No abstract

e Rapid diagnosis is essential for the management of Staphylococcus aureus infections. A host recognition protein from S. aureus bacteriophage phiSLT was recombinantly produced and used to coat streptavidin latex beads to develop a latex agglutination test (LAT). The diagnostic accuracy of this bacteriophage-based test was compared with that of a conventional LAT, Pastorex StaphPlus, by investigating a clinical collection of 86 S. aureus isolates and 128 coagulase-negative staphylococci (CoNS) from deep tissue infections. All of the clinical S. aureus isolates were correctly identified by the bacteriophage-based test. While most of the CoNS were correctly identified as non-S. aureus isolates, 7.9% of the CoNS caused agglutination. Thus, the sensitivity of the bacteriophage-based LAT for identification of S. aureus among clinical isolates was 100%, its specificity was 92.1%, its positive predictive value (PPV) was 89.6%, and its negative predictive value (NPV) was 100%. The sensitivity, specificity, PPV, and NPV of the Pastorex LAT for the identification of S. aureus were 100%, 99.2%, 98.9%, and 100%, respectively. Among the additionally tested 35 S. aureus and 91 non-S. aureus staphylococcal reference and type strains, 1 isolate was false negative by each system; 13 and 8 isolates were false positive by the bacteriophage-based and Pastorex LATs, respectively. The ability of the phiSLT protein to detect S. aureus was dependent on the presence of wall teichoic acid (WTA) and corresponded to the production of ribitol phosphate WTA, which is found in most S. aureus clones but only a small minority of CoNS. Bacteriophage-based LAT identification is a promising strategy for rapid pathogen identification. Finding more specific bacteriophage proteins would increase the specificity of this novel diagnostic approach.

Livestock-associated methicillin-resistant (LA-MRSA) isolates are increasingly migrating from livestock into human and animal health care settings. Alternative substances are needed to overcome the drawbacks of currently available drugs used for MRSA eradication. The recombinant bacteriophage endolysin HY-133 has proved to be an active agent against Here, the activity of HY-133 was studied against a large collection of genetically diverse LA-MRSA isolates revealing its high activity against-, -, and-positive LA-MRSA.

Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent’s stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.

Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 − log10 decrease in CFU/mL within 1 h compared to oxacillin, which required 4–24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.