Molecular study on methicillin-resistant Staphylococcus aureus strains isolated from dogs and associated personnel in Jordan

Yaser Hamadeh Tarazi，Ahmed Mahmoud Almajali，Mustafa Mohammad Kheer Ababneh，Humam Shawket Ahmed，Adnan Saleem JaranDepartment of Basic Veterinary Medical Sciences，Faculty of Veterinary Medicine，Jordan University of Science and Technology，P.O.Box 3030，Irbid，0，Jordan

2Department of Biological Sciences，Faculty of Science，Al Al-Bayt University，Al-Mafraq，Jordan

Molecular study on methicillin-resistant Staphylococcus aureus strains isolated from dogs and associated personnel in Jordan

Yaser Hamadeh Tarazi1*，Ahmed Mahmoud Almajali1，Mustafa Mohammad Kheer Ababneh1，Humam Shawket Ahmed1，Adnan Saleem Jaran2

1Department of Basic Veterinary Medical Sciences，Faculty of Veterinary Medicine，Jordan University of Science and Technology，P.O.Box 3030，Irbid，22110，Jordan

2Department of Biological Sciences，Faculty of Science，Al Al-Bayt University，Al-Mafraq，Jordan

ARTICLE INFO

Article history：

Accepted 1 Jul 2015

Available online 18 Aug 2015

Staphylococcus aureus

Methicillin-resistant

Staphylococcus aureus

Methicillin-susceptible

Staphylococcus aureus

Dogs

Associated personnel

Jordan

Objective：To determine the prevalence，genetic relatedness，and pattern of antimicrobial susceptibility in methicillin-resistant Staphylococcus aureus（S.aureus）（MRSA）isolated from household dogs，farm dogs，and stray dogs，compared to isolates from their associated personnel.

Methods：MRSA was isolated from 250 nasal swabs（150 swabs from dogs and 100 swabs fromhumans）.PCR assays were usedtodetectthe presenceof both the nuc and mecA genes，which confirmed the identity of S.aureus isolates and the presence of methicillin resistance，respectively.Disk diffusion was used to determine the antibiotic susceptibility against 15 antimicrobial agents along with an E-test that determined the minimum inhibitory concentration for oxacillin.Pulsed field gel electrophoresis was conducted to determine the genetic relatednessofMRSAisolatesfromdogstothosefromassociatedandunassociatedpersonnel. Results：The prevalence of S.aureus in dogs and humans was 12.7%and 10.0% respectively，while the prevalence of MRSA isolates in dogs and humans was 5.3%and 5.0%，respectively.The prevalence of MRSA isolates in household dogs，farm dogs，and stray dogs was 7.8%，4.7%，and 0.0%，respectively.MRSA isolates demonstrated a significantly higher rate of multi-resistance against three or more antimicrobial agents than methicillin-susceptible S.aureus（MSSA）.Trimethoprim-sulphamethoxazole and chloramphenicol were the most effective antibiotics against all MRSA isolates.Pulsed field gel electrophoresis revealed a strong association between dog MRSA isolates and MRSA isolates from strongly associated personnel.

Conclusions：MRSA is prevalent in house dogs，as well as in dog rearing centers and among their strongly associated personnel.A strong association was found between the MRSA isolates from dogs and those from humans who are in close contact.In addition，MRSA isolates showed a high rate of multi-resistance compared to MSSA isolates.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.06.015

1.Introduction

Methicillin-resistantStaphylococcusaureus（S.aureus）（MRSA）isamajorhealthcare-associatedpathogenworldwideand has increased in incidence dramatically over the last decade［1，2］. Companion animals have been implicated more frequently aspotential reservoirs of MRSA than other livestock［3，4］.In several studies，a 0%-4%prevalence rate of MRSA in dogs has been reported［5-7］.Other reports demonstrated MRSA at a higher prevalence（～9%）in pets and veterinary staff［8，9］，and the nasal carriage of MRSA plays a key role in the epidemiology and pathogenesis of community-associated infections［10，11］.

