Amoxicillin-Clavulanate Therapy Increases Childhood Nasal Colonization by Methicillin-Susceptible Staphylococcus aureus Strains Producing High Levels of Penicillinase

Survey design.In December 1995, we randomly selected 20 French playschools in an administrative department of central France with 600,000 inhabitants, where more than 99.5% of 3- to 6-year-old children attend playschools. This department was chosen as typical of French departments in terms of sociodemographic characteristics, medical activity of practitioners not affiliated with hospitals, and social security coverage. The 3- to 6-year age group was chosen to enhance the feasibility of recruiting a random population-based sample; their inclusion was based on registration at the beginning of the school year. The parents gave informed consent to the study, and the children gave oral consent to bacterial screening.

At the beginning of the study, the parents were given a questionnaire on which to record sociodemographic characteristics, together with their child's drug consumption (trade name, unit dose, daily frequency, and duration of treatment). The study was conducted over a 6-month period, from December 1995 to May 1996, with the help of the teachers. Because the ecological niche of S. aureus is the anterior nares (15), the children were screened for anterior nasal carriage of S. aureus at the end of the 6-month follow-up period. Children whose parents could not remember treatment details were excluded.

Ethical considerations.This project was sponsored by the Institut National de la Santé et de la Recherche Médicale (INSERM). It was examined by the institutional review board of the Creteil teaching hospital and approved by the French Ministry of Health. It was also approved by the appropriate French computer watchdog committees (Comité Consultatif sur le Traitement de l'Information en Matière de Recherche dans le Domaine de la Santé and Commission Nationale de l'Informatique et des Libertés). The legally required scientific informed-consent forms were signed by the parents of enrolled children.

Sample collection and microbiological methods.After sampling, swabs were immediately placed in TVG-AER transport medium (Bio-Rad, Marnes-La-Coquette, France) and transported within 12 h to the central laboratory (BIO VSM Lab). On reception, the swabs were plated on selective mannitol salt agar (bioMérieux, Marcy-l'Etoile, France) and incubated for 24 and 48 h at 37°C. Mannitol-fermenting colonies were Gram stained and tested for catalase and free coagulase by using rehydrated rabbit plasma (Bio-Rad) according to the currently recommended procedure (11). S. aureus strain ATCC 25923 was used as a control for culture and identification procedures. Mannitol-fermenting staphylococci that tested positive for free coagulase were identified as S. aureus and were stored at −70°C in brain heart infusion (BHI) with 15% glycerol. They were then transported to the Microbiology Department of Robert-Debré Hospital for further studies.

Detection of penicillin and methicillin resistance and measurement of penicillinase production.Penicillin resistance was detected by the penicillin disk susceptibility test and the nitrocefin-based test (7). MICs of oxacillin were determined by the standard agar dilution method, as recommended by the French Microbiology Society (7).

All S. aureus isolates were screened for the mecA gene (encoding methicillin resistance) by PCR, as reported by Vannuffel et al. (31).

Penicillinase production was measured for penicillin-resistant, methicillin-susceptible S. aureus isolates. Strains were grown overnight in 10 ml of BHI medium, collected by centrifugation, and stored at −20°C. Cell pellets were resuspended in 0.5 ml of 50 mM phosphate buffer, pH 7.4, containing lysostaphin at a final concentration of 0.1 mg/ml. The suspensions were incubated at 37°C for 15 to 20 min and then sonicated for 1 min on ice. The disrupted cells were centrifuged at 17,000 × g rpm for 10 min at 4°C. Penicillinase activity in cleared extracts was determined by using nitrocefin as the substrate, and the protein concentration was determined by using the Bio-Rad Bradford dye-binding assay. For penicillinase assays, 20 to 50 μl of extract was added to a cuvette containing 0.1 mM nitrocefin in 50 mM phosphate buffer, pH 7.4, and hydrolysis was monitored at 482 nM for 5 min. Activity was linearly dependent on the amount of extract added, and specific penicillinase activity in the extracts was recorded as moles of nitrocefin hydrolyzed per minute per milligram of protein. All activity assays were run in duplicate.

