- Volume 67, Issue 2, 2018

Purpose. Staphylococcus epidermidis colonies often display several morphologies and antimicrobial susceptibility patterns when cultured from device-related infections, and may represent one or multiple genotypes. Genotyping may be helpful in the clinical interpretation, but is time consuming and expensive. We wanted to establish a method for rapid discrimination of S. epidermidis genotypes for use in a routine microbiology laboratory.

Methodology. A real-time PCR targeting eight discriminatory class I or II single-nucleotide polymorphisms (SNPs) in six of the seven housekeeping genes was constructed. Post PCR, high-resolution melt (HRM) analysis using EvaGreen as fluorophore discriminated amplicons based on their percentage GC content.

Results. In silico, 42 representative sequence types (STs), including all major MLST group and subgroup founders, were separated into 23 different cluster profiles with a Simpson’s index of diversity of 0.97. By HRM-PCR, 11 commonly encountered hospital and outbreak STs were separated into eight HRM patterns.

Conclusion. This method can rapidly establish whether S. epidermidis strains belong to different genotypes. It can be used in patients with S. epidermidis infections, as an aid in outbreak investigations and to select strains for investigation with more discriminatory methods, saving workload and costs. Results may be obtained the same day as culture results. Its strength lies mainly in indicating differences, as some STs may have the same melt profile. Changes in S. epidermidis epidemiology may warrant alterations in the inclusion of SNPs. We believe this method can reduce the threshold for performing genotyping analysis on an increasingly important nosocomial pathogen.

Purpose. The aim of this study was to assess if use of the β LACTA test (BLT) for extended-spectrum beta-lactamase (ESBL) detection and/or early bacterial identification by mass spectrometry (MALDI-TOF MS) improves therapeutic decision-making when combined with advice from the antimicrobial stewardship team (AMST) for the management of Gram-negative bacillary (GNB) bacteraemia.

Methods. Prospective observational theoretical study that included patients with GNB bacteraemia during a 6-month period. We compared, against the antimicrobial choice of the local AMST as informed of the Gram-stain result, a hypothetical choice, i.e. one AMST would have made had it been informed of the MALDI-TOF MS results only (option H) with the actual choice AMST made after being informed of the combined MALDI-TOF MS and BLT results (option A).

Results/Key findings. A total of 131 episodes of GNB bacteraemia were included. Options H and A led to virtually the same rate of efficient antimicrobial therapy (in 120/131 and 123/131 episodes, respectively, P=0.63). Compared to the gold standard, options H and A did not lead to a significant reduction of carbapenem prescription (9/131, 6/131 and 12/131, P=0.57 and P=0.65, respectively).

Conclusions. Under our test conditions, BLT, when used in conjunction with MALDI-TOF MS and AMST advice, did not allow a significant optimization of the antimicrobial prescription made on the basis AMST advice only. However, the impact of BLT should be evaluated in a population with high prevalence of ESBL-producing Enterobacteriaceae and/or when treatment choices are not made by infectious disease specialists.

Purpose. Staphylococcus epidermidis is the predominant contaminant of platelet concentrates (PCs), a blood product used to treat patients with platelet deficiencies. This microorganism is able to form surface-attached aggregates (biofilms) in human skin. Herein, the abundance of S. epidermidis biofilm-producers in contaminated PCs compared to skin isolates was explored. Furthermore, the potential positive selection of S. epidermidis biofilm-producers during the blood donation process and PC manufacturing was investigated.

Methodology. Twenty-four S. epidermidis isolates obtained from contaminated PCs and 48 S. epidermidis isolates obtained from the venipuncture area of human volunteers were compared for their ability to form biofilms in laboratory media and in PCs using a semi quantitative crystal violet assay. Also, the presence of the biofilm-associated icaA and icaD genes was assessed by PCR-amplification.

Results/Key findings.Biofilm production in laboratory media showed a higher number of S. epidermidis biofilm-producers in the skin-derived group (43.7 %) compared to the PC-derived isolates (25 %). However, all skin and PC isolates formed biofilms in PCs. The prevalence of ica-positive biofilm-producer isolates was similar in PC and skin isolates (16.6 and 18.8 %, respectively). In contrast, the abundance of ica-negative biofilm-producers was lower in PC isolates compared to skin isolates (8.3 vs 25 %, respectively).

Conclusion. Positive selection of S. epidermidis biofilm-producers during blood donation and PC manufacturing was not observed. Interestingly, ica-negative biofilm-producers seem to be negatively affected by skin disinfection, blood processing and PC storage. Furthermore, this study shows that S. epidermidis adopts a biofilm-forming phenotype in PCs regardless of its genetic background or origin.

Purpose. The lack of novel antifungal drugs and the increasing incidence and severity of fungal infections are major concerns worldwide. Herein, we tested the activity of the Blad-containing oligomer (BCO), a new antifungal molecule already in use for agriculture, on Malassezia spp. and dermatophytes, the causal agents of human tinea versicolor and tinea pedis. Given the lack of a standard method for Malassezia susceptibility testing and the plethora of published methods, we also developed an improved method for this genus.

Methodology. The efficacy of BCO was assessed in vitro and compared to that of the drugs currently utilized in the treatment of tinea versicolor (fluconazole and itraconazole) and tinea pedis (itraconazole and terbinafine). For dermatophytes, the standard microdilution broth-based method was used, with small adjustments, and several broth formulations and inocula sizes were tested to develop an improved susceptibility method for Malassezia spp.

Results. We successfully developed a microdilution broth-based method with considerable advantages over other available methods, and used it for all in vitro susceptibility tests of Malassezia spp. isolates. We report that, on a molar basis, BCO was more effective than fluconazole or itraconazole on most strains of Malassezia spp. isolated from clinical samples (n=29). By contrast, BCO was less effective than itraconazole or terbinafine on the common dermatophytes Trichophyton rubrum and Trichophyton interdigitale.

Conclusion. These data place BCO as a promising drug for the treatment of Malassezia-associated skin diseases. Further in vivo studies are now required to ascertain its applicability in the clinical setting.

Purpose. Previous research demonstrated that IL-10 was up-regulated in Chlamydia trachomatis-infected cells and that exogenous IL-10 was able to inhibit the secretion of pro-inflammatory cytokines by infected cells. However, the mechanisms are not well understood. The aim of this study was to investigate the mechanisms for up-regulation of IL-10 and inhibition of pro-inflammatory cytokine secretion in C. trachomatis-stimulated peripheral blood mononuclear cells (PBMCs).

Methodology. Human PBMCs were isolated from the blood of healthy human donors by standard Ficoll–Hypaque density gradient centrifugation. Cells were exposed to C. trachomatis in the presence or absence of MEK inhibitor U0126, the p38 inhibitor SB203580, the STAT3 inhibitor Ruxolitinib or anti-human IL-10 antibody. Cytokines were measured from culture supernatants using ELISA kits. Cells were harvested for real-time quantitative PCR to determine IL-10 mRNA levels and for Western blot assay to detect the expression of ERK1/2, p-ERK1/2, p38, p-p38, STAT3 and p-STAT3.

Results. Both mRNA and protein levels of IL-10 were up-regulated in stimulated cells, and the production of IL-10 was reduced when cells were treated with U0126 or SB203580. The expression of cytokines IL-6, IL-8 and TNF-α was enhanced in stimulated cells treated with anti-human IL-10 antibody. Moreover, neutralization of IL-10 resulted in a significant decrease of phosphorylated STAT3 in stimulated cells. Ruxolitinib caused a significant increase in the production of IL-6, IL-8 and TNF-α in stimulated cells.

Conclusion. IL-10 is up-regulated in an ERK- and p38-dependent fashion in stimulated human PBMCs. IL-10 inhibits the production of pro-inflammatory cytokines by activating the JAK/STAT signalling pathway.

Purpose. Glycopeptides are widely used for the treatment of meticillin-resistant Staphylococcus aureus (MRSA) infections. Although difficult to detect, isolates with reduced (GISA), hetero (hGISA) or complete (GRSA) resistance to glycopeptides are increasingly reported. Optimal therapy for such strains is unknown. We compared the in vitro and in vivo activity of tedizolid (TED), a recently licensed oxazolidonone, with vancomycin (VAN) and teicoplanin (TEIC) combined with fusidic acid (FD) or rifampicin (RIF) against S. aureus (SA) with reduced susceptibility to glycopeptides.

Methods. Susceptibility was determined for six (GISA, hGISA and GRSA) reference strains and 72 clinical MRSA isolates screened for hGISA/GISA-like phenotypes. Synergy and bactericidal activity were assessed using chequerboard and time-kill assays. The G. mellonella wax moth caterpillar model was used to measure the activity of TED and the combinations in vivo.

Results. Glycopeptide MICs (VAN/TEIC) ranged from 0.5–8/4 and 0.125–1 for TED. No significant synergy was noted when VAN/TEIC were combined with either RIF or FD. Time-kill assays confirmed that TED was bacteriostatic but superior to VAN and TEIC against GISA strains. In G. mellonella TED was more effective than TEIC monotherapy versus GISA strains. The combination of TEIC with RIF was the most effective combination overall, both in vitro and in vivo.

Conclusions. TED had good in vitro activity versus MRSA including those with reduced susceptibility to glycopeptides. Although bacteriostatic, it was effective in the G. mellonella model and superior to TEIC in the treatment of GISA. Although this supports the use of TED for MRSA and GISA, the TEIC/RIF combination also warrants further study.