Longitudinal survey of Staphylococcus aureus in cystic fibrosis patients using a multiple-locus variable-number of tandem-repeats analysis method

BMC Microbiology Longitudinal survey of Staphylococcus aureus in cystic fibrosis patients using a multiple-locus variable-number of tandem-repeats analysis method Hoang Vu-Thien Katia Hormigos 0 Galle Corbineau 0 Brigitte Fauroux Harriet Corvol Didier Moissenet Gilles Vergnaud 0 Christine Pourcel 0 0 Universite Paris Sud 11, CNRS, UMR 8621, Institut de Genetique et Microbiologie , Orsay, 91405 , France Background: Staphylococcus aureus infection in patients with cystic fibrosis (CF) is frequent and may be due to colonization by a few pathogenic lineages. Systematic genotyping of all isolates, methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) is necessary to identify such lineages and follow their evolution in patients. Multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) was used to survey S. aureus clinical isolates in a French paediatric CF centre. Results: During a 30 months period, 108 patients, aged 2 to 21 years, regularly followed up at the centre, provided sputum for culture. From 79 patients, a total of 278 isolates were genotyped by MLVA, resolving into 110 genotypes and 19 clonal complexes (CC) composed of similar or closely related isolates. 71% of the strains were distributed into four main CCs, in term of number of isolates and number of genotypes. Spa (Staphylococcus protein A) typing was performed on representative samples, showing an excellent concordance with MLVA. In 17 patients, strains from two to four different CCs were recovered over time. On six occasions, S. aureus isolates with the same genotype were shared by 2 different patients and they belonged to one of the four main clusters. Methicillin-resistance was observed in 60% of the isolates, 90% of which belonged to the main clonal complexes CC8, CC45 and CC5. In 5 patients, methicillin-resistance of S. aureus isolates was not associated with the mecA gene: for four patients, it was due to overproduction of b-lactamase, leading to BOR-SA (borderline S. aureus) isolates, while a strain showing probably a new modified penicillin-binding capacity (MOD-SA) was observed from one patient. Conclusion: Systematic genotyping of S. aureus isolates recovered from sputum of CF children allows a thorough analysis of the strains responsible for sporadic as well as chronic colonization and the follow up of their evolution over time. We show here that more than 70% of these strains belong to 4 major CCs. MSSA as well as MRSA, BORSA and MOD-SA isolates can persist over several years, despite antibiotic treatments. - Background Cystic fibrosis (CF) is caused by a mutation in the CFTR-gene leading to dysfunction of the exocrine glands. The disease is responsible for chronic airway obstruction in the lung, a favourable condition for pulmonary infections during childhood. In different studies investigating pathogens in CF, S. aureus was observed in 4 to 60% of patients frequently in association with other bacteria, such as Pseudomonas aeruginosa [1-3]. Since the introduction of methicillin in 1959, methicillin-resistant S. aureus (MRSA) clones have rapidly emerged and spread worldwide and account for 10 to 30% of S. aureus infections [4,5]. Molecular epidemiology studies using Multi Locus Sequence Typing (MLST) on clinical strains of S. aureus have shown that they are distributed into 11 major clonal complexes (CC) [6]. MRSA strains represent the most threatening challenge as they are frequently resistant to many antibiotics and there is evidence that antibiotic treatments not only facilitate the spreading of these clones but also enhance their pathogenicity [7]. Patients with CF are at particular risk for pulmonary colonization of MRSA, both because of their difficulty in clearing mucus and because of their frequent hospital visits, which can increase exposure to MRSA. Several studies reported that 20 to 35% of CF patients harbored a MRSA strain and described the emergence of community-acquired MRSA (CA-MRSA) [8-11]. Methicillin-susceptible strains (MSSA) also constitute a risk in CF patients, particularly because of the existence of biofilms in the infected lung in which they can escape from antibiotic treatment [12]. The epidemiology of S. aureus in CF patients has been investigated in different studies, but mostly MRSA were analysed and the role of MSSA was not assessed. In order to extend the knowledge of the population of S. aureus chronically infecting CF patients, all the isolates should be systematically genotyped with a high degree of discrimination which is difficult using the currently available techniques. The polymorphism of the Staphylococcus protein A gene (spa), first used by Frenay et al. [13] to genotype S. aureus and further evaluated by Shopsin et al. [14] has proven to be very useful to investigate S. aureus genetic diversity. Subsequently MLST became the most widely used technique to analyse the epidemiology of S. aureus and to perform phylogenetic studies [15]. Although the combined discriminatory power of spa typing and MLST is high, these techniques do not sufficiently discriminate within the major CCs and their cost is elevated. New approaches have been developed which use Variable Number of Tandem Repeats (VNTR) either to produce a multiple band pattern in a technique called MLVF [16,17] or to perform Multiple loci VNTR analysis (MLVA). MLVA consists in the analysis of individual VNTRs allowing the description of a strain in the form of a code easily exchangeable between laboratories [18]. MLVA with 6 VNTRs could correctly assigned isolates to outbreaks or identified isolates as unlinked [19]. Schouls et al. using 8 VNTRs showed that MLVA was at least as discrimina tory as Pulse Field Gel electrophoresis (PFGE) and twice as discriminatory as spa-sequence typing [20]. Finally we recently described a very informative MLVA scheme which makes use of 14 VNTRs (MLVA-14) and demonstrated that its discriminatory power was much higher than those of MLST and spa typing [21]. In the present work we used the proposed MLVA-14 assay to genotype S. aureus isolates recovered from firstly as well as chronically colonized CF patients, over a period of 30 months. The aim of our study was to investigate the genetic diversity of MRSA and MSSA present in the sputum of CF children whether sporadically or chronically. The longitudinal survey of genotypes provided information on the variations in those strains recovered from some patients over a maximum period of 24 months. Results Clinical characteristics of S. aureus colonization From a total number of 143 patients attending the Armand Trousseau CF centre during the 30 months study period, 108 provided sputum of which 79 showed one or several cultures positive for S. aureus. It is likely that most were community-acquired S. aureus contaminations as the majority of patients were outpatients. In addition there was no outbreak episode during the study period. Although this study was not designed to (...truncated)