Elsevier

Biomaterials

Volume 25, Issue 18, August 2004, Pages 4117-4125
Biomaterials

Search for the insertion element IS256 within the ica locus of Staphylococcus epidermidis clinical isolates collected from biomaterial-associated infections

https://doi.org/10.1016/j.biomaterials.2003.11.027Get rights and content

Abstract

Staphylococcus epidermidis biofilm-forming strains produce a polysaccharide intercellular adhesin (PIA), which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants. PIA synthesis is regulated by the icaADBC locus. In vitro, by repeated subcultures of a biofilm-producing strain, the loss of the ability to produce biofilm appears associated with the insertion of the IS256 element into the ica locus. This study was aimed (i) to investigate if the five genes of ica locus are always all present in different strains of S. epidermidis, and (ii) to search if IS256 insertion naturally occurs in ica locus without making recourse to the experimental procedure of repeated subcultures of strains. 120 S. epidermidis clinical isolates from peri-prosthesis infections were investigated both by an original multiplex PCR analysis of the ica genes and by PCR amplification of the IS256 element. Also two reference strains (the biofilm-negative S. epidermidis ATCC 12228 and the biofilm-forming ATCC 35984 [RP62A]) and two biofilm-negative RP62A-derived acriflavin mutants (D9 and HAM892) were analysed. D9 e HAM892 were for the first time shown to contain in ica locus, at the base 3319, a 1300-bp insertion with a DNA sequence corresponding to IS256. Among the 120 clinical isolates, 51 (43%) turned out completely ica-positive, 69 completely ica-negative (57%).

The genes of the ica locus appear, in all cases of the present collection, strictly linked each other, so they are either all present or all absent.

In this collection, IS256 was present in eight out of the 69 ica-negative strains and in 34 out of the 51 ica-positive strains. IS256, also when present in bacterial genomic DNA, was never found inside the ica locus, thus suggesting that insertion/excision of this element is not a natural occurring mechanism for off/on switching of biofilm production.

Introduction

Staphylococcus epidermidis, an opportunist micro-organism, is now recognised as a real “new” pathogen, in particular as etiologic agent of infections associated with indwelling medical devices [1], [2].

Among the pathogenetic mechanisms exploited by S. epidermidis in causing prosthesis-associated infections, of outstanding importance is the production of a polysaccharide biofilm, which wraps up and shields bacterial colonies on the surface and within anfractuosities of a biomaterial. Coagulase negative staphylococci produce biofilm on indwelling medical devices after a primary adhesion. Bacterial adherence and biofilm production proceed in two steps. First, an attachment to an hydrophobic surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step, which occurs onto hydrophobic and not to hydrophilic superficies, requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. This protein is composed of two bacteriolytically active domains, a 60 kDa amidase domain, implied in the last step of cell division, and a 52 kDa glucosaminidase domain. AtlE protein exhibits a vitronectin-binding activity and is thought to have a role in staphylococcal adherence to hydrophobic surfaces (see [3], [4] for reviews). The second step in bacterial adhesion is associated with the production of biofilm, the polysaccharide intercellular adhesin (PIA), a polymer of β-1,6-linked N-acetylglucosamine [3].

In S. epidermidis, PIA production is mediated by the ica operon, which comprises icaA, icaD, icaB, icaC genes and the regulatory gene icaR [5], [6]. The enzyme N-acetylglucosaminyl transferase, which synthesises the N-acetylglucosamine polymer, is encoded by icaA. While the sole expression of icaA brings about a low enzymatic activity, the co-expression of icaA with icaD remarkably increases the N-acetylglucosaminyl transferase activity [5].

The functional interaction between icaA and icaD could be necessary by the unusual β-1, six link between the N-acetylglucosamine residues. Glycopolymers composed of β-1, six linked monomers exhibit a three-dimensional structure much different from that of polymers composed of β-1, four linked monomers, the glycopolymers whose synthesis is promoted by the majority of glycosyltransferase. The specific role of icaB is not yet known, although it is suggested to encode for a deacetylase which removes some acetyl groups giving positively charged glucosamine residues [3]. Co-expression of icaA, icaD, icaC brings about the synthesis of poly-N-acetylglucosamine chains longer than those synthesised by the sole co-expression of the couple of icaA and icaD genes [5].

In some biofilm-producer strains, PIA synthesis has been shown to undergo in vitro to a phase variation process, which leads to biofilm-negative variants. As a possible mechanism of the variation, a process of insertion/excision of the insertion sequence IS256 in icaA gene, or in icaB, or, more frequently in icaC has been proposed [7].

In consideration of these intriguing observations on ica locus, the need arises of a molecular method able to detect with certainty the ica operon in S. epidermidis different strains. The method should be capable to determine whether all genes of the operon be always conjointly present, or otherwise different strains do exist with different gene assortments of the ica operon, with one or more ica-genes lacking, or with insertion elements present.

Multiplex PCR proves to be a useful method both for ica-gene screening in a collection of clinical isolates and for a close examination of the gene arrangement of single strains.

The aim of this study was to search the natural occurrence of the IS256 insertion element either in ica locus or in genomic DNA of clinical strains of S. epidermidis, just as they were isolated and not subjected to artificial manoeuvrings to induce phase variations. The search for ica genes was carried out in 120 S. epidermidis isolates from prosthesis associated infections and in four S. epidermidis reference strains and compared with the phenotypic biofilm-forming ability, evaluated onto Congo red agar (CRA) plate test [8], [9].

Section snippets

Bacterial strains

Two S. epidermidis reference strains were used, the biofilm-negative forming ATCC 12228 and the biofilm-forming RP62A. Besides these, two biofilm-negative mutants, D9 and HAM892 [10], [11], derived by acriflavin mutation from S. epidermidis RP62A, were investigated. D9 was from the Laboratory of Ultrastructures, Istituto Superiore di Sanità, Rome, Italy and HAM892 was kindly provided by G. Pier and E. Muller, Harward University, Boston, Mass. The present study focused on 120 clinical isolates

Multiplex PCR design

In the present study multiplex PCR allowed to detect, by three sets of amplification reactions, the presence of all ica-genes in order to detect insertion elements. Single representative fragments for each the icaR, icaA, icaD and icaB genes were amplified. Since the icaC gene may harbour IS256 element in two highly probable known positions (base 3285 and base 3742 [7]), five different amplification segments were selected. Two of them (icaCβ and icaCγ) contained each one of the two known

Discussion

S. epidermidis biofilm-forming strains produce PIA, a β-1,6-linked N-acetylglucosamine polymer, which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants. PIA synthesis is regulated by the icaADBC locus [5], [6], [14]. In recent reports, ica locus emerges as a strong marker of virulence in clinical isolates of staphylococci from biomaterials-centered infections [15], [16]. Studies suggest that ica locus is more prevalent in clinical S. epidermidis isolates

Conclusions

A multiple PCR method for the detection of all genes of the ica operon, responsible for biofilm production in S. epidermidis, has been developed. In a collection of 120 S. epidermidis clinical isolates from peri-prosthesis infections the genes of the ica locus were found strictly linked each other, so they are either all present or all absent. The presence of the insertion element IS256 in ica locus was searched by this method and by direct PCR amplification of IS256 sequence. The insertion

Acknowledgements

This work was supported by a grant from Italian Ministry of Health (reference number SVE 225/2001) “Pathogenesis and molecular epidemiology of implant-associated infections and strategies of prevention”.

We are grateful to Mrs. C. Vescovini for the skilled assistance in preparing manuscript and to Mr.V. Pirini for his excellent technical assistance.

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