Phenotypic detection of nosocomial mecA-positive coagulase-negative staphylococci from neonates

M. De Giustia, L. Pacificob,c, D. Tufia, A. Panerob, A. Bocciaa and C. Chiesab,c,*

a Department of Experimental Medicine, Hygiene Chair b Institute of Pediatrics, La Sapienza University of Rome c Institute of Experimental Medicine, National Research Council, 00161-Rome, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over a 3-year period, we screened antimicrobial resistance genotype (mecA-positive or -negative) in clinically significant coagulase-negative staphylococci isolated from patients residing in our neonatal intensive care unit. For the 152 study strains, the accuracy of standard methods (agar dilution MIC, disc diffusion and agar screen tests) in detecting oxacillin resistance during 48 h of incubation was evaluated. Using mecA gene PCR and Southern blot hybridization as the gold standard, the differential in MICs of additional antibiotics selected for their relevant clinical use in our setting was also compared with mecA status of the isolates. The frequency of mecA was 48.6% among study strains. When applying the previous (1998) and most current (1999) NCCLS interpretive criteria, the specificities of oxacillin agar dilution MICs in detecting the 78 mecA-negative isolates were 100 and 89.7%, respectively, at 24 h, and 100 and 80.7%, respectively, at 48 h. In this respect, the sensitivities of oxacillin agar dilution MICs in detecting the 74 mecA-positive strains were 75.6 and 97.2%, respectively, at 24 h, and 86.4 and 100%, respectively, at 48 h. When applying the previous and most current NCCLS zone size interpretive criteria, oxacillin zone diameters were in false-susceptible error for 13.5 and 8.1%, respectively, of the 74 mecA-positive strains tested at 24 h, and for 6.7 and 2.7%, respectively, at 48 h. Accordingly, when the 78 mecA-negative strains were considered, oxacillin zone diameters were in false-resistant error for 2.5 and 8.9%, respectively, at 24 h, and for 8.9 and 15.3%, respectively, at 48 h. The oxacillin salt agar screen assay accurately identified all mecA-negative strains at both 24 and 48 h. However, 26 (35.1%) and 7 (9.4%) of the mecA-positive strains were misinterpreted as susceptible by the agar screen test at 24 and 48 h, respectively. Using the presence of mecA as the reference standard for interpreting oxacillin susceptibility results, strains lacking mecA were more likely to be susceptible to ampicillin, ceftazidime, gentamicin, netilmicin and rifampicin than were mecA-positive strains. Vancomycin was the only antibiotic tested for which all strains, regardless of mecA status, remained susceptible.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, coagulase-negative staphylococci (CoNS) have been recognized as a major cause of nosocomial infections. Isolates from hospitals and other health care settings are often resistant to the penicillinase-resistant penicillins oxacillin or methicillin, and this indicates cross-resistance to all classes of ß-lactam antibiotics including cephalosporins. Resistance to oxacillin can have a substantial impact on health care by adversely affecting morbidity and mortality, enhancing opportunity for disease transmission because of treatment failure and increasing costs. Nowhere is this phenomenon a potentially greater challenge than in the newborn intensive care unit (NICU) where the burden of nosocomial CoNS infections is as high or higher than in any other patient group, adult or pediatric.1 Furthermore, the nearly unrestricted use of penicillins, expanded-spectrum cephalosporins and aminoglycosides as empiric antibiotic therapy in neonates increases the risk of a shift toward more antibiotic-resistant CoNS strains.2

The frequent resistance of CoNS to multiple antibacterial agents has prompted the use of glycopeptides for empirical therapy and even prophylaxis. However, both the selection of glycopeptide resistance and the potential transmission of such resistance between species has led to calls for the restricted use of glycopeptides.3,45 In this setting, rapid and reliable identification of the resistance pattern in CoNS may allow more judicious use of glycopeptides. Standardized methods of susceptibility testing have been developed for this purpose. However, phenotypic expression of oxacillin resistance is usually heterogeneous. In addition, oxacillin resistance is frequently influenced by culture conditions such as inoculum size, incubation time, temperature, PH and salt concentration of the medium. These factors complicate the detection, especially for strains with low-level resistance, and emphasize the need to use a rapid, accurate and sensitive method for detection of oxacillin resistance which is not dependent on growth conditions. With the advent of molecular techniques, a new gold standard could be detection of the mecA gene. Although other mechanisms have been implicated, production of PBP 2a, a characteristic penicillin-binding protein with extremely low affinity for ß-lactam antibiotics, is by far the most common mechanism responsible for phenotypic expression of oxacillin resistance. The mecA gene encoding PBP 2a6,7,8 is absent from susceptible staphylococcal isolates and, unlike the protein PBP 2a, can be detected independently of growth conditions. Therefore, the mecA gene is considered a useful molecular marker of oxacillin resistance in all staphylococci. This has prompted several investigators to test different methods for the detection of mecA. In particular, the polymerase chain reaction (PCR) appears to be a rapid, sensitive and specific assay for such detection.9,10

In the present study, the application of PCR and Southern blot hybridization for screening clinically significant CoNS isolates from NICU patients provided an opportunity to evaluate the accuracy of standard methods for detecting oxacillin resistance and the usefulness of recently recommended oxacillin breakpoints. The survey also provided the opportunity to evaluate, among mecA-positive and -negative clinical CoNS isolates, the likelihood of susceptibility to additional antimicrobial agents selected for their relevant clinical use in this patient population.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All infants admitted to the NICU in 1995, 1996 and 1997 were included in the study. Neonates were eligible for the study if they remained hospitalized for >2 days (i.e. they were at risk of nosocomial infection). Cultures were taken only when indicated by clinical signs suggestive of infection, never for surveillance purposes. Common indications were apnea, bradycardia, respiratory deterioration (as manifested by increased requirements for respiratory support and oxygen), temperature instability, lethargy or gastrointestinal problems including abdominal distension and feeding difficulties. To obtain data that were independent of arbitrary clinical interpretation, cultures were considered as significant if they yielded a single morphological type or species of CoNS. To avoid introducing artifacts resulting from varying numbers of cultures obtained during the workup for a clinical episode of suspected nosocomial infection, only the first positive culture obtained in each workup was considered.11 This resulted in 152 consecutive isolates from potential cases of nosocomial infection. Bacteria were isolated from the following clinical specimens: blood (n = 73), indwelling intravascular catheters (n = 25), pleural fluid (n = 3), bronchoalveolar lavage (n = 45) and tracheal aspirates (n = 6).

Isolates were identified as CoNS by colony and microscopic morphology, Gram's stain characteristics, coagulase reactions and the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur, Marnes la Coquette, France). CoNS were identified to the species level with the ID 32 Staph Api System (bioMérieux, la Balme-Les Grottes, France) by following the manufacturer's instructions. Additional biochemical tests were used when necessary.12 All strains were stored at -70°C in skimmed milk (Difco Laboratories, Detroit, MI, USA). For testing, isolates were removed from storage, streaked onto a Trypticase soy agar plate supplemented with 5% sheep blood (Beckton Dickinson Microbiology Systems, Cockeysville, MD, USA) and incubated under aerobic conditions at 35°C for 24 h. An isolated colony was picked from each plate, streaked on to a new Trypticase soy agar plate and incubated for 24 h. All inocula for antimicrobial susceptibility testing were prepared from this subculture. The control strains used in this study were Staphylococcus aureus American Type Culture Collection (ATCC) 29213, ATCC 25923 and ATCC 43300, Streptococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922 and ATCC 35218.

