We determined the frequency of isolation of staphylococcal small-colony variants (SCVs) from 31 culture-positive patients undergoing revision of total hip prosthesis for aseptic loosening or presumed prosthetic-joint infection (PJI). We analysed auxotrophy of cultured SCVs, their antimicrobial susceptibility profiles and their biofilm-forming capacity. Eight SCV strains were cultivated from six (19 %) patients. All SCVs were coagulase-negative staphylococci (CNS) with Staphylococcus epidermidis as the predominant species; there was also one Staphylococcus warneri SCV. The SCVs were auxotrophic for haemin, with one strain additionally auxotrophic for menadione. We noted the presence of two phenotypically (differences concerning antimicrobial susceptibility) and genetically distinct SCV strains in one patient, as well as the growth of two genetically related SCVs that differed in terms of their morphology and the type of auxotrophy in another. Seven out of eight SCVs were resistant to meticillin and gentamicin. In addition, antibiotic sensitivity testing revealed three multidrug-resistant SCV-normal-morphology isolate pairs. One S. epidermidis SCV harboured icaADBC genes and was found to be a proficient biofilm producer. This paper highlights the involvement of CNS SCVs in the aetiology of PJIs, including what is believed to be the first report of a S. warneri SCV. These subpopulations must be actively sought in the routine diagnosis of implant-associated infections. Moreover, in view of the phenotypic and genetic diversity of some SCV pairs, particular attention should be paid to the investigation of all types of observed colony morphologies, and isolates should be subjected to antimicrobial susceptibility testing.

The purpose of the study was to evaluate the usefulness of sonication for the diagnosis of prosthetic joint infections (PJIs) by its comparison with periprosthetic tissues (PTs) and synovial fluid (SV-F) cultures. The study groups included 54 patients undergoing exchange of total hip prostheses for so called "aseptic" loosening occurring without clinical manifestations of an accompanying PJI and 22 patients who developed a sinus tract communicating with the prosthesis which was indicative of an ongoing infectious process. Significant positive culture results were obtained among 10 (18.5%) patients with "aseptic" implant failure and in 18 (81.8%) patients who developed a sinus tract. Sonicate-fluid (S-F) yielded bacterial growth in all culture-positive patients with "aseptic" loosening vs. 15 patients with presumed PJIs. There was a concordance in terms of bacterial species isolated from S-F and conventional cultures from individual patients. Coagulase-negative staphylococci were isolated most frequently. Sensitivity of sonication (75%) exceeded that estimated for PTs (69%) and SV-F (45%) cultures. We conclude that identification of causative agents of PJIs which is critical to further therapeutic decisions is aided by the combination of sonication and conventional culture.

Staphylococcus epidermidis small colony variants can survive inside macrophages and their survival has been proposed as a pivotal process in the pathogenesis of biomaterial associated infections. In the present study the intracellular location of clinical isolates of SCV and parental wild type strains inside macrophages was determined. Furthermore, the effect of IFN-γ and rapamycin on the level of SCV/WT as well as lysosomes colocalisation and iNOS induction in THP-activated macrophages in response to WT and SCV strains of Staphylococcus epidermidis were examined. It was demonstrated that SCV strain of S. epidermidis can survive and persist inside macrophages and its intracellular survival is supported by the induction of phagosomal acidification. The ability to reduce the high proportion of LysoTracker positive SCV containing phagosomes was exclusively found when IFN-γ was used. The findings suggest that IFN-γ mediates SCV killing via two distinct mechanisms, phagosome alkalisation and an increased iNOS synthesis, so the cytokine may control S. epidermidis WT and SCV infection in macrophages. Staphylococcus epidermidis SCV is a less potent stimulus of iNOS than the WT strain and the feature may help SCV to persist in hostile environment of macrophages. Rapamycin treatment did not influence the iNOS synthesis but reduced the percentage of both bacterial strains within acidic organelles. However, the percentage of SCV within LysoTracker positive organelles, even though reduced comparing to non-primed cells, was higher than in the WT strain indicating that Staphylococcus epidermidis possesses unique metabolic features allowing SCV to survive within macrophages.

Staphylococcus epidermidis is commonly involved in biomaterial-associated infections. Bacterial small colony variants (SCV) seem to be well adapted to persist intracellularly in professional phagocytes evading the host immune response. We studied the expression of PD-L1/L2 on macrophages infected with clinical isolates of S. epidermidis SCV and their parent wild type (WT) strains. The cytokine pattern which is triggered by the examined strains was also analysed. In the study, we infected macrophages with S. epidermidis WT and SCV strains. Persistence and release from macrophages were monitored via lysostaphin protection assays. Moreover, the effect of IFN-γ pre-treatment on bacterial internalisation was investigated. Expression of PD-L1/L2 molecules was analysed with the use of FACS. Inflammatory reaction was measured by IL-10, TNF-α ELISAs, and transcriptional induction of TNF-α. Our study revealed that clinical SCV isolates were able to persist and survive in macrophages for at least 3 days with a low cytotoxic effect and a reduced proinflammatory response as compared to WT strains. Bacteria upregulated PD-L1/L2 expression on macrophages as compared to non-stimulated cells. The results demonstrated that the ability of S. epidermidis SCVs to induce elevated levels of anti-inflammatory cytokine, IL-10, and reduced transcriptional induction of TNF-α, together with expression of PD-L1 on macrophages and the ability to persist intracellularly without damaging the host cell could be the key factor contributing to chronicity of SCV infections.

Bacterial small colony variants represent an important aspect of bacterial variability. They are naturally occurring microbial subpopulations with distinctive phenotypic and pathogenic traits, reported for many clinically important bacteria. In clinical terms, SCVs tend to be associated with persistence in host cells and tissues and are less susceptible to antibiotics than their wild-type (WT) counterparts. The increased tendency of SCVs to reside intracellularly where they are protected against the host immune responses and antimicrobial drugs is one of the crucial aspects linking SCVs to recurrent or chronic infections, which are difficult to treat. An important aspect of the SCV ability to persist in the host is the quiescent metabolic state, reduced immune response and expression a changed pattern of virulence factors, including a reduced expression of exotoxins and an increased expression of adhesins facilitating host cell uptake. The purpose of this review is to describe in greater detail the currently available data regarding CoNS SCV and, in particular, their clinical significance and possible mechanisms by which SCVs contribute to the pathogenesis of the chronic infections. It should be emphasized that in spite of an increasing clinical significance of this group of staphylococci, the number of studies unraveling the mechanisms of CoNS SCVs formation and their impact on the course of the infectious process is still scarce, lagging behind the studies on S. aureus SCVs.