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Beta-Lactamase Detection in Staphylococcus Aureus and Coagulase-Negative Staphylococcus Isolated from Bovine Mastitis1

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Beta-Lactamase Detection in Staphylococcus Aureus and Coagulase-Negative Staphylococcus Isolated from Bovine Mastitis1 Pesq. Vet. Bras. 34(4):325-328, abril 2014 Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis1 2 2 2 2 2* Bruno F. Robles , Diego B. Nóbrega , Felipe F. Guimarães , Guido G. Wanderley ABSTRACT.- and H. Langoni Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylo- coccus isolatedRobles from B.F., bovine Nóbrega mastitis. D.B., Guimarães Pesquisa Veterinária F.F., Wanderley Brasileira G.G. &34(4):325-328. Langoni H. 2014. De- partamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista, Distrito de Rubião Junior s/n, Botucatu, SP- 18618-900, Brazil. E-mail: [email protected] bacteriaThe objectives type (Staphylococcus of the study aureus were to versus evaluate coagulase-negative the presence/production Staphylococcus of beta-lactama - CNS) and ses by both phenotypic and genotypic methods, verify whether results are dependent of- S. aureus. verify the agreement between tests. A total of 200 bacteria samples from 21 different her ds were enrolled, being 100 CNS and 100 Beta-lactamase presence/detectionS. aureuswas performed byS. aureus different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro- resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or - ). Several beta-lactamase producing isolates were from the same herd. Phe notypic tests excluding in vitro resistance to penicillin showed a strong association measu red by the kappa coefficient for bothblaZ bacteria, coagulase-negative species. Nitrocefin Staphylococcus and CLT are, Staphylococcus more reliable aureustests for detecting beta-lactamase production in staphylococci. INDEX TERMS: Beta-lactamase, RESUMO.- [Detecção . de beta-lactamase em Staphylo- coccus aureus e Staphylococcus coagulase negativa iso- lados de mastite bovina.] Os objetivos do presente estu- dutores de beta-lactamase eram de um mesmo rebanho. do foram avaliar a presença/produção de beta-lactamases Testes fenotípicos excluindo resistência in vitro à penicilina mostraram uma forte associação medida pelo coeficiente se os resultados são dependentes do tipo de bactéria (Sta- kappa para ambas as espécies de bactérias. Nitrocefina e por ambos os métodos fenotípicos e genotípicos, verificar CLT são testes mais confiáveis para detectar a produção de phylococcus aureus contra Staphylococcus coagulase nega- blaZ, Staphylococcus coagulasebeta-lactamase negativa em, Staphylococcus estafilococos. aureus. total de 200 amostras bactérianas oriundas de 21 rebanhos TERMOS DE INDEXAÇÃO: Beta-lactamase, distintostiva - CNS) foram e verificar incluídos, a concordância sendo 100 CNS entre e 100 os S.testes. aureus Um INTRODUCTION presença/detecção de beta-lactamase foi realizada por di- ferentes testes (PCR, teste trevo (clover leaf test) - CLT, disco. A Staphylococcus aureus (S. aureus) is one of the most com- de todos os testes não foram dependentes do tipo de bac- Staphylococcus (CNS) are com- tériasNitrocefin (CNS e ouresistência S. aureus in vitro à penicilina). S.Os aureus resultados pro- monlymon causes isolated of contagious from mastitis bovine cases mastitis in several (Melchior countries et al. 2006). Coagulase-negative - 1 ). Vários isolados de (Pitkala et al. 2004, de Freitas Guimarães et al. 2013), with 2 Received on January 4, 2014. limited knowledge and published papers regarding antimi Accepted for publication on March 1, 2014. crobialS. aureus, resistance CNS can mechanisms. harbor resistance In recent genes years, to several treatment an- Departamento de Higiene Veterinária e Saúde Pública, Universidade of CNS-caused infections has become an important topic. Estadual Paulista (Unesp), Distrito de Rubião Júnior s/n, Botucatu, SP As 18618-900, Brazil. *Corresponding author: [email protected] timicrobials (Hammad et al. 2012, Silva et al. 2013). 325 326 Bruno F. Robles et al. Table 1. Absolute (n), relative (%) and cumulative (Cf) one of the most frequently used drugs in veterinary medici- frequencies of coagulase-negative Staphylococcus (CNS) and Beta-lactam compounds such as penicillin continues to be Staphylococcus aureus isolates enrolled in the present study Staphylococcus according to herd number expressionne (Pitkala etof al.beta-lactamase 2007). Two primary enzymes resistance encoded mechanisms by the blaZ gene,to beta-lactams and production are noteworthy of the penicillin-binding in protein spp.: 2a there- CNS 1 11 11 11 S. aureus 1 - - - sulting in a higher-level of resistance encoded by the mecA Bacteria Herd N % Cf Bacteria Herd N % Cf in staphylococci causing animal diseases is most commonly 2 - - - 2 14 14 14 duegene to (Fuda the blaZ et al. 2005). Prevalence of penicillin resistance 53 26 26 1917 53 14 14 2018 64 11- 11- 30- 64 1 1 2119 Different tests can be performed to evaluate beta-lacta- gene (Malik et al. 2007, Pitkala et al. 2007). 