Antimicrobial susceptibility testing

Five colonies were transferred into a tube containing 9 mL of Mueller–Hinton broth (Difco Laboratories) to prepare a suspension equivalent in density to that of a 0.5 McFarland barium sulphate standard. All three susceptibility tests were performed with the adjusted inocula. Quantitative subcultures demonstrated that the final inocula contained approximately 108 cfu/mL for oxacillin disc diffusion as well as for oxacillin agar screen plate procedure, and 104 cfu/spot for agar dilution tests. All testing was performed in duplicate. Antimicrobial agents, including oxacillin, ampicillin, ceftazidime, gentamicin, netilmicin, rifampicin, teicoplanin and vancomycin, were obtained as sterile powders from Sigma Chemical Co. (St Louis, MO, USA) or their respective manufacturers. Oxacillin discs were purchased from Becton Dickinson Microbiology Systems.

Agar dilution tests

Agar dilution MIC testing was performed using an inoculum-replicating device and according to NCCLS guidelines.13 Organisms were tested on Mueller–Hinton agar (Difco Laboratories) plates containing each drug tested in concentrations ranging from 0.125 to 256 mg/L. Oxacillin and ceftazidime plates were supplemented with 2% NaCl.13,14 Plates containing oxacillin were incubated at 30°C for 24 and 48 h; other plates were incubated at 35°C for 24 h.

Oxacillin disc testing

The disc test was performed as described by NCCLS15 with a 1 µg oxacillin disc and Mueller–Hinton agar without additional NaCl. The plates were incubated in ambient air at 35°C, with readings at 24 and 48 h of incubation. The resulting zone of inhibition was measured, and the plates were examined closely (with transmitted light) for evidence of colonies or a fine haze of growth within the zone of inhibition. Questionable colonies were confirmed by using either a hand magnifying lens or microscopically.

Oxacillin agar screen method

Agar screen tests for susceptibility to oxacillin were performed as described by NCCLS.13 For each isolate, Mueller–Hinton agar plates containing 4% NaCl and oxacillin (6 mg/L) were spot inoculated with a cotton swab that had been dipped directly into the colony suspension. Plates were then incubated at 35°C, with readings at 24 and 48 h of incubation. If individual colonies appeared or if a light haze of growth appeared, the strain was considered positive.

Detection of the mecA gene by PCR

Bacterial DNA was obtained as described by Murakami et al.9 On the basis of the DNA sequences of the mecA gene, the following two primers were designed to amplify staphylococcal DNA. One of the primers (5'-AAAATCGATGGTAAAGGTTGGC-3') corresponded to nucleotides 1282–1303, and the other (5'-AGTTCTGCAGT- ACCGGATTTGC-3') was complementary to nucleotides 1793–1814. These primers gave rise to a PCR product of 533 bp. To verify that the fragment amplified by PCR represented the mecA gene, an oligonucleotide probe for Southern hybridization, 5'-ATCTGTACTGGGTTAATC-3', corresponding to nucleotides 1581–1598 of the PBP 2a coding frame, was synthesized. The PCR amplification was carried out by using 5 µL of template DNA, each primer at 0.25 µM and the Gene Amp DNA amplification kit for PCR (Perkin-Elmer Cetus, Norwalk, CT, USA). Forty cycles were repeated as follows: denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min with a final extension at 72°C for 5 min. Ten microlitres of each of the amplified samples was analysed by agarose gel electrophoresis. Primers targeting the 16S rRNA gene were used as an internal control for each amplification reaction to identify potential false-negative results.10 Southern blot hybridization of PCR products was carried out as described previously.9 Positive control strains included S. aureus ATCC 43300, and negative control strains included S. aureus ATCC 25923 and ATCC 29213.

