Laboratory detection methods for methicillin resistance in coagulase negative Staphylococcus isolated from ophthalmic infections.
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.
Oliveira
Adália Dias Dourado
AD
Departamento de Oftalmologia, Universidade Federal de São Paulo, São Paulo, SP, Brasil. adalia@uol.com.br
d'Azevedo
Pedro Alves
PA
de Sousa
Luciene Barbosa
LB
Viana-Niero
Cristina
C
Francisco
Waldemar
W
Lottenberg
Cláudio
C
Martino
Marines Dalla Valle
MD
Höfling-Lima
Ana Luisa
AL
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
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