In Jordan，MRSA is widely prevalent in Jordanian hospitals and represents a serious public health problem.The nasal carriage rate of S.aureus among the Jordanian healthy young population was 40%，and 19%of the nasal S.aureus，and 57% of clinical isolates were resistant to oxacillin［12］.A retrospective study conducted at King Abdullah University Hospital in North Jordan showed that 152 S.aureus isolates collected from different infections revealed that the overall rate of MRSAwas 34%，with a rate of 57%-70%in the adult intensive care unit［13］.Toourknowledge，nostudiesonMRSAin companion animals in Jordan were found in the literature that demonstrated its prevalence and health hazard.Therefore，this study was conducted to document the prevalence of MRSA in dogs and their associated personnel，as well as to determine their genetic relatedness and antimicrobial resistant profile.

2.Materials and methods

2.1.Sample collection，transportation，and preparation

Intotal，250nasalswabswerecollectedfrom150dogsand100 humans during a period between March and the end of October 2009.The 150 dog samples were collected from household dogs，stray dogs，and farm dogs from the middle and northern parts of Jordan.Thetotalnumbersofhouseholddogs，straydogs，andfarm dogs were 77，30，and 43，respectively，as illustrated in Table 1.

The 100 human nasal swab samples were collected from personnel strongly associated with dogs（5 from dog owners，50 from employees who feed，take care of，treat，and train dogs daily at the Spana Welfare Center，Humane Center for Animal Welfare，and K-9 Center，25 from intermediately associated personnel，including veterinarians working in clinics and veterinary students，and 20 from unassociated personnel who have never been in contact with dogs）.A sterile cotton swab moistened with normal saline was inserted into the nares and gently rotated to make contact with the nasal septum.For dogs，smaller swabs were inserted to a distance of about 0.5-1.0 cm.All swabs were placed in a transport medium and stored at 4°C until cultured within 6-h collection at the Microbiology Research Laboratory，Faculty of VeterinaryMedicine，JordanUniversityofScienceand Technology.

2.2.Isolation and identification of S.aureus

All nasal swabs were cultured on mannitol salt agar（Oxoid，UK）and incubated aerobically at 37°C for 24-48 h.The cultures were then examined for the presence of S.aureus（yellow colonies）and for a microscopic appearance after Gram staining.The presumptive S.aureus isolates were further examined for pigments and coagulase production，by using the tube method［14］.

Table 1 Distribution of the dogs'nasal swab samples according to the dogs' locations and type of rearing system in Jordan.

2.3.Molecular identification of S.aureus isolates

2.3.1.DNA extraction and identification of the nuc gene

The extraction protocol was done according to the Wizard genomic DNA purification kit（Promega cooperation，Technical manualgenomicDNApurificationpartTM0580，Madson，USA）. Then，presumptive S.aureus isolates were tested by PCR amplificationofthenucgene［15］.PCRamplificationwasconductedata final volume of 25μL［12.5μL of Go Taq master mix（Promega，USA），5μL（2.5 pmol）of each primer F（GCGATTGATGGTGA TACGGTT）as well as R（AGCCAAGCCTTGACGAACT AAAGC），2.5μL of a bacterial DNA sample and 5μL nuclease free water］.The PCR amplification was conducted as follows: 5 min at 94°C，35 cycles for 30 s at 94°C，45 s at a corresponding annealing temperature of 55°C，and 45 s at 72°C，and a final extension of 10 min at 72°C.The PCR products were observed on 1.5%agarose gels.

2.4.Identification of MRSA by E-test and antimicrobial susceptibility testing

MRSA isolates were identified by E-test（Oxoid，UK），which is a gradient antibiotic stabilized on a plastic strip with 30 graduations to provide an accurate minimum inhibitory concentration（MIC）over a range of 256-0.015μg/mL.This test was conducted for oxacillin only，according to the manufacturers'instructions（Oxoid，UK）and guidelines.Mueller-Hinton agar（Difco，Detroit，MI，USA）supplemented with 2%NaCl was used for this purpose［16］.Samples for the E-test were prepared according to Clinical and Laboratory Standards Institute［17］. Isolates showed MICs equaled to or greater than 4μg/mL，which were considered MRSAs［18］.

The agar disk diffusion susceptibility test of 15 antimicrobials［cefoxitin（10μg），penicillin（10 IU），cephalexin（30μg），kanamycin（30μg），gentamicin（10μg），tobramycin（10μg），amikacin（30μg），ciprofloxacin（5μg），azithromycin（15μg），erythromycin（15μg），tetracycline（15μg），amoxicillin-clavulanic acid（20/ 10μg），trimethoprim-sulphamethoxazole（1.25/23.75μg），nalidixic（30μg），and chloramphenicol（30μg）］was carried out by using the Clinical and Laboratory Standard Institute guidelines［19］.The S.aureus ATCC 25923 strain was used as a control.

2.5.Molecular identification of MRSA

For further confirmation，MRSA isolates were tested by PCR amplification of the mecA gene.PCR amplification was conducted atafinalvolumeof25μL［12.5μLofGoTaqmastermix（Promega，USA），5μL（2.5 pmol）ofeachprimer F（5′-GCAATC GCTAAA GAACTAAG）aswellasR（5′-GGGACCAACATAACCTAA TA）［20］，2.5μLofabacterialDNAtemplateand5μLnucleasefree water］.PCR amplification was conducted as follows:denaturation atonecycleof94°Cfor3min，30cyclesat94°Cfor45s，annealing at53°Cfor2min，extensionat72°Cfor30s，andafinalextension at 72°C for 5 min.The PCR products were observed by electrophoresis on 1.5%agarose gels（Nusieve Bioproducts，Maine，USA）.

2.6.Pulsed field gel electrophoresis（PFGE）

PFGE was used to study the genetic relatedness between the S.aureus isolates from dogs and human S.aureus isolates. PFGE was performed according to a Canadian standard protocolby using the restriction enzyme SmaI［21］.Electrophoresis was done with a CHEF-DR III apparatus（Bio-Rad，USA）by using the auto-algorithm program and switch times of 5.3-34.9 for 18 h at a 6.0 V/cm gradient and 14°C in 0.5μL Tris-Borate-EDTA.The gel was stained for 20 min with 0.5 mg of ethidium bromide per liter and destained with Millipore-filtered H2O for at least 30 min with water changes for three times. PFGE gels were viewed under ultraviolet light.

2.7.Data analysis

Gels were photographed and digitized with the FOTO/Analyst Archiver system，and an analysis with BioNumerics version 2.0 software was conducted（Applied Maths，Belgium）.The S.aureus ATCC 43300 and ATCC 25923 strains were used as reference standards for MRSA and methicillin-susceptible S.aureus（MSSA），respectively.A Wilcoxon test（paired）was used to compare the antibiotic susceptibility of MRSA and MSSA isolates against different antimicrobial agents，and it was also used to compare the MICs of MRSA and MSSA isolates against oxacillin.

3.Results

3.1.Conventional and molecular identification of S.aureus

In total，46 isolates were confirmed as Staphylococci；among them，29 isolates（19 from dogs and 10 from humans）were conventionally identified as S.aureus and confirmed by amplifying the thermonuclease gene（nuc）using PCR（Figure 1）.The prevalence rate of S.aureus in dogs was 12.7%（19/150）and in humans，it was 10%（10/100）.

3.2.MRSA and susceptibility test

Out of 29 S.aureus strains，13 were MRSA；among them，8 were from dogs（prevalence rate was 5.3%）and 5 were from humans（prevalence rate was 5.0%）.Six MRSA isolates were from 77 household dogs［one from each of the Swelieh city，Spana Welfare Center，Humane Center for Animal Welfare，and K-9 Center/Ramtha and two from the K-9 Center/Amman（prevalence rate was 7.8%）］.The other two MRSA isolates were isolated from 43 farm dogs from the Al-Zarqa and Irbid governorates（prevalence rate was 4.7%）.No MRSA was isolated from stray dogs.One human MRSA isolate was from a dog owner whose dog had MRSA，and the other four MRSA isolates were from strongly associated personnel（two from K-9，one from Spana Welfare Center，and one from Humane Center for Animal Welfare）.

All MRSA isolates were found to be resistant to cefoxitin.In contrast，all MRSA isolates were susceptible to trimethoprimsulphamethoxazole and chloramphenicol.MRSA was also found to be susceptible to azithromycin，cephalexin，aminoglycoside，kanamycin，gentamicin，tobramycin，and amikacin in a range of 62%-85%.The antibiotics resistant to all MRSA and MSSA isolates are shown in Table 2，and the MRSA isolates were found to be significantly more resistant to the tested antibiotic than MSSA isolates（P＜0.001）.In addition，the MRSA isolates of humans were significantly more resistant than the MRSA isolates of dogs against erythromycin，tetracycline，and amoxicillin-clavulanic acid（Table 3）.

3.3.MIC

An E-test was used to evaluate the MIC of oxacillin.Out of 29 S.aureus isolates，all MRSA（13）isolates showed oxacillin resistance（oxacillin MIC≥4μg/mL）.

In addition，MRSA MICs were significantly higher than MSSA MICs in both humans and dogs（P≤0.001），and MRSAMICs were significantly higher in humans than MRSA MICs in dogs（P≤0.001），as shown in Table 4.

Table 2 Antibiotic resistance pattern of 13 methicillin resistant and 16 susceptible S.aureus isolates.n（%）.

Table 3 Comparison of antibiotic resistance pattern of 8 MRSA isolates from dogs and 5 MRSA from humans.n（%）.

Table 4 MIC of MRSA and MSSA isolates in dogs and humans against oxacillin by E-test.μg/mL.

3.4.PCR for the identification of the mecA gene

The results of PCR showed that 13（45%）of 29 S.aureus isolates were positive for the presence of the mecA gene（Figure 2）.Five MRSA isolates were from humans and eight were from dogs.

3.5.PFGE

The SmaI macrorestriction fragment profiles of 29 S.aureus isolates were determined by PFGE（Figure 3）.A dendrogram of a percent similarity and DNA relatedness were calculated based on the Dice coefficient，which revealed five major clusters of isolates:Jordan A，Jordan B，Jordan C，Jordan D，and Jordan E（Figure 4）.All isolates are grouped into the above-mentioned five clusters，as shown in Table 5.

Table 5 Groups of the 29 S.aureus isolates in different clusters determined by PFGE dendogram.

4.Discussion

In this study，the prevalence of the S.aureus nasal carriage isolates in dogs was 12.7%and of MRSA，it was 5.3%.To the best of our knowledge，this is the first report about the nasal carriage of S.aureus and MRSA in dogs in Jordan.The S.aureus nasal carriage of dogs in Jordan is more prevalent than 8.8%which was reported in a Hong Kong study［22］，similar to the other study in Austria［23］.The human nasal carriage rate was 10%，which is similar to other reports in Jordan［11］，but it is not in agreement with others［12］，which reported a 40%nasal carriage rate in Jordan.However，the variable in the nasal carriage of S.aureus may be different due to populations，geographical locations，and the influence of genetic and environmental factors［10］.In addition，the cell-wall lipoteichoic acid，hormonal status，and antimicrobial activity of nasal secretions play a role［24］.

In this study，the prevalence rate of MRSA in dogs was 5.3% and most dogs carrying MRSA are either household dogs or reared at rearing centers（prevalence rate was 7.9%），where they areexposeddailytoclosecontactwiththeirpersonalassociates.In contrast，stray dogs did not have MRSA，and this could be explained by that those dogs are not exposed to the human population and do not receive veterinary care.Two MRSA isolates were from farm dogs and a study may be needed to demonstrate the prevalence of MRSA in farm animals in Jordan.The MRSA prevalence in dogs was similar to other studies［7，8，25］.

The MRSA nasal carriage rate in humans was 5%，and that represents 50%of the 10 S.aureus strains isolated from 100 human nasal swabs，which may be explained by daily close contact with dogs that transmit MRSA between owners and personnel strongly associated with dogs easily.It may also be due to the continued use of antibiotics［9，22，26］.The close contact between household pets and humans offers favorable conditions for the transmission of MRSA by direct contact（petting，licking，and physical injuries）or through the domestic environment（contamination of food，water，and plates）and physical contact withdogs，aswellasthroughcontactwithhousehold environments contaminated by pets（floors，furniture，and carpets）［27］.

In the current study，the oxacillin resistance in human isolates was significantly higher than that in dog isolates.This may indicate the unwise use of antibiotics in humans，wherein people obtain antibiotics without a prescription.In other countries，this is not always the case，as a higher oxacillin resistance in canine isolates compared to that in human isolates was reported［22］.

The present investigation also demonstrated MRSA isolates were significantly more resistant to the tested antibiotics than those of MSSA.However，all MRSA isolates were susceptible to trimethoprim-sulphamethoxazole and chloramphenicol.The results of the current work showed similar findings to that of Al-Zu'bi et al.［12］，where all nasal isolates were susceptible to chloramphenicol，while clinical isolates showed some resistance.