In-vitro selection for oxacillin resistance

In-vitro selection for oxacillin resistance was performed using the pour plate technique.16,17 Test isolates were grown in Mueller–Hinton broth overnight at 35°C to an approximate concentration of 1 x 109 cfu/mL. One hundred microlitres of inoculum (approximately 1 x 108 cfu/mL) was added to 15 mL of melted Mueller–Hinton agar cooled to 50°C. Oxacillin concentrations ranging from 0.06 to 256 mg/L were added, and the agar was vortexed and immediately poured into sterile Petri plates. After solidification at room temperature, colonies visible at 48 h of incubation in agar containing the highest concentrations of oxacillin were selected and grown in antibiotic-free Mueller–Hinton broth. The pour plate procedure was repeated until the concentration of oxacillin at which the organism grew did not increase during five passages. Colonies were selected from the plates containing the highest concentration of oxacillin, and MICs were determined by the agar dilution method as outlined above.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Four species of CoNS were found in the 152 clinical strains investigated. Staphylococcus epidermidis was the predominant species, accounting for 74% of all isolates. Other isolates were identified as Staphylococcus haemolyticus (11%), Staphylococcus hominis (11%) and Staphylococcus warneri (4%).

Antimicrobial resistance genotype (mecA status)

The mecA gene was found in all of the four CoNS species (Table I). Seventy-four (48.6%) of the 152 CoNS strains were found to carry the mecA gene on the basis of the hybridization analysis, and their distribution among the different species was as follows: S. epidermidis,55 (48.6%) of 113 strains; S. haemolyticus, nine (52.9%) of 17 strains; S. hominis, eight (50.0%) of 16 strains; and S. warneri, two (33.3%) of six strains.


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Table I. Distribution of mecA in 152 CoNS strains
 
Oxacillin agar dilution MICs

While this manuscript was under review, we learned that in January 1999 the NCCLS publication on performance standards for antimicrobial susceptibility testing included revised oxacillin MIC breakpoints for CoNS: >=0.5 mg/L (instead of >=4 mg/L18) indicated resistance,19 and <=0.25 mg/L (instead of <=2 mg/L18) indicated susceptibility.19 We therefore report the interpretation of oxacillin agar dilution MIC results obtained from the survey in terms of both the previous (1998) and revised (1999) NCCLS criteria.

Using the previous NCCLS MIC breakpoint (>=4 mg/L) for oxacillin resistance in CoNS,18 among the 74 mecA-positive isolates, 56 (75.6%) tested resistant at 24 h (Table II) with MIC50and MIC90 values of 64 and >=256 mg/L, respectively. Incubation for an additional 24 h showed no change in the MICs for these isolates. For the remaining 18 mecA-positive strains, eight (including three isolates from blood, two isolates from bronchoalveolar lavage and three from intravascular catheters) tested oxacillin-susceptible at 24 h but oxacillin-resistant at 48 h, while 10 [all of which were blood isolates identified as S. epidermidis(n = 6) or S. hominis (n = 4)] remained oxacillin-susceptible at 48 h, as judged by MICs of 1 or 2 mg/L (Table II). All 10 strains were also categorized as susceptible to ceftazidime.18 For the 78 mecA-negative strains, MICs of oxacillin remained <=2 mg/L at both 24 and 48 h of incubation (Table II).


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Table II. Comparison of presence of mecA with oxacillin MIC
 
To confirm that a susceptible CoNS isolate possessing the mecA genotype is capable of expressing oxacillin resistance, all of the 10 mecA-positive isolates (for which 48 h oxacillin MICs were <=2 mg/L) were studied for in-vitro expression of resistance to oxacillin. Ten randomly selected mecA-negative control strains (48 h MICs, 0.25–2 mg/L) were included. Oxacillin resistance, as defined by the previous NCCLS interpretive criteria,18 could be selected for all 10 mecA-positive strains. For these isolates, after five passages, oxacillin MICs increased during 48 h of incubation two- to six-fold into the resistant range, compared with MICs recorded for the original isolates after the same time of incubation. Results of the agar resistance selection method showed that three, two, two and three of the 10 mecA-positive strains developed 48 h oxacillin MICs of 8, 16, 32 and 64 mg/L, respectively. All these oxacillin-resistant laboratory-derived strains were identified as mecA-positive with a biochemical pattern as well as a colony morphology identical to the respective parent strains. In contrast, for the 10 mecA-negative clinical strains, despite five passages, 48 h MICs remained largely unaffected.

