Methicillin-resistant Staphylococcus aureus nasal colonization in a department of pediatrics: a cross-sectional study

The Bambino Gesù Children’s Hospital, located in Rome, Italy, is a 607-bed comprehensive medical centre for pediatric healthcare and research. The Department of Pediatrics includes 132 beds in one single building divided into 4 floors by level of assistance.

Over a 3-month period between February 8, 2010 and May 14, 2010 we conducted a cross-sectional study enrolling patients admitted to 3 wards belonging to the Department of Pediatrics. Children admitted to the wards were affected by general pediatric acute or chronic illnesses, rheumatologic diseases, infectious diseases (excluding HIV), bronchopneumopathies, cystic fibrosis, endocrinological diseases and genetic diseases.

No age or pathology constraints were applied in the selection of the population. Patients not speaking Italian, English, Spanish and French were excluded. Written, informed consent was obtained from patients older than 18 years of age, or from parents of patients younger than 18 years of age, together with patient assent, or written, informed consent if patient was older than 12 years.

Enrolled patients underwent an anterior nasal swab in one of the nares within 12 h of admission. A questionnaire was administered to the parents or to the patient to collect demographic and epidemiological data, including: birth date, sex, parents’ data (age, level of education and employment), number of siblings, number of household members, number of bedrooms, presence of smoking people at home, presence of domestic animals, presence of an household member working in a healthcare facility, current breastfeeding (for children < 2 years), daycare center or school attendance, extra-school sport activity (for children > 4 years), nail biting (for children > 4 years), diagnosis of a chronic underlying disease, past diagnosis of pneumonia or of an invasive infection, emergency room visit during last 6 months, hospital admission during last 6 months, administration of antibiotic therapy during last 6 months, upper respiratory tract infection during last week. The study was approved by the Bambino Gesù Children’s Hospital Ethical Committee.

Samples were collected from anterior nares using the ESwab (Copan, Brescia, Italy) collection and transport device, composed of a flocked swab adsorbed into 1.0 ml of Amies liquid transport medium. For each nasal sample two aliquots (10 μl each) of ESwab liquid medium were streaked onto mannitol salt agar, Chapman (bioMérieux, Marcy l’Etoile, France) and on chromogenic agar MRSASelect (Bio-Rad, Nazareth Eke, Belgium) respectively, and incubated at 37°C for 18–24 h. Colonies grown on Chapman agar having morphology suggestive of S. aureus, or mauve in color presumptively MRSA positive on chromogenic agar were subcultured onto 5% sheep blood agar. Phenotypical identification was assessed by Vitek2 system (bioMérieux Marcy l’Etoile, France) according to the manufacturer’s instructions.

In vitro susceptibility testing for oxacillin, erythromycin, clindamycin, gentamicin, ciprofloxacin, rifampicin, trimethoprim-sulfamethoxazole, mupirocin, vancomycin, teicoplanin (Sigma Chemical, St. Louis, MO, USA) and linezolid (Pfizer, Groton, CT, USA), was further performed by the broth microdilution method to determine the minimum inhibitory concentrations (MICs), following the CLSI guidelines. The EUCAST guidelines were also used for comparison [14, 15]. In particular the oxacillin susceptibility was interpreted according to CLSI recommendations, and in cases of discrepancy susceptibility testing was repeated.

The penicillin-binding protein (PBP2a) latex agglutination test (SLIDEX MRSA Detection, bioMérieux Marcy l’Etoile, France) was used as an additional phenotypic confirmatory test for presumptively detected MRSA strains.

Genomic DNA was extracted from all bacterial cultures and used as a template for amplification.

All isolates were screened for the presence of the mec A, luk S-PV/luk F-PV genes and ACME-locus. PCR experiments were carried out following procedures previously reported [16‐18]. The SCCmec (Staphylococcal-Chromosomal-Cassette mec) types were first determined by a multiplex-PCR protocol previously described, and assigned to the corresponding types [19]. Moreover, for the characterization of the SCCmec IV subtype an alternative multiplex-PCR protocol was performed [20], the results were confirmed with other different multiplex-PCR protocols, focusing on the mec gene complex and the ccr gene complex [21] and hsd RMS operon [16].

The sequences of the seven housekeeping genes used for MLST, corresponding to the allelic profile arc C-aro E-glp-gmk-pta-tpi-yqi L, were obtained by comparing the sequences obtained for all MRSA strains in study with those in the MLST database http://​saureus.​mlst.​net[22]. All sequence-types (STs) described in the study were deposited on the MLST website. PCR products were analyzed by electrophoresis on 1% or 2% agarose gels (Sigma Aldrich, Saint Louis Mo, USA).

Sample size calculation was based on the assumption of a carriage rate of 2%. Around 785 subjects needed to be screened, in order to estimate the carriage rate with adequate precision (95% confidence intervals between 1% and 3% for 785 subjects).

Proportions of the social-demographic characteristics of each group of colonized/not-colonized children were compared through χ² or Fisher exact test where appropriate. Statistical tests were considered significant at a p-value <0.05 level.

In order to study the association between potential risk factors and colonization status at the time of hospital admission, we performed a univariate analysis through a logistic regression model in which odds ratios and 95% CI were calculated. Potential risk factors included in the analysis as independent variables were all those investigated in the questionnaire, plus age at admission.

In each model colonization status was included as a dependent variable, as follows: colonized patients vs non-colonized; MSSA colonized vs non-colonized patients and MRSA colonized vs non-colonized patients. At the univariate level, the analysis was restricted to children aged ≤4 years for “sport”; ≥ 3 years for “nail biting”; <2 years for “breastfeeding”.

Variables with a p-value <0.20 at the univariate analysis were selected to be included in the multivariate analysis using a logistic regression model, with nasal carriage being the dependent variable as reported above. Since none of the variables analyzed on a restricted population at the univariate level had a p value <0.20, the multivariate model included the entire population. Statistical software STATA 11 was used for data analysis.

We enrolled 785 patients from 3 pediatric wards of the Department. Mean age of patients was 4.62 years (median: 2.66, range: 0.03-27.7), of which 403 (51%) were males. Other characteristics of the population are reported in Table 1. Among the enrolled patients, no one had been admitted for a symptomatic S. aureus infection or was diagnosed with a nosocomial S. aureus infection during hospitalization.

Among the MRSA colonized patients, 5 had an underlying condition (mucopolysaccharidosis, arthrogryposis, neurofibromatosis, type 1 diabetes, osteoid osteoma). The remaining 4 patients had been admitted with the following diagnoses: bronchiolitis, rickettsiosis, chickenpox, sepsis. Six of the MRSA-colonized patients had already been hospitalized in the last 6 months. Combining information on underlying diseases and previous hospitalizations, 3 out of 9 MRSA colonized patients did not have risk factors for previous healthcare exposures.

Risk factors for MSSA and MRSA carriage identified by multivariate analysis are reported in Table 2.

According to the multivariate analysis, compared to non-colonized patients, MRSA colonized patients were more likely to have been hospitalized in the previous 6 months. Antibiotic therapy in the last 6 months was found as a protective factor for MRSA colonization. The multivariate analysis did not identify age as a risk factor for MRSA colonization, while MSSA-colonized patients were more likely to belong to the 6–13 age group compared to non-colonized children.

Antimicrobial susceptibility and molecular characteristics of the isolates are reported in Table 3.

Nine MRSA were isolated, all characterized by phenotypic and molecular methods, as described in the Methods section.

All isolates were oxacillin resistant, 4/9 were erythromycin resistant, 3/9 ciprofloxacin and rifampicin resistant, 1/9 gentamicin resistant, and only one isolate was resistant to cotrimoxazole. All strains were susceptible to clindamycin, mupirocin, linezolid and glycopeptides.

MLST analysis showed 7 different STs associated with different variants of SCCmec IV and only one with SCCmec V.

Only 1 strain carried lukS-PV/lukF-PV genes, and did not harbour the arginine catabolic mobile element (ACME), associated toUSA300 clone.