Phenotypic methods for determination of methicillin resistance in Staphylococcus spp . from health care workers

Introduction: Staphylococcus spp. is an important healthcare-associated pathogen and the identification of methicillin-resistant strains in samples of colonization may provide data to assist in the antimicrobial therapy success. Objectives: To determine the occurrence of colonization by methicillin-resistant Staphylococcus spp. (MRS), through the detection of the mecA gene and to evaluate different phenotypic methods for the presumptive detection of methicillin resistance in samples of the anterior nasal cavity and hands of the health care personnel of a university hospital in the state of Pernambuco, Brazil. Methods: We selected the 28 isolates of Staphylococcus spp ., which showed an intermediate or resistant phenotypic profile for oxacillin, detected by the Kirby Bauer technique. The methods used were disk-diffusion tests for cefoxitin, minimal inhibitory concentration by E-test for oxacillin, screening for oxacillin resistance and mecA gene detection by polymerase chain reaction (PCR). Results: About the phenotypic methods utilized, only the E-test of oxacillin did not show a statistically significant difference in relation to PCR for the mecA gene detection, considered the gold standard. Conclusion: The E-test of oxacillin was the best of the phenotypic methods utilized. It is necessary to correctly detect MRS in healthy individuals, because they can act as carriers and can therefore be a potential source of microorganisms involved in hospital infections.


introDuCtion
In the last decade, Staphylococcus has emerged as predominant microorganism in nosocomial infections (6,21) .The main staphylococcal infections reported include folliculitis and furuncles and even serious toxic shock syndrome and sepsis (6,18,21,22) .Coagulase-negative staphylococci (CNS) can be found in the normal microbiota of the skin and in mucosal membranes and have received special attention as potent pathogens, specifically in catheter-related bacteremias (6,27,28) .
At the end of the 90, the main change in antimicrobial resistance was the increase in resistance to methicillin among Staphylococcus aureus and CNS in all regions.Resistance to methicillin in Staphylococcus results resistance to all beta-lactam antibiotics, leaving few therapeutic options (21) .High rate of isolation of staphylococci resistant to methicillin leads to the need to use on a large scale more expensive or toxic antimicrobials such as vancomycin (7,21,26,27) .
Methicillin resistance in Staphylococcus is made by two main mechanisms: the betalactamase production and/or the presence of the PBP2a.This latter mechanism encoded by SCCmec cassette, a transposon, is the most important and worrying cause of transfer between staphylococcal species.The transposon SCCmec has two components, the mec to resistance and the ccr to mobility of the transposon, and they are used to classify the SCCmec types.There are others antibiotics groups of resistance in some types of SCCmec, which makes a multidrug resistant strain.The common element between SCCmec types is the mecA gene that encodes the PBP2a, therefore the detection of this gene is considered the gold standard for methicillin resistance detection (2,34) .There is a homologue of this gene, the mecA LGA251 , described in cattle and, more recently, in clinical samples from patients in Germany, which can be ambiguous the mecA gene detection in some places (10) .Moreover the detection of the mecA gene by polymerase chain reaction (PCR) requires inaccessible and sophisticated equipment to most laboratories.
The Clinical and Laboratory Standard Institute (CLSI) standardized the disk diffusion method for cefoxitin as a presumptive method of the mecA gene, but the phenotypic expression of this gene is still a challenge for microbiology laboratories (2,3,9,24,28) due to the discrepancies observed between laboratories that participate in the Antimicrobial Surveillance Program (23) .Currently, one of the main objectives for the control of hospital infections is the rational use of antimicrobials, which makes the evaluation of the accuracy of phenotypic methods used for determining the susceptibility profile essential for guaranteeing the most appropriate choice of therapeutic antimicrobials (19) .
Another important factor in the context of nosocomial infections is the transmission chain in the hospital environment (27) , in which health care professional are included as disseminating sources of this microorganism (5,32) .Thus, considering that Staphylococcus spp.are an important pathogenic agent associated with nosocomial infections and that studies about the colonization of health care workers are scarce, our objectives were to determine the occurrence of colonization by methicillin-resistant Staphylococcus spp., through the detection of the mecA gene and to evaluate different phenotypic methods for the presumptive detection of methicillin resistance in samples of the anterior nasal cavity and hands of the health care personnel from a university hospital in the state of Pernambuco, Brazil.