Retrospective review of treatment decisions related to these 10 mecA-positive strains with 48 h MICs (1 and 2 mg/L) falling into the `susceptible range' (as previously defined by NCCLS18), revealed that seven patients were treated with glycopeptides alone or in combination with other drugs, while the remaining three were treated with cephalosporins alone. The last subgroup of patients were all identified as being truly infected with S. epidermidis on the basis of clinical and ancillary laboratory (a positive sepsis screen,20 and demonstration of the repeat isolation of S. epidermidis from blood cultures taken on day 3 to 4 after initiation of therapy) studies. In these three patients, initial treatment with a 4- to 5-day course of cephalosporins resulted in no improvement (n = 1) or worsening (n = 2) of their clinical signs and symptoms. After the three neonates had received vancomycin, their clinical conditions improved rapidly and their blood cultures were sterile within 48 h. Sterilization of the blood cultures did not coincide with line changes or other identifiable factors.

When applying the updated NCCLS MIC breakpoint for detecting oxacillin resistance in CoNS, 72 of the 74 mecA-positive isolates were categorized as resistant (sensitivity, 97.2%) after 24 h of incubation (Table II). No false-susceptible error occurred after 48 h of incubation (sensitivity, 100%). Use of the new interpretive breakpoint MICs was also examined for the 78 mecA-negative strains. Of these, eight (specificity, 89.7%) and 15 (specificity, 80.7%) isolates were categorized as oxacillin-resistant at 24 and 48 h of incubation, respectively (Table II).

Oxacillin disc diffusion susceptibility results

The revised (1999) NCCLS documents recommended the following zone diameter breakpoints with a 1 µg oxacillin disc: <=17 mm (instead of <=10 mm18) indicates resistance, and >=18 mm (instead of >=13 mm18) indicates susceptibility of CoNS.19

When applying the previous NCCLS oxacillin zone-size interpretive criteria,18 among the 74 mecA-positive isolates, 64 (sensitivity, 86.4%) and 69 (sensitivity, 93.2%) strains tested as oxacillin-resistant by the disc diffusion method after 24 and 48 h of incubation, respectively. Among the 78 mecA-negative isolates, two (specificity, 97.4%) and seven (specificity, 91.0%) strains produced a resistant zone diameter after incubation for 24 and 48 h, respectively. None of the mecA-positive or -negative strains had zone sizes that classified them at either time of incubation in the intermediate category.

Using the most current NCCLS oxacillin disc diffusion interpretive criteria,19 six (sensitivity, 91.8%) and two (sensitivity, 97.2%) of the 74 mecA-positive isolates produced, respectively, a false-susceptible oxacillin disc test zone diameter after 24 and 48 h of incubation. Use of the new zone size interpretive criteria was also examined for the 78 mecA-negative strains. Of these, seven (specificity, 91.0%) and 12 (specificity, 84.6%) isolates produced a false-positive oxacillin disc test zone diameter at 24 and 48 h of incubation, respectively.

Oxacillin salt agar screen

The oxacillin salt agar screen plate method accurately identified all mecA-negative strains at 24 and 48 h. However, 26 (35.1%) and seven (9.4%) of the mecA-positive strains were misinterpreted as susceptible by the agar screen test at 24 and 48 h, respectively.