8 15 15 53 8 - - - for detecting production of beta-lactamase is the usage of 7 8 8 38 7 - - - mase production in staphylococci. A qualitative procedure 119 1- 1- 54- 119 3- 3- 25- 1210 20- 20- 74- 1210 1 1 2622 Nitrocefin disks. The reaction is based on the production- of a colored compound when the substrate (nitrocefin) is exposed to a beta-lactamase-producing bacteria. The clo 1513 13- 13- 87- 1513 36 36 3632 ver leaf test (CLT) is an alternative with high sensitivity and 14 - - - 14 1 1 33 specificityStudies forregarding investigating beta-lactamase beta-lactamase production production in Sta in- 16 - - - 16 1 1 37 phylococcusstaphylococci (Bergan et al. 1997). 17 2 2 89 17 2 2 39 Comparison of beta-lactamase activity in coagulase positi- 2018 - - - 2018 591 591 10040 isolates and comparison of tests are scarce. 2119 11- 11- 100- 2119 1- 1- 41- Total 100 100 100 100 ve and negative isolates would prove to be valuable, adding presence/productionmore results to available of beta-lactamases literature. by both phenotypic The objectives of the present study were to evaluate the- S. aureus dent of bacteria type (coagulase positive versus coagulase al. 2005). ExtractionS. aureus and purification were performed by physical- methods (boiling and centrifugation) (Malik et al. 2007). negativeand genotypic Staphylococcus methods, verify whether results are depen- ATCCStatistical 29213 and analysis. 25923 were used as positive and nega tive control respectively. Staphylococcus ) and verify the agreement betwe- Descriptive analyses were performed en tests. Our hypotheses were that more coagulase-positive using the PROC FREQ procedure of SAS software (SAS 2008).- isolates would present beta-lactamase enzy Agreement between tests was achieved by Kappa coefficient and- mes and testsMATERIALS would have a highAND agreement METHODS coefficient. McNemar test. Differences in results frequencies regarding bacte Samples. ria type were determined by hierarchical models.P Farm and ani mal effect were first assessed using the chi-square test. Variables- and 100 StaphylococcusA total of aureus 200 bacteria samples from 21 different individually associated with result test at a -value ≤ 0.25 were herds located in São Paulo State were enrolled, being 100 CNS selected to build a final generalized mixed model. Farm was in (Table 1). Isolates were taken from cluded as an explanatory variable whereas animal did not enter cowsMicrobiological with diagnosed procedures.intramammary In infectionvitro antimicrobial (IMI) from 2009suscep to- the final models. To verify whether farm should be included as a 2012 and stored at -80°C. fixed or random effect, individual generalized mixed models were constructed for each result test including farm as a random effect. tibility testing was conducted by disc diffusion method using the A compound symmetry covariance structure was used to account two following antimicrobial agents: oxacillin and penicillin (Oxoid- for clustering of isolates from same farm. In cases of estimated V- Ltd., Basingstoke, Hampshire, England), and interpreted according correlation values ≥0.20, models were building considering farm- to theNitrocefin Clinical and® Disks. Laboratory Standards® Institute (CLSI 2008). Be- cillinas a randomsusceptibility effect. results, This applies the model to all hierarchical beta-lactamases structure tests assu re- ta-lactamases detection was performed by three methods. sults (PCR, clover leaf test and Nitrocefin disk), whereas to peni acquired and stocked at Nitrocefin-10o commercial disks (Becton Di- ckinson Microbiology Systems, Cockeysville, United States) were med farm as a fixed effect (estimated V correlation value = 0.16). C until usage. Disks were initially em Each final modelP provided the best fit for the data, assessed using bedded in saline solution, and with a sterile loop, colonies were- Akaike and Bayesian information criteria. Statistical significance streaked onto its surface. Disks wereS. aureus observed within 60 minutesS. aureus at was defined as ≤ 0.05. All statistical analyses were performed ambient temperature. A positive reaction was considered as a chan with the SAS 9.2 software. RESULTS ge ofClover colour Leaf from Test yellow (Hodge to pink. Test). ATCC 29213 and - PCR detection of blaZ 25923 were used as positive and negative control respectively. Staphylococcus aureus - The clover leaf test was per S. aureus gene was not performed in four isolates 29213formed andaccording S. aureus to Bergan et al. (1997). Presence of an irregular inhibition zone was considered as a positive result.
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