title: Laboratory detection methods for methicillin resistance in coagulase negative Staphylococcus isolated from ophthalmic infections.
abstract: OBJECTIVE
  To evaluate different methods of oxacillin susceptibility testing of ocular isolates, considering polymerase chain reaction (PCR) as the 'gold standard', and to compare the in vitro susceptibility to oxacillin with that of other antimicrobials used in ophthalmologic practice.
  
  
  METHODS
  The Vitek gram-positive identification card was used to identify ocular coagulase negative Staphylococcus species. The presence of the mecA gene was determined by the polymerase chain reaction assay with a combination of two primer sets (mecA and 16S rRNA) in a single region. Results were analyzed and compared with other oxacillin susceptibility methods: PBP2a detection by rapid slide latex agglutination test (SLA); oxacillin E-test; the Vitek automated gram-positive susceptibility card (GPS-105); the oxacillin salt agar screening test (OSAS) at a concentration of 6.0, 1.0 and 0.75 microg oxacillin per ml and the cefoxitin disk diffusion test (CDD). Automated susceptibility was also determined to other antimicrobial agents (fluoroquinolones, penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin and rifampin.
  
  
  RESULTS
  Of the 69 CoNS isolates tested, 71% were mecA-positive and 29% mecA-negative. All methods tested had a statistically significant agreement with polymerase chain reaction. There was a tendency of positive polymerase chain reaction predomination among the S. epidermidis isolates in comparison to non-epidermidis isolates, although this was not statistically significant (78.3% vs. 56.5%; chi2= 2.54; P= 0.11). The oxacillin salt agar screening test (0.75 microg oxacillin/ml) showed the best performance, with 100% sensitivity and negative predictive value; 95% specificity and 98% positive predictive value. Using the E-test, the mecA-positive isolates were statistically significantly more resistant to ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin (P= 0.002; P= 0.008; P= 0.002 and P= 0.003, respectively). There was a statistically significant higher proportion of resistance of the coagulase negative Staphylococcus mecA-positives for: penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin and tetracycline (P< or =0.05). All coagulase negative Staphylococcus species were susceptible to vancomycin and there was no statistically significant correlation between the mecA-positive isolates and resistance to trimethoprim-sulfamethoxazole or to rifampin.
  
  
  CONCLUSIONS
  In the present study, we found that the E-test and the oxacillin salt agar screening test S (0.75 microg oxacillin per ml), when compared with polymerase chain reaction, were the most accurate currently available methods to phenotypically detect oxacillin resistance of coagulase negative Staphylococcus species. This study demonstrated that a good option for screening of ocular isolates for oxacillin resistance in the microbiology laboratory is the cefoxitin disk diffusion test and the automated Vitek system. We believe it is important to have available methods that accurately detect methicillin resistance of the less commonly encountered species, chiefly because of their increasing importance as opportunistic pathogens.
authors: Departamento de Oftalmologia, Universidade Federal de São Paulo, São Paulo, SP, Brasil. adalia@uol.com.br
authors: Adália Dias Dourado
authors: AD
authors: Oliveira
authors: Pedro Alves
authors: PA
authors: d'Azevedo
authors: Luciene Barbosa
authors: LB
authors: de Sousa
authors: Cristina
authors: C
authors: Viana-Niero
authors: Waldemar
authors: W
authors: Francisco
authors: Cláudio
authors: C
authors: Lottenberg
authors: Marines Dalla Valle
authors: MD
authors: Martino
authors: Ana Luisa
authors: AL
authors: Höfling-Lima
mesh_terms: D000362:Agar; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002440:Cefoxitin / Q000494:pharmacology; D003030:Coagulase; D006801:Humans; D007841:Latex Fixation Tests; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods*; D010068:Oxacillin / Q000494:pharmacology; D046915:Penicillin-Binding Proteins; D010641:Phenotype; D016133:Polymerase Chain Reaction; D013203:Staphylococcal Infections / Q000382:microbiology; D013210:Staphylococcus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003030:Coagulase; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus; D002440:Cefoxitin; D000362:Agar; D008712:Methicillin; D010068:Oxacillin