In the current study，the MRSA isolates showed 92%resistance to penicillin，which is similar to another study in Jordan that showed all human MRSA isolates were resistant to penicillin and 23%of MRSA isolates were resistant to cephalexin，which is not in agreement with other reports［11，28］.MRSA in the current study showed 23%resistance to aminoglycoside，gentamicin，kanamycin，tobramycin，and amikacin，in contrast to the studies of others where higher resistance to kanamycin（98%），tobramycin（97%），and amikacin（89%）was reported［11，28，29］.In total，77%and 23%of MRSA isolates were susceptible to ciprofloxacin and nalidixic acid，respectively，which is not in agreement with other reports that demonstrated 100%and93%ofMRSAisolatesweresusceptibleto ciprofloxacin and nalidixic acid，respectively［9，11］.The MRSA in the current work revealed a 54%resistance to erythromycin，which is in agreement with other reports［11，28，30］.The MRSA susceptibilitytotetracyclinewas46%，whichisnotin agreement with other studies that reported all MRSA isolates were susceptible to tetracycline［9，11］.The lowest resistance was observed against azithromycin（15%）and 46%of MRSA isolates were resistant to amoxicillin-clavulanic acid，which is in contrast with another study in Korea，wherein 97%and 98% ofMRSAisolateswereresistanttoazithromycinand amoxicillin-clavulanicacid，respectively［29］.Gentamicin，ciprofloxacin，azithromycin，amoxicillin-clavulanic acid，and tetracycline are human medicines，and the emergence of strainsresistant to these antibiotics demonstrates the potential public health risk of MRSA.

The resistance patterns of MSSA are significantly less than those of MRSA（P＜0.001），except for trimethoprimsulphamethoxazole，which is similar.All MSSA isolates were susceptible to all antimicrobial agents tested in this study in a range of 69%-100%，and this is in agreement with another study wherein multi-drug resistance was found to be less common amongst MSSA isolates［30］.In the current study，the MSSA isolates showed no resistance to ciprofloxacin，azithromycin，or trimethoprim-sulphamethoxazole，followed by aminoglycoside and chloramphenicol.These results are similar to the findings of another report in India and are in contrast to the results of a UK study that stated all MSSA isolates were susceptible to gentamicin and tetracycline［30，31］.

ThediscrepanciesofMRSAantibioticsusceptibilityresultsmay be due to the use of different methods for susceptibility testing，different breakpoints for the evaluation of the results，and the misuse of antimicrobial agents，resulting in microbial fitness［27］.

Two of five human MRSA isolates showed significantly higher oxacillin MICs than those in dog MRSA isolates（Table 4）.This may be due to the high and uncontrolled use of antimicrobial agents in human medicines in Jordan.In addition，the authors'observations indicated that dogs included in this study were less exposed to antimicrobial treatment.In case of MRSA with oxacillin MIC≥8μg/mL，multiple-drug resistance was observed.

PFGE revealed five major clusters designated as pulsed-field types:Jordan A，Jordan B，Jordan C，Jordan D，and Jordan E. These clusters showed a percentage of similarities among different isolates.Isolates in clusters Jordan A，Jordan B，Jordan D，and Jordan E showed that the genetic relatedness between dog and human MRSA isolates were as follows.In cluster Jordan A，the relatedness between MRSA isolates from dogs and those from humans was 80%-90%，and in clusters Jordan B and Jordan E，the relatedness was 100%.In cluster Jordan D，the relatedness was 80%-90%.This may be because most human and dog MRSA isolates were collected from the same rearing centers where dogs and personnel are in daily close contact for months or even years.MRSA isolates in the Jordan E cluster are from one dog and his owner in Swelieh city.These results agree with other study findings that MRSA isolate relatedness is high when dogs and humans share the same place［32］.The MRSA isolates from either dogs or humans shared mostly similar antibiotic resistance patterns，and this agreed with another study［32］.Based on these data，we assume that the dogs are colonized with the same strain or clone as their owners or strongly associated personnel.This is in agreement with other reports about the possibility of MRSA transmission between dogs and humans［3，9，32，33］.The current study concludes and confirms the presence of MRSA in the nasal cavities of dogs，dog owners，and personnel strongly associated with dogs in Jordan，and that MRSA is highly prevalent in household dogs rather than in stray dogs.A genetic relatedness among the MRSAisolatesofdogsandthoseofhumanswas demonstrated，suggestingastrongpossibilityofMRSA transmission between them，and MRSA isolates showed a high rate of multi-resistance compared to MSSA isolates.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

This research was funded by the Deanship of Research at the Jordan University of Science and Technology（Project No.50/ 2009）.