PFGE.The 20 S. aureus isolates with the highest penicillinase production (>0.237 μmol/min/mg) were analyzed by pulsed-field gel electrophoresis (PFGE) of SmaI-digested total DNA, as described previously (9).

Descriptive and analytical methods.Only exposure to systemically administered antibiotics was taken into account. We first examined factors possibly associated with nasal carriage of S. aureus. The analysis of factors associated with penicillinase production was restricted to penicillin-resistant, methicillin-susceptible strains. After the homogeneity of variance was tested, a t test was used to compare penicillinase production according to the oxacillin MIC of the corresponding strain. Penicillinase values were dichotomized around the median for univariate and multivariate analyses.

The chi-square test was used to compare binary variables. Multivariate analyses used logistic regression models which were constructed with variables with P values of <0.2 in univariate analysis, followed by backward stepwise regression. P values of <0.05 were considered statistically significant. Stata SE 8 software was used.

Participation rate and population characteristics.Based on school registers, 832 children were eligible. Nonparticipation was due to lack of questionnaire completion (7.6%), refusal by parents (4.7%), refusal by children (3.9%), usual absence of children such as for an afternoon nap at home (2.3%), absence of children from the school because of parents' holidays (1.4%), and other reasons (1.4%). The parents of 648 children (77.9%) completed the questionnaire, and these children were sampled for S. aureus carriage. The mean age was 4.9 years (95% confidence interval [95% CI], 4.8 to 5.0), and the mean body weight was 18.3 kg (95% CI, 18.0 to 18.5].

Antibiotic exposure during the previous 3 months.At least one course of antibiotics was prescribed to 42.8% of the children during the 3 months before nasal sampling. All the antibiotics were given orally. The patterns of different antibiotic classes are presented in Table 1. Among the cephalosporins, narrow-spectrum drugs were most frequently used (9.3% of children); expanded- and broad-spectrum drugs accounted for 0.9 and 4.5% of prescriptions, respectively.

Risk factors for S. aureus carriage.One hundred sixty-six children (25.6%) were colonized by S. aureus. The carriage rate was not influenced by gender or by amoxicillin or macrolide use. Age was the only factor associated with S. aureus colonization: the risk of colonization increased with age. Cephalosporin use during the previous 3 months was associated with a lower risk of S. aureus colonization in univariate analysis but not in multivariate analysis (Table 1).

Beta-lactam susceptibility.Seven strains (4.2%) were susceptible to penicillin G. One strain (0.6%) was resistant to methicillin and harbored the mecA gene. This strain was susceptible to erythromycin, clindamycin, trimethoprim-sulfamethoxazole, rifampin, and gentamicin. The carrier was a 4.7-year-old girl with no predisposing factors such as hospitalization during the previous 6 months or parents close to sick persons.

Level of penicillinase production and antibiotic use.Among penicillin-resistant, methicillin-susceptible strains (n = 158), the pattern of penicillinase production (Fig. 1) was clearly associated with the oxacillin MICs for the strains (P < 0.001) (Fig. 2). Upon analysis of penicillinase production dichotomized around the median, two factors were associated with increased penicillinase production: an age of ≥6 years (odds ratio [OR], 2.7; P = 0.02) and amoxicillin-clavulanate exposure (OR, 3.7; P = 0.03). Exposure to amoxicillin alone was not associated with the level of penicillinase production; neither was exposure to cephalosporins or macrolides (Table 2).

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FIG. 1.

Pattern of penicillinase production by penicillin-resistant, methicillin-susceptible strains of S. aureus (n = 158). The median value was 0.05 μmol/min/mg.

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FIG. 2.

Penicillinase production according to the oxacillin MIC (n = 158).

Total DNAs from the 20 strains with the highest levels of penicillinase production were analyzed by PFGE. Only two pairs of isolates (isolates 52 and 70 and isolates 463 and 481) were indistinguishable (Fig. 3).

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FIG. 3.

PFGE patterns of the 20 S. aureus strains with the highest levels of penicillinase production. The identification numbers of the strains are shown above the gel.