Detection of Staphylococcus aureus by 16S rRNA directed in situ hybridisation in a patient with a brain abscess caused by small colony variants.

A 45 year old man was admitted to hospital with a right sided facial paralysis and three month history of seizures. Computed tomography showed a left temporal mass including both intracerebral and extracerebral structures. Ten years earlier the patient had undergone a neurosurgical intervention in the same anatomical region to treat a subarachnoid haemorrhage. In tissue samples and pus obtained during neurosurgery, Staphylococcus aureus was detected by a 16S rRNA-directed in situ hybridisation technique. Following long term cultivation, small colony variants (SCV) of methicillin resistant S aureus were identified. The patient was treated successfully with a combination of vancomycin and rifampin followed by prolonged treatment with teicoplanin, with no sign of infection on follow up nine months after discharge. This is the first report in which S aureus SCV have been identified as causative organisms in a patient with brain abscess and in which in situ hybridisation has been used to detect S aureus in a clinical specimen containing SCV. Antimicrobial agents such as rifampin which have intracellular activity should be included in treatment of infections caused by S aureus SCV.

Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization.

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.

A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256.

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.