Biological samples
A cross-sectional study, in which, the biological samples of hands and anterior nasal cavity of 202 health workers from surgical clinics, intensive care units (IUCs), hemodyalisis and nephrology services, of the Hospital das Clínicas of the Federal University of Pernambuco, Brazil, were collected in the period of March to July 2007.This study was approved by the Ethics Committee on Research of the Federal University of Pernambuco -CAAE nº 0275.0.172.000-06.A sample of each anatomical site, from each professional, was obtained using sterile and dry swabs and, taken to the bacteriology laboratory, in tubes containing brain heart infusion (BHI) and cultivated in agar sheep blood 5% and incubated at 35 o C for 24 hours.Staphylococcus spp.suspect colonies were analyzed by Gram staining, catalase test and coagulase tests, growth in mannitol salty agar and DNAse activity; novobiocin proof for coagulase negative Staphylococcus.The antibiogram was performed by disc diffusion test (9) , the isolates selected were that showed resistance or intermediate profile to oxacillin and/or cefoxitin, that are considered presumptive techniques by CLSI, of low cost and routine use for methicilin resistance detection.So that, were used 28 isolates for presumptive detection of mecA and PCR.The mecA gene were detected by PCR.
For quality control in these tests, the standard strain ATCC 33591 S. aureus was used for the positive control (MRSA) and the standard strain ATCC 29213 (MSSA) for the negative control.

Methicilin and oxacillin susceptibility
The susceptibility to methicillin was tested by disk-diffusion method: the Staphylococcus isolates were cultivated in 5% blood agar for 24 hour at 35ºC, and subsequently, colonies were suspended in saline to a turbidity equivalent to 0.5 on the McFarland scale (10 8 CFU/ml).The bacterial suspension obtained was seeded in a homogeneous manner, with the help of a sterile swab, on the surface of plates containing Mueller-Hinton agar.Next, with the aid of flamed tweezers, a 30 µg cefoxitin disk was placed on the surface, where an inhibition halo ≤ 21 mm for S. aureus and ≤ 24 mm for CNS was considered the presumptive test (9) .

Determination of the minimal inhibitory concentration (MIC) by the oxacillin E-test
Cultures of S. aureus and CNS were grown in nutrient agar plates, and picked colonies were placed in a tube containing sterile saline (0.85% NaCl) to a turbidity of 0.5 on the McFarlands scale (9) .The suspension obtained was evenly seeded with the help of a sterile swab, on the surface of Mueller-Hinton agar plates, after which oxacillin strips were placed on the surface (E-test -Probac do Brazil ® ), using flamed tweezers.The plates were then incubated at 35ºC for 24 hour.The MIC was determined following the manufacturer's specifications, corresponding to the standards proposed by CLSI (9) .

Screening for oxacillin resistance
Colonies from subcultures on nutrient agar plates were resuspended to obtain a turbidity equivalent to 0.5 on the McFarland scale.A platinum wire loop of 1 μl was dipped in the suspension, and the bacteria were seed in an area with a diameter of 10 to 15 mm in plates with Mueller-Hinton agar medium containing NaCl (4% v/v; 0.68 mol/l) and 6 μg/ml oxacillin.These plates were incubated at 35ºC for 24 hour and then read, considering that > 1 colony = resistance (9) .

Deoxyribonucleic acid (DNA) preparation and identification by PCR of the mecA gene
Total DNA was extracted from individual colonies after growth in brain heart infusion (BHI) broth for 24 hour at 37ºC, following the protocol described by Freitas et al. (16) .PCR was performed utilizing the primers described by Petinaki et al. (29) .The amplification reaction mixture was prepared in a total volume of 25 µl containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl 2 , 200 mM dNTP (Promega), 20 pmol of each primer, 20 ng of genomic DNA, 1U Taq DNA polymerase (Promega).The reactions were carried out in a thermocycler (Biometra), programmed initially for 30 thermal cycles, with denaturation of 1 min at 94ºC, annealing of 1 min at 50ºC and extension of 1 min at 72ºC, followed by a final step of 10 minutes at 72ºC.The negative control contained all the components of the reaction mixture except DNA.The ATCC 33591 S. aureus was used for the positive control (MRSA).The amplification product was submitted to 1% agarose gel electrophoresis with ethidium bromide staining, and visualized in a UV transilluminator and digitized (Kodak Digital Science).

Statistical analysis
Differences between the susceptibility methods and the level of significance of the results were calculated by the chi-squared test and Fisher's exact test.p < 0.05 was considered statistically significant.Validity tests including sensitivity, specificity, and positive and negative predictive values were calculated.Sensitivity was defined as the percentage of mecApositive isolates determined to be non-susceptible by phenotypic testing, and specificity was defined as the percentage of mecA-negative isolates determined to be susceptible by phenotypic methods.

rESuLtS
It was obtained 404 samples of 202 health professionals, in which 14.6% (59/404) were S. aureus isolates and 6.2% (25/404) were classified as S. epidermidis by the novobiocin test.Thus, after the susceptibility testing, twenty-eight (14%) Staphylococcus spp.isolates (14 isolates of S. aureus and 14 S. epidermidis) were selected from the 202 health care professionals, which showed a resistant or intermediate phenotypic profile to methicillin by the disk-diffusion method.
The presumptive tests of detection of the mecA gene showed that 71.43% (20/28) of these were from the neonatal intensive care unit (ICU), where 57.14 % (16/28) of the total samples examined were from the professional class of nursing aids and technicians and mecA gene was detected in 15 isolates (nine S. epidermidis and six S. aureus), as can be seen in the Table 1.
Of the phenotypic methods utilized, only the oxacillin E-test did not show a statistically significant difference in relation to the gold standard (PCR).
The results of the validity tests for all the techniques studied are presented in Table 2. Screening for oxacillin sensitivity showed better results in relation to the other drugs, and the E-test showed the best specificity.Similarly, screening for oxacillin resistance and the disk-diffusion test for cefoxitin showed 11 false positives, while the oxacillin E-test showed five false negatives.

