Search

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

Multidrug-resistant bacteria cause difficult-to-treat infections and pose a risk for modern medicine. Sources of multidrug-resistant bacteria include hospital, municipal and slaughterhouse wastewaters. In this study, bacteria with resistance to 3rd generation cephalosporins were isolated from all three wastewater biotopes, including a maximum care hospital, municipal wastewaters collected separately from a city and small rural towns and the wastewaters of two pig and two poultry slaughterhouses. The resistance profiles of all isolates against clinically relevant antibiotics (including β-lactams like carbapenems, the quinolone ciprofloxacin, colistin, and trimethoprim/sulfamethoxazole) were determined at the same laboratory. The bacteria were classified according to their risk to human health using clinical criteria, with an emphasis on producers of carbapenemases, since carbapenems are prescribed for hospitalized patients with infections with multi-drug resistant bacteria. The results showed that bacteria that pose the highest risk, i. e., bacteria resistant to all β-lactams including carbapenems and ciprofloxacin, were mainly disseminated by hospitals and were present only in low amounts in municipal wastewater. The isolates from hospital wastewater also showed the highest rates of resistance against antibiotics used for treatment of carbapenemase producