Genomic characterization of a paediatric MRSA outbreak by next-generation sequencing.

INTRODUCTION: Twelve strains of meticillin-resistant Staphylococcus aureus (MRSA) isolated during a suspected outbreak in a paediatric intensive care unit were analysed by whole-genome sequencing (WGS). AIM: To define the clonality of MRSA strains to a high discriminative power, and to evaluate the presence of genetic determinants responsible for antibiotic resistance and virulence. RESULTS: Ten out of 12 strains belonged to multi-locus sequence type ST2625, while the other two strains were ST8. Among the ST2625 strains, analysis based on 1126 genes showed that they were clonal, sharing more than 98.3% of allelic identities, and one strain was isolated from a healthcare worker. All ST2625 strains were characterized by the SCC-Mec cassette IVa, and resistoma analysis indicated correspondence between phenotypic and genotypic characteristics. The study of 63 genes associated with virulence was correlated with the pattern of clonality shown. CONCLUSION: This analysis confirmed the occurrence of an outbreak. As such, standard infection control measures were strictly enforced, and this led to prompt termination of the outbreak.

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

An improved method for HLA-B and -C supratyping.

A growing body of evidence links the analysis of the KIR genotype and the presence of their HLA-B and -C ligands to a wide repertoire of human diseases. We noticed that, using a panel of 184 Caucasoid donors, a limited number of HLA alleles were incorrectly supratyped by previously described pyrosequence-based assays. Here we describe a simple implementation of the reported methods that corrects all the discrepancies found with HLA-B and -C molecular typing and allows establishing a quick and high-throughput method for the determination of HLA-Bw4 I(80), Bw4T(80), Bw6 and HLA-C1 or -C2 supratype.

A comparison of two blood culture procedures for the isolation of staphylococci in a paediatric intensive care unit.

Blood culture results obtained between January 2000 and July 2003 were reviewed for 1360 patients in a paediatric intensive care unit (PICU). The BacT/Alert FA aerobic medium was used with a blood volume of 1.5 mL for the first 23 months, and the BacT/Alert PF paediatric medium was used with a 0.5-mL volume for the remaining 18 months. The isolation rates were similar during both periods (13.4% vs. 13.1%), and staphylococci were the most common isolates (72.8%). There was a shorter time to detection of staphylococci with the smaller-volume (PF) procedure, which thus seems suitable for use in the diagnosis of staphylococcal bacteraemia in the PICU.

Use of the E test to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis.

Treatment of Burkholderia cepacia-complex infections in cystic fibrosis patients is problematic, since the microorganism is often resistant to most antimicrobial agents. In this study, the Epsilometer test, or E test, was used to assess the activity of antimicrobial combinations against Burkholderia cepacia-complex. In a preliminary evaluation, the E test was compared to the checkerboard method using 10 test organisms. Synergy testing by the E test was then performed on 131 clinical isolates of Burkholderia cepacia-complex using various combinations of antimicrobial agents. Agreement between the E test and the checkerboard method was 90%. The rate of resistance to individual agents ranged from 48% for meropenem to 100% for tobramycin, chloramphenicol, and rifampin. In 71.6%, 15.6%, and 12.6% of the test evaluations performed, the combinations tested resulted in additivity/indifference, synergism, and antagonism, respectively. The highest rates of synergy were observed with combinations of ciprofloxacin-piperacillin (44%), rifampin-ceftazidime (33%), chloramphenicol-ceftazidime (22%), cotrimoxazole-piperacillin/tazobactam (22%), and ciprofloxacin-ceftazidime (21%). Rates of antagonism for cotrimoxazole and chloramphenicol in combination with beta-lactam agents were higher than those observed for ciprofloxacin plus beta-lactam agents. These results suggest that the E test is a valuable and practical method to be considered for improving the identification of possible therapeutic options in cystic fibrosis patients infected with organisms belonging to the Burkholderia cepacia-complex.

Erythromycin resistance in Streptococcus pyogenes in Italy.

In a prospective study of acute pharyngitis in Italian children, 69 (38.3%) of 180 isolates of Streptococcus pyogenes were resistant to macrolides. S. pyogenes was eradicated in 12 (63.1%) of 19 patients with erythromycin-resistant S. pyogenes treated with clarithromycin and in 22 (88%) of 25 patients with erythromycin-susceptible strains. The constitutive-resistant phenotype was correlated with failure of macrolide treatment.

[Epidemiology and sensitivity to antibiotics in paediatric respiratory infections over a 4-year period. Retrospective study of 460 H. influenzae, S. pneumoniae and M. catarrhalis strains] / Epidemiologia e sensibilita agli antibiotici in infezioni respiratorie di pazienti pediatrici in un periodo di 4 anni. Studio retrospettivo su 460 ceppi di H. influenzae, S. pneumoniae e M. catarrhalis.

H. influenzae, S. pneumoniae and M. catarrhalis are the most common bacterial pathogens causing respiratory infections in children. Resistance to antibiotics may vary according to the geographical area. It is therefore important that the resistance pattern of such pathogens is determined by surveillance studies carried out both on a national scale and by individual laboratories. In this study, we determined retrospectively the prevalence of H. influenzae, S. pneumoniae and M. catarrhalis in upper respiratory tract infections involving subjects of paediatric age, with reference to the type of clinical sample (pharingeal swab and nasal swab), symptomatology and age group. Moreover, for the above micro-organisms the pattern of sensitivity to antibiotics was assessed. In the observation period (January 1996-December 1999), at the day hospital of the Paediatric Pneumology Division of the Gaslini Institute in Genova, in 476 patients between 0 and 15 years of age a total of 460 respiratory pathogens were isolated: 164 S. pneumoniae strains, 163 of H. influenzae (96 belonging to type B and 67 non-attributable to any type) and 133 of M. catarrhalis. As regards sensitivity to antibiotics, ceftriaxone and amoxycillin/clavulanic acid proved to be the most active molecules in all the studied strains.

The effect of fresh human serum on ofloxacin bactericidal activity.

We have studied the possibility of an increase in ofloxacin bactericidal activity when it is combined with fresh human serum. The tested strains were 10 clinical isolates of Klebsiella pneumoniae. From among our strains 5 were susceptible to serum bactericidal activity and 5 were found to be resistant. We selected two serum concentrations (15 and 35%) to test against susceptible strains and two (55 and 75%) to test against resistant strains in combination with the minimum inhibitory concentration (MIC), 1/2MIC and 1/4MIC of ofloxacin. The results show a slight variability among the tested strains depending on microbiological characteristics of single strains, however, the serum + ofloxacin combination was advantageous. Only one exception was observed: a resistant strain that had an increased survival percentage against ofloxacin and serum in combination.