Agar dilution MICs of antimicrobial agents other than oxacillin

>All isolates were also tested for susceptibility to additional antimicrobial agents more widely used in our NICU (i.e. ampicillin, ceftazidime, gentamicin, netilmicin, rifampicin, teicoplanin and vancomycin) using the agar dilution MIC method. Interpretive criteria were those of the current NCCLS documents.18,19 Susceptibility profiles were tabulated for each of the above seven drugs according to mecAstatus of the isolates (Table III). Vancomycin was the only antimicrobial agent to which all isolates, regardless of mecA status, remained susceptible. The numbers of teicoplanin-nonsusceptible isolates were too few to permit comparison between mecA-positive and -negative strains. Of the four teicoplanin-nonsusceptible isolates, two were identified as mecA-positive (one S. haemolyticus and one S. epidermidis; teicoplanin MICs, 32 and 16 mg /L, respectively) and two as mecA-negative (one S. haemolyticus and one S. epidermidis; MICs, 32 and 16 mg /L, respectively). In contrast, the likelihood of susceptibility to the remaining five antimicrobial agents appeared to be related to the mecA status of the strains. A significantly higher percentage of strains lacking mecAwas found to be susceptible to ampicillin (23.0% versus 0; P < 0.0001, Fisher's exact test), ceftazidime (79.4% versus 16.2%; P< 0.0001), gentamicin (94.8% versus 27.0%; P< 0.0001), netilmicin (100% versus 62.1%; P < 0.0001) and rifampicin (97.4% versus 64.8%; P < 0.0001), compared with that of mecA-positive strains.


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Table III. In-vitro agar dilution susceptibility results to seven antibiotics according to mecA status of CoNS strains
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The reported prevalence of resistance to oxacillin or methicillin among disease isolates of CoNS has varied from 44 to 94% in NICU patients.2,21,22,23 There are various reasons which may in part account for these different prevalence rates in such patients. Antimicrobial prescribing practices may vary with the result that antimicrobial resistance would also vary in different types of NICU.24 Other potential factors affecting prevalence of resistance in CoNS are artefacts resulting from inclusion of more than one clinical culture per workup of a single patient. Accordingly, in the present study, only the first positive culture obtained in each clinical workup was counted. Finally, the different efficiencies of methodologies used for detection of oxacillin resistance may affect the reported prevalences.

We used PCR amplification of the mecA gene as the gold standard to compare the efficiencies of widely used methodologies for the detection of oxacillin resistance in CoNS isolates from our NICU. In the last decade, a number of modifications to MIC techniques have been advocated to facilitate detection of oxacillin resistance in CoNS.25,26 Moreover, many studies have indicated the need to revise the MIC interpretive breakpoints for oxacillin when testing CoNS.27,28,29,30,31,32,33 In January 1999, the NCCLS documents recommended <=0.25 mg/L as a susceptible breakpoint concentration of oxacillin, corresponding to an inhibition zone diameter of >=18 mm with a 1 µg disc, and >=0.5 mg/L as a resistant breakpoint concentration, corresponding to an inhibition zone diameter of <=17 mm.19 Our study and others27,28,30.31,32,33 indicate that the previous NCCLS MIC breakpoint (>=4 mg/L) for oxacillin resistance significantly underestimated the degree of true oxacillin resistance among CoNS strains. Although we extended the incubation time from 24 to 48 h with MIC techniques,25,28,34,35,36 we found that 10 (13.5%) of the 74 mecA-positive study strains still produced MICs (1–2 mg/L) that fell into the susceptible range as previously defined by NCCLS,18 and we suspected that these strains might have been falsely classified as susceptible to oxacillin. In our in-vitro studies, we were able to select in all 10 mecA-positive strains subpopulations of organisms for which MIC values were beyond the `susceptible' breakpoint. In addition, in-vivo findings from three neonates supported our concern. These neonates who were truly infected with CoNS strains that had 48 h MICs (1–2 mg/L) apparently below the NCCLS resistant breakpoint but were mecA-positive, failed to respond clinically and microbiologically to ß-lactam antibiotic therapy.