DiSCuSSion
The majority of colonized health care professionals, are transitory carriers, but can become persistent carriers, especially when they have skin lesions.The preventive strategy would be the identification and treatment of these colonized individuals, who could act as a reservoir in endemic situation (5,18) .Various investigators have proposed methods aimed at providing accuracy in the determination of MRS colonization status (15,20) .
Screening for oxacillin susceptibility can be utilized for the detection of Staphylococcus resistance, generally with very good accuracy (9,24) .The advantage of the test is the economic reasons and in diagnostic laboratories due to its reproducibility and cost-effectiveness (31) .
Several studies have demonstrated PCR as a sensitive method for the detection of resistance in staphylococci (1,11,24,25) .With the advantage of PCR over phenotypic methods with rapid turnaround time, measures to control infection can be taken more quickly (31) .Some studies have shown discrepancies in accuracy between the phenotypic tests and molecular detection of the mecA gene by PCR, considered the gold standard method (3,24,30) .
In our study, the oxacillin E-test was shown to be the most efficacious in the phenotypic presumptive detection of the mecA gene, when compared to PCR.In some studies (14,31) , the presence of the mecA gene was correlated with E-test, which showed variations in the conditions of analysis or pointed to other mechanisms utilized by bacteria for resistance to methicillin, for example betalactamases production (10,29,34) .
By other hand, in another study, the cefoxitin disk diffusion test was the most effective among those analyzed (33) .For the routine detection of methicillin resistance in Staphylococcus spp., the diskdiffusion tests are the most widely utilized, where the cefoxitin disk test is preferred than the oxacillin disk test to detect this resistance, because the latter is comparable in accuracy and shows a better reading (9) .Cefoxitin is regarded as the most reliable way to identify even strains that show a level of methicillin resistance, which is not possible using a 1-µg methicillin disk (28,34) .In our study, the methicillin disk was utilized as selection criterion of the samples, showing in this manner false positive samples when compared to PCR results, reinforcing the possibility of other mechanisms of resistance.
Antunes et al. (3) considered that the treatment of staphylococcal infections depends on the detection of methicillin resistance, usually done using drug susceptibility tests with oxacillin/methicillin disks, but as the results of these assays are inconsistent, complications during the treatment of these infections is to be expected due to inadequate therapy.A false-positive result would lead to an increase in the costs and unnecessary use of some last generation antibiotics, for example, vancomycin (8) .

ConCLuSion
To evaluate a prevalence of MRS among health care professionals, it is important to determine preventive measures against hospital infections, including making these professionals aware of precautionary measures for the control of infections, considering them as a potential source of infection, especially those colonized by MRSA.
The oxacillin E-test for phenotypic detection of resistance to methicillin combined with PCR for detection of the mecA gene, which encodes PBP2a, is recommended for the correct determination of the susceptibility to methicillin in samples of staphylococci.The rapid and correct identification of the gene of methicillin resistance assures the appropriate planning of antibiotic therapy and of epidemiological control measures for MRS.

ACknowLEDgEMEntS
This work was supported by Fundação de Amparo Ciência do Estado de Pernambuco (FACEPE) grant nº APQ 0579-2.12/07.According to some authors, due to the existence of other mechanisms of resistance, the test to determine the MIC of oxacillin for staphylococci by the agar gradient diffusion test (E-test ® ) should be utilized as a confirmatory and/or adjuvant test on bacterial isolates in which the susceptibility pattern is doubtful or difficult to interpret (33) .Other authors believe that the detection of methicillin resistance in coagulase-negative Staphylococcus is a challenge for clinical laboratories because many false negative results can be attributed to heterogeneous resistance to methicillin expressed by these microorganisms (8,15,27) .Baddour and coworkers (4) suggest the combination of two phenotypic methods for greater sensitivity.
In cases of heterogeneous resistance, subpopulations of resistant and sensitive S. aureus can coexist in the same culture, because all bacteria with resistance genes in their genomic DNA have been shown to express them in routine susceptibility tests performed in the laboratory (7) .

tABLE 1 -
Phenotypic test results and mecA gene detection

tABLE 2 -
Validity test results (PCR is the gold standard)Phenotypic methods for determination of methicillin resistance in Staphylococcus spp.from health care workers PCR: polymerase chain reaction; PPV: positive predictive value; NPV: negative predictive value.