PREVALENCE OF ICA-OPERON AMONG COAGULASE-NEGATIVE STAPHYLOCOCCI (CONS) ISOLATED FROM ORTHOPAEDIC PATIENTS. REDUCED EXPRESSION OF ICAA AND ICAC GENES INFLUENCE THE BIOFILM PRODUCTION

Abstract

Introduction The aim of this study was firstly to investigate the prevalence of icaABCD-operon which codes the production of the polysaccharide intracellular adhesin(PIA), responsible for biofilm production, in a collection of clinically significant staphylococci isolated from orthopaedic infections and secondly to assess the relationship between biofilm production and the presence or not of ica-operon.

First Step – Material & Methods Between 1/2003 and 12/2005 200 CoNS were isolated from orthopaedic patients associated with soft tissue and bone infections(group I) and 200 CoNS from blood cultures of hospitalized patients from different wards of the same Hospital(group II). Identification was carried out by Gram-stain, catalase and coagulase tests and the API Staph System. Detection of icaADBC genes was performed by PCR. Production of biofilm was tested by the method of Christensen.

Results In group I, 62(31.37%) carried the entire ica-operon; from these isolates biofilm formation was detected in 35(17.5%). 5 isolates, despite biofilm production, did not carry any gene of ica-operon. In group II, 70(35.5%) carried entire the ica-operon; biofilm formation was detected in 37(18.5%) of these isolates. 3 S. capitis, 1 S. epidermidis and 1 S. hominis carried only the icaADB, icaA and icaB genes respectively.

Second Step – Material & Methods Based on the observation of PIA-production only in (50%) of ica(+) CoNS, 20 S. epidermidis isolates recovered from clinical specimens (pus) of orthopaedic patients and belonging to distinct PFGE clones, were selected on the basis of the presence of the entire ica operon. Nevertheless, only 10 of them produced biofilm. Nucleotide sequence analysis of ica-operon was carried out in all isolates; expression of icaADBC genes was also tested by RT-PCR.

Results Sequencing analysis revealed that all isolates carried an intact ica-operon, without point mutations. Concerning icaADBC mRNA production, all genes of ica-operon were expressed in biofilm-producing isolates, whereas in the no-biofilm producing strains the icaA and icaC genes were not expressed, while a faint expression was observed for the icaB and icaD genes.

Discussion Biofilm-forming capacities of CoNS from orthopaedic infections was not significantly greater than those from other infections (p> 0,05). The capacity of ica-operon(+) staphylococcal isolates to form biofilm seems to be dependent on the expression of ica-genes, specifically of icaA and icaC. The inability of ica(+) isolates to produce biofilm emphasizes that some unknown mechanisms influence icaADBC expression. Finally, the recognition of biofilm-producing CoNS without carrying any gene of ica operon underlined the existence of unidentified also mechanisms controlling biofilm production, apart from icaADBC expression.