Prior to the most current NCCLS publication on the MIC interpretive standards for CoNS, resistant breakpoints of >=0.5 mg/L,30,32,33 >=1 mg/L27 or >=2 mg/L28 had been proposed for oxacillin or methicillin. Against this background, our study indicates that using MIC breakpoint concentrations of >=0.5 mg/L (as accepted in 1999 by NCCLS), >=1 mg/L and >=2 mg/L for oxacillin resistance in CoNS, the sensitivities of agar dilution MICs in detecting mecA-positive isolates are 97.2, 91.8 and 81.0%, respectively, at 24 h, and 100, 100 and 91.8%, respectively, at 48 h. In this respect, the specificities of agar dilution MICs in detecting strains lacking mecA are 89.7, 92.3 and 97.4%, respectively, at 24 h, and 80.7, 83.3 and 88.4%, respectively, at 48 h. From the neonatologist's standpoint, use of the most current NCCLS breakpoint (>=0.5 mg/L) for detecting oxacillin resistance among CoNS may result in a biologically relevant and more clinically useful test. However, one can look at the problem from the other side.37 From our data, the application of such a breakpoint even for 24 h of incubation would probably be associated with increased use of vancomycin. This would increase costs, the risk of adverse reactions and the emergence of glycopeptide-resistant organisms.

The 1999 NCCLS susceptibility testing documents still recommended the disc diffusion test but no longer included the salt agar screen method for phenotypic detection of oxacillin resistance in CoNS.19 In the present evaluation, when applying the previous NCCLS oxacillin disc diffusion interpretive criteria,18 the disc diffusion method was slightly more sensitive than the agar screen assay in detecting the 74 mecA-positive isolates (69 versus 67; P= 0.7) after 48 h of incubation, but significantly less specific in detecting the 78 mecA-negative isolates (seven false-positive results versus no false-positive result; P= 0.01) within the same time interval. When adhering to the most recent NCCLS oxacillin disc diffusion interpretive criteria, oxacillin disc zones gave false-susceptible errors for 8.1 and 2.7% of the mecA-positive strains tested at 24 and 48 h, respectively. On the other hand, the false-resistant error rates were 8.9 and 15.3%. The Food and Drug Administration criterion is that false-susceptible errors attributable to a new test's performance should be <1.5%.38 Furthermore, the rate of false-resistant errors cannot exceed 3%. Accordingly, in our hands, use of the new NCCLS oxacillin disc diffusion interpretive criteria failed to assure acceptable test accuracy.

Strains with the mecA gene are not only resistant to ß-lactams but are also commonly resistant to fluoroquinolones, aminoglycosides, tetracyclines, macrolides and trimethoprim–sulphamethoxazole.36 Similarly, previous studies utilizing standardized susceptibility testing methods showed that oxacillin-susceptible CoNS strains were more likely to be susceptible to ß-lactam and non-ß-lactam antimicrobials than were oxacillin-resistant strains.33,39 Using mecA gene PCR analysis as the reference genotypic method for interpreting oxacillin susceptibility results, our study showed that strains lacking mecA were more likely to be susceptible to ampicillin, ceftazidime, gentamicin, netilmicin and rifampicin than were mecA-positive strains. Vancomycin was the only antibiotic tested for which all strains, regardless of mecA status, remained susceptible by NCCLS criteria.

For oxacillin-resistant CoNS, a correct result is the only result compatible with an adequate standard of care for patients, including those residing in a neonatal intensive care setting. The present study demonstrates a need for a commercially manufactured kit for detection of the mecA gene that should be available to most clinical microbiology laboratories. Of equal importance is the need to determine, in prospective studies, the therapeutic and epidemiological implications of our findings as previous studies have used antimicrobial susceptibility assessed by disc diffusion or MIC techniques to evaluate the clinical significance of CoNS isolates from patients residing in intensive care nurseries.2,22,40,41,42


    Notes
 
* Correspondence address: Institute of Pediatrics, La Sapienza University of Rome, Viale Regina Elena, 324, 00161-Rome, Italy. Tel: +39-06-49-218-480; Fax: +39-06-70-96-349. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 11 December 1998; returned 26 February 1999; revised 23 March 1999; accepted 28 April 1999