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Carbapenem antibiotics constitute the mainstay therapy of nosocomial infections with extended spectrum beta-lactamase producing Gram-negative bacteria; however, resistance against these compounds is increasing. This study was designed to demonstrate that carbapenemase-producing bacteria are disseminated from hospitals into the environment. To this end, resistant bacteria were isolated from a clinical/urban and from a rural catchment system in Germany in 2016/17. The study followed the dissemination of resistant bacteria from the wastewater through the wastewater treatment plant (WWTP) into the receiving surface waters. The bacteria were cultivated on selective agar and characterized by antibiotic testing, real-time PCR targeting carbapenemase genes and typing. Bacteria with resistance to third generation cephalosporins were isolated from all sample sites. 134 isolates harboring carbapenemase genes encoding VIM, NDM and OXA-48 and 26 XDR (extensively drug-resistant) strains with susceptibility to only one or two antibiotics were isolated from the clinical/urban system. The rural system yielded eight carbapenemase producers and no XDR strains. In conclusion, clinical wastewaters were charged with a high proportion of multidrug resistant bacteria. Although most of these bacteria were eliminated during wastewater treatment, dissemination into surface waters is possible as single carbapenemase producers were still present in the effluent of the WWTP.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

Summary Background The current increase in nosocomial infections caused by vancomycin-resistant enterococci (VRE) warrants improvement of detection methods and hygiene measures. Knowledge of the local epidemiology is important for monitoring compliance of medical personnel with hygiene measures. Aim To evaluate semi-automated repetitive element palindromic polymerase chain reaction (rep-PCR) for rapid molecular typing of VRE. Methods Primary VRE isolates were collected during an observation period of one year and retrospectively typed by rep-PCR. Molecular typing was performed on isolates from two departments with elevated VRE rates and patients with increased risk for systemic VRE infections. Typing results were correlated with temporal and spatial information on patient moves, VRE laboratory results and multi-locus sequence typing (MLST). Findings Approximately 70% of VRE isolates within a department could be assigned to similarity clusters. Spread of VRE was limited to the individual departments. There was no evidence for spread of endemic VRE strains within the geographical catchment area of the hospital. Our results demonstrate the utility of rep-PCR typing on a department level. However, a Diversilab® threshold of ≥98% had to be applied to claim similarity, and suspected transmissions needed to be confirmed by vanA/B genotyping and compiled information on spatial and temporal patient contact. MLST verified the findings. Conclusion Spread of predominantly detected vancomycin-resistant Enterococcus faecium was limited to the department level with no evidence for wider dissemination within the hospital. Well-standardized and validated (semi-)automated rep-PCR systems are useful for rapid detection of possible VRE transmission. However, suspected transmissions need to be confirmed by clinical and microbiological parameters.

The assembly of the bacterial cell wall requires synchronization of a multitude of biosynthetic machineries and regulatory networks. The eukaryotic-like serine/threonine kinase PknB has been implicated in coordinating cross-wall formation, autolysis and cell division in Staphylococcus aureus. However, the signal molecule sensed by this kinase remained elusive so far. Here, we provide compelling biochemical evidence that PknB interacts with the ultimate cell wall precursor lipid II, triggering kinase activity. Moreover, we observed crosstalk of PknB with the two component system WalKR and identified the early cell division protein FtsZ as another PknB phosphorylation substrate in S. aureus. In agreement with the implied role in regulation of cell envelope metabolism, we found PknB to preferentially localize to the septum of S. aureus and the PASTA domains to be crucial for recruitment to this site. The data provide a model for the contribution of PknB to control cell wall metabolism and cell division.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice., This work was supported by a grant of the Deutsches Zentrum für Infektionsforschung (DZIF) to Gabriele Bierbaum (TTU 08.902) and the Fundação para a Ciência e a Tecnologia (FCT) to Luís D.R. Melo (SFRH/BD/66166/2009) as well as by the BONFOR funds of the Medical Faculty of the University of Bonn and funds of the Centre International de Recherches Médicales de Franceville (CIRMF) and the Institut de Recherche pour le Développement (IRD).

Background Nasal carriage with Staphylococcus aureus is a common risk factor for invasive infections, indicating the necessity to monitor prevalent strains, particularly in the vulnerable paediatric population. This surveillance study aims to identify carriage rates, subtypes, antimicrobial susceptibilities and virulence markers of nasal S. aureus isolates collected from children living in the Ashanti region of Ghana. Methods Nasal swabs were obtained from children < 15 years of age on admission to the Agogo Presbyterian Hospital between April 2014 and January 2015. S. aureus isolates were characterized by their antimicrobial susceptibility, the presence of genes encoding for Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) and further differentiated by spa-typing and multi-locus-sequence-typing. Results Out of 544 children 120 (22.1%) were colonized with S. aureus, with highest carriage rates during the rainy seasons (27.2%; p = 0.007), in females aged 6–8 years (43.7%) and males aged 8–10 years (35.2%). The 123 isolates belonged to 35 different spa-types and 19 sequence types (ST) with the three most prevalent spa-types being t355 (n = 25), t84 (n = 18), t939 (n = 13), corresponding to ST152, ST15 and ST45. Two (2%) isolates were methicillin-resistant S. aureus (MRSA), classified as t1096 (ST152) and t4454 (ST45), and 16 (13%) were resistant to three or more different antimicrobial classes. PVL and TSST-1 were detected in 71 (58%) and 17 (14%) isolates respectively. Conclusion S. aureus carriage among Ghanaian children seems to depend on age, sex and seasonality. While MRSA rates are low, the high prevalence of PVL is of serious concern as these strains might serve not only as a source for severe invasive infections but may also transfer genes, leading to highly virulent MRSA clones.