［1］Chambers HF，Deleo FR.Waves of resistance:Staphylococcus aureus in the antibiotic era.Nat Rev Microbiol 2009；7（9）:629-41.

［2］European Center for Disease Prevention and Control.Annual report of European antimicrobial resistance surveillance network（EARSNet）2010.Stockholm:European Center for Disease Prevention and Control；2011.［Online］Available from:http://repositorio.insa.pt/ handle/10400.18/702［Accessed on 26th January，2015］

［3］Morris DO，Lautenbach E，Zaoutis T，Leckerman K，Edelstein PH，Rankin SC.Potential for pet animals to harbor methicillin-resistant Staphylococcus aureus when residing with human MRSA patients. Zoonoses Public Health 2012；59（4）:286-93.

［4］Leonard FC，Markey BK.Methicillin-resistant Staphylococcus aureus in animals:a review.Vet J 2008；175（1）:27-36.

［5］Loeffler A，Lloyd DH.Companion animals:a reservoir for methicillin-resistant Staphylococcus aureus in the community？Epidemiol Infect 2010；138（5）:595-605.

［6］Bramble M，Morris D，Tolomeo P，Lautenbach E.Potential role of pet animals in household transmission of methicillin-resistant Staphylococcus aureus:a narrative review.Vector Borne Zoonotic Dis 2011；11（6）:617-20.

［7］Hanselman BA，Kruth S，Weese JS.Methicillin-resistant staphylococcal colonization in dogs entering a veterinary teaching hospital.Vet Microbiol 2008；126（1-3）:277-81.

［8］Gandolfi-Decristophoris P，Regula G，Petrini O，Zinsstag J，Schelling E.Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs.J Vet Sci 2013；14（4）:449-56.

［9］Morris DO，Boston RC，O'Shea K，Rankin SC.The prevalence of carriageofmethicillin-resistantStaphylococcibyveterinary dermatology practice staff and their respective pets.Vet Dermatol 2010；21（4）:400-7.

［10］Shetty V，Trumbull K，Hegde A，Shenoy V，Prabhu R，K S，et al. Prevalence of community-acquired methicillin-resistant Staphylococcus aureus nasal colonization among children.J Clin Diagn Res 2014；8（12）:DC12-5.

［11］El-Jalil HA，Jallad M，Thwaini AJ.Nasal carriage of methicillin resistant Staphylococcus aureus in individuals exposed and not exposed to hospital environments.Eur J Sci Res 2008；22（4）:570-4.

［12］Al-Zu'bi E，Bdour S，Shehabi AA.Antibiotic resistance patterns of mecA-positive Staphylococcus aureus isolates from clinical specimens and nasal carriage.Microb Drug Resist 2004；10（4）:321-4.

［13］Alzoubi KH，Hayajneh WA，Ayoub AM，Al-Safi SA，A-azzam SI，Mhaidat NM.Prevalence of methicillin-resistant Staphylococcus aureus（MRSA）at a tertiary hospital in North Jordan.Jordan J Pharm Sci 2010；3（1）:37-43.

［14］Forbes BA，Sahm DF，Weissfeld AS.Baily and Scott's diagnostic microbiology.12th ed.Amsterdam:Elsevier Mosby；2007，p.1056.

［15］da Silva WP，Silva JA，de Macedo MRP，de Ara´ujo MR，Mata MM，Gandra EA.Identification of Staphylococcus aureus，S. intermedius and S.hyicus by PCR amplification of coa and nuc genes.Braz J Microbiol 2003；34（Suppl 1）:125-7.

［16］Clinical and Laboratory Standards Institute.Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals；approved standard.3rd ed.Wayne:Clinical and Laboratory Standards Institute；2008.［Online］Available from: http://www.docin.com/p-64884924.html［Accessedfrom13th February，2015］

［17］Clinical and Laboratory Standards Institute.Surveillance for methicillin-resistant Staphylococcus aureus：principles，practices，and challenges；a report.Wayne:Clinical and Laboratory Standards Institute；2010.［Online］Available from:http://shop.clsi.org/ microbiology-documents/M55.html［Accessed on 7th May，2010］

［18］Rowe F，Vargas Superti S，Machado Scheibe R，Dias CG.Agar diffusion，agar dilution，Etest，and agar screening test in the detection of methicillin resistance in staphylococci.Diagn Microbiol Infect Dis 2002；43（1）:45-8.

［19］Clinical and Laboratory Standards Institute.Performance standards for antimicrobial susceptibility testing；M100-S18 informational supplement.Wayne:Clinical and Laboratory Standards Institute；2008.

［20］Ouchenane Z，Agabou A，Smati F，Rolain JM，Raoult D.Staphylococcal cassette chromosome mec characterization of methicillinresistant Staphylococcus aureus strains isolated at the military hospital of Constantine/Algeria.Pathol Biol Paris 2013；61（6）: 280-1.

［21］Mulvey MR，Chui L，Ismail J，Louie L，Murphy C，Chang N，et al. Development of a Canadian standardized protocol for subtyping methicillin-resistant Staphylococcus aureus using pulsed-field gel electrophoresis.J Clin Microbiol 2001；39（10）:3481-5.

［22］Boost MV，O'Donoghue MM，James A.Prevalence of Staphylococcus aureus carriage among dogs and their owners.Epidemiol Infect 2008；136（7）:953-64.

［23］LoncaricI，K¨unzelF，LickaT，SimhoferH，SpergserJ，Rosengarten R.Identification and characterization of methicillinresistant Staphylococcus aureus（MRSA）from Austrian companion animals and horses.Vet Microbiol 2014；168（2-4）:381-7.

［24］Weidenmaier C，Goerke C，Wolz C.Staphylococcus aureus determinants for nasal colonization.Trends Microbiol 2012；20（5）: 243-50.

［25］Jordan D，Simon J，Fury S，Moss S，Giffard P，Maiwald M，et al. Carriage of methicillin-resistant Staphylococcus aureus by veterinarians in Australia.Aust Vet J 2011；89（5）:152-9.

［26］Zhang W，Hao Z，Wang Y，Cao X，Logue CM，Wang B，et al. Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and veterinary staff in China.Vet J 2011；190（2）:e125-9.

［27］Umber JK，Bender JB.Pets and antimicrobial resistance.Vet Clin North Am Small Anim Pract 2009；39（2）:279-92.

［28］RajaduraipandiK，ManiKR，PanneerselvamK，ManiM，Bhaskar M，Manikandan P.Prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus:a multicentre study.Indian J Med Microbiol 2006；24（1）:34-8.

［29］Kim HB，Jang HC，Nam HJ，Lee YS，Kim BS，Park WB，et al. In vitro activities of 28 antimicrobial agents against Staphylococcus aureus isolates from tertiary-care hospitals in Korea:a nationwide survey.Antimicrob Agents Chemother 2004；48（4）:1124-7.

［30］Al-Khulaifi Manal M，Amin Aref Nagwa M，Al Salamah AA. Phage typing，PCR amplification for mecA gene，and antibiotic resistance patterns as epidemiologic markers in nosocomial outbreaks of methicillin resistant Staphylococcus aureus.Saudi J Biol Sci 2009；16（1）:37-49.

［31］Marchese A，Gualco L，Maioli E，Debbia E.Molecular analysis and susceptibility patterns of methicillin-resistant Staphylococcus aureus（MRSA）strains circulating in the community in the Ligurian area，a northern region of Italy:emergence of USA300 and EMRSA-15 clones.Int J Antimicrob Agents 2009；34（5）:424-8.

［32］McDougalLK，StewardCD，KillgoreGE，ChaitramJM，McAllister SK，Tenover FC.Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States:establishing a national database.J Clin Microbiol 2003；41（11）:5113-20.

［33］Loeffler A，Pfeiffer DU，Lloyd DH，Smith H，Soares-Magalhaes R，Lindsay JA.Methicillin-resistant Staphylococcus aureus carriage in UK veterinary staff and owners of infected pets:new risk groups.J Hosp Infect 2010；74（3）:282-8.

27 Mar 2015

Yaser Hamadeh Tarazi，Department of Basic Veterinary Medical Sciences，Faculty of Veterinary Medicine，Jordan University of Science and Technology，P.O.Box 3030，Irbid，Jordan.

Tel:+962 795423348

Fax:+962 2 7201081

E-mail:tarazi@just.edu.jo

Peer review under responsibility of Hainan Medical University.

Foundation Project:Supported by the Deanship of Research at the Jordan University of Science and Technology（Project No.50/2009）.

inrevisedform24Jun2015