typical growth pattern and colony characteristics, which include beta hemolytic activity on blood agar (Fig.  1) [12] mentioned that S. aureus is found hemolytic on blood agar, whereas S. epidermis is no hemolytic, thus it confirms that bacterial colonies observed during this study were of S. aureus, which has ability to produce hemolysin enzyme that lyses the red blood cells. Morphologically S. aureus was found gram positive cocci often arranged in single, double or grapes like (Fig. 2). Habib et al. [13] defined morphological properties as gram positive cocci arranged in single, pairs or in chain and S.aureus were produced golden yellow and shiny colonies on nutrient agar. The bacterial isolates of this study is confirmed by the findings of above studies. The Staphylococcus aureus were confirmed by its biochemical properties, which included catalase, methyl red, urease, gelatin liquification, coagulase, oxidase positive, indole oxidase negative (Table 3). Rusenova & Rusenov [14] reported that the biochemical properties of S. aureus as catalase coagulase urease positive, indole and oxidase negative. The above biochemical result is in agreement with present study.
The bacterial susceptibility to methicillin was confirmed through minimum inhibitory concentration (MIC) through using micro broth 2-fold dilution method. A concentration of 1280μg/ml of methicillin was used. The 20μg/ml concentration of methicillin was break point for MIC were observed in this study. Out of 37 isolates of S. aureus only 08 isolates were shown resistant to methicillin phenotypically ( Table 4) 5(23.80%) isolates were found moderate level of resistance to methicillin in diseased dogs. Out of 16, 3(18.75%) isolates were found low level of resistant to methicillin in diseased cats (Table 5) [15]. stated that, Methicillin Resistant S. aureus was treated with different antibiotic, but Methicillin was the most effective antibiotic against all MRSA isolates they mentioned that some samples showed resistance to the drug even at low concentration, which indicated that bacteria are methicillin resistance. The PCR results of the present study suggest that all methicillin resistant samples did not carry mec-A gene (Fig. 3). The DNA fragment of 533bp was amplified and confirmed as mec-A in 25% of human isolates by [16]. The findings of this study are in disagreement with our study, as we found that no mec-A gene, But in this study no band was seen at any stage of gel electrophoresis methicillin resistant isolates were only found phenotypically resistant to methicillin and the level of resistant was moderate, this phenotypical resistance may be due to the Penicillin binding protein (PBP), Beta-lactmase enzyme and methicillinase enzyme production and different strain of Staphylococcus aureus. Absence of the mec-A gene in this study can be attributed to many causes subsequently there are other mechanisms that are nonmec dependent mechanism that donate individually or in combination with different antibiotic resistance in Staphylococci isolates [17].

Conclusion
On the basis of present finding it is concluded that S. aureus is prevalent in nasal swabs of diseased dogs and cats. The prevalence Percentage is higher in diseased dogs as compared to diseased cats and S. aureus showed moderate resistant to methicillin when MIC test done but this resistant were recorded more in dogs as compared to cats. This resistance was due to phenotypically and moderate level of the resistance against methicillin with absence of mec-A gene shows that the examined pets are not the reservoir of mec-A gene. However, more studies at the larger scale at (country level) are recommended to further confirm that the pets are reservoir for mec-A gene or not.