An advanced methicillin-resistant Staphylococcus aureus (MRSA) detection PCR approach targeting SCCmec-orfX along with mecA and mecC was evaluated for S. aureus and coagulase-negative staphylococci. The possession of mecA and/or mecC was correctly confirmed in all cases. All methicillin-susceptible S. aureus strains (n ‫؍‬ 98; including staphylococcal cassette chromosome mec element [SCCmec] remnants) and 98.1% of the MRSA strains (n ‫؍‬ 160, including 10 mecC-positive MRSA) were accurately categorized.

BackgroundVancomycin resistant enterococci (VRE) occur with enhanced frequency in hospitalised patients. This study elucidates the prevalence of VRE on admission among surgical intensive care unit (SICU) patients, whether these patients are at special risk for VRE acquisition and which risk factors support this process.MethodsPatients admitted to SICUs of the University Hospital Münster were examined during August–October 2017. VRE screening was performed within 48 h after admission and directly prior to discharge of patients. In parallel risk factors were recorded to estimate their effect on VRE acquisition during SICU stay.ResultsIn total, 374 patients (68% male) with a median age of 66 years were admitted to one of the SICUs during the investigation period. Of all, 336 patients (89.8%) were screened on admission and 268 (71.7%) on discharge. Nine patients were admitted with previously known VRE colonisation. Twelve (3.6%) further patients were VRE positive on admission. During ICU stay, eight (3.0%) additional patients turned out to be VRE colonised. Risk factors found to be significantly associated with VRE acquisition were median length of stay on the ICU (14 vs. 3 days; p = 0.01), long-term dialysis (12.5% vs. 2.0% of patients; p = 0.05), and antibiotic treatment with flucloxacillin (28.6% vs. 7.2% of patients; p = 0.01) or piperacillin/tazobactam (57.1% vs. 26.6% of patients; p = 0.01).ConclusionsSICU patients are not at special risk for VRE acquisition. Previous stay on a SICU should therefore not be considered as specific risk factor for VRE colonisation.

Objectives: Increasing resistance of microorganisms and particularly tolerance of bacterial biofilms against antibiotics require the need for alternative antimicrobial substances. S. aureus is the most frequent pathogen causing vascular graft infections. In order to evaluate the antimicrobial efficacy, quantification of the bacterial biofilms is necessary. Aim of the present study was the validation of an in vitro model for quantification of bacterial biofilm on vascular graft surfaces using three different assays.Methods: Standardized discs of vascular graft material (Dacron or PTFE) or polystyrene (PS) as control surface with 0.25 cm2 surface area were inoculated with 10−3 diluted overnight culture of three biofilm-producing S. aureus isolates (BEB-029, BEB-295, SH1000) in 96-well PS culture plates. After incubation for 4 and 18 h, the biofilm was determined by three different methods: (a) mitochondrial ATP concentration as measure of bacterial viability (ATP), (b) crystal violet staining (Cry), and (c) vital cell count by calculation of colony-forming units (CFU). The experiments were performed three times. Quadruplicates were used for each isolate, time point, and method. In parallel, bacterial biofilms were documented via scanning electron microscopy.Results: All three methods could quantify biofilms on the PS control. Time needed was 0:40, 13:10, and 14:30 h for ATP, Cry, and CFU, respectively. The Cry assay could not be used for vascular graft surfaces due to high unspecific background staining. However, ATP assay and CFU count showed comparable results on vascular graft material and control. The correlations between ATP and CFU assay differed according to the surface and incubation time and were significant only after 4 h on Dacron (BEB-029, p = 0.013) and on PS (BEB-029, p < 0.001). Between ATP and Cry assay on PS, a significant correlation could be detected after 4 h (BEB-295, p = 0.027) and after 18 h (all three strains, p < 0.026). The reproducibility of the ATP-assay presented as inter-assay-variance of 2.1 and as intra-assay variance of 8.1 on polystyrene.Conclusion: The in-vitro model reproducibly quantifies biofilm on standardized vascular graft surfaces with ATP assay as detection system. The ATP assay allows accelerated microbial quantification, however the correlation with the CFU assay may be strain- and surface-dependent.

d HY-133 is a recombinant bacteriophage endolysin with bactericidal activity against Staphylococcus aureus. Here, HY-133 showed in vitro activity against major African methicillin-susceptible and methicillin-resistant S. aureus lineages and ceftaroline/ceftobiprole-and borderline oxacillin-resistant isolates. HY-133 was also active against Staphylococcus schweitzeri, a recently described species of the S. aureus complex. The activity of HY-133 on the tested isolates (MIC 50 , 0.25 g/ml; MIC 90 , 0.5 g/ml; range, 0.125 to 0.5 g/ml) was independent of the species and strain background or antibiotic resistance. Bacteriophage endolysins are peptidoglycan hydrolases with strong species-specific bactericidal activity (1). Endolysins are considered a potential new class of antimicrobial agents which could be used both for decolonization of methicillin-resistant Staphylococcus aureus (MRSA) and invasive infections, as animal models have revealed (1). Indeed, there is a need for the development of new antimicrobial agents, in particular against S. aureus infections, as resistance rates are rising not only in industrialized countries but also in so-called developing regions, such as subSaharan Africa (2).Similar to its progenitor PRF-119 (3), HY-133 (owned by Hyglos GmbH, Bernried, Germany) is an optimized, recombinantly produced chimeric bacteriophage endolysin which has a pronounced bactericidal activity against S. aureus and an increased proteolytic stability due to a shortened linker peptide (4). These chimeric endolysins consist of an enzymatic active domain (EAD), a cysteine-and histidine-dependent aminopeptidase/hydrolase (CHAP) from the endolysin of phage K, and a cell wallbinding domain (CBD) from lysostaphin (3, 4).While the progenitor molecule PRF-119 demonstrated activity against all clinical S. aureus isolates collected from multiple centers in a European country (3), the present study aimed at challenging the optimized bacteriophage endolysin HY-133 with isolates belonging to the S. aureus complex from sub-Sahara Africa, including the recently described species Staphylococcus schweitzeri (5, 6). This species shows a peptidoglycan type which is distinct from that of S. aureus sensu stricto (5).Here, we tested the in vitro activity of HY-133 against a highly diverse collection of African S. aureus complex lineages (n ϭ 61). This collection consists of major African methicillin-susceptible S. aureus (MSSA), as well as methicillin-, ceftaroline/ceftobiprole-, and borderline oxacillin-resistant S. aureus from humans (colonization and infection) and animals (colonization) ( Table 1). The most common African MSSA and MRSA included in this study represent lineages which have been found to be predominant in sub-Saharan Africa based on a recent review (7). We also included 25 isolates from different clonal lineages of S. schweitzeri. The multilocus sequence types (ST) of the isolates included in this study are given in Table 1. The activity of HY-133 was determined using the broth microdilution method in accordance wi...

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Health care-associated methicillin-resistant (MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs ( = 1,103) and flocked swab (ESwab) nasal specimens ( = 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens.

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We developed a methodology for antimicrobial susceptibility testing (AST) based on the BacterioScanTM216R laser scattering technology, using methicillin resistance in Staphylococcus aureus and vancomycin resistance in enterococci as exemplar for important resistance phenotypes. Fifty methicillin-resistant (MRSA) and 50 methicillin-susceptible (MSSA) S. aureus, as well as 50 vancomycin-resistant enterococci (VRE) and 50 vancomycin-susceptible enterococci (VSE) isolates were used for the study. Optimal test conditions were derived by investigating the effects of inoculum size, medium, incubation temperature and broth filtration. We proposed four different statistical approaches for rapid discrimination between resistant and susceptible bacteria. The statistical approach based on raw measurements of bacterial concentrations delivered sensitivity of 100% and specificity of 94% for discrimination between MRSA and MSSA already after 3 hours of incubation. Categorical agreement of ≥90% was achieved after 140 min with this approach. Differentiation between VRE and VSE was possible with 98% sensitivity and 92% specificity after 3 hours, using a sophisticated statistical approach based on concentration slopes derived from the raw concentration measurements. This approach provided categorical agreement of ≥90% after 165 min. The sensitivity and specificity estimates were confirmed by leave-one-out cross validation. In conclusion, the phenotypic AST methods developed in this study are promising for rapid detection of MRSA and VRE. The development and application of this technology would allow early detection of the resistant pathogens, thus facilitating swift change to the targeted antimicrobial treatment as well as timely initiation of appropriate infection control measures. Further studies are warranted to validate this approach for the detection of other resistance phenotypes, including direct testing from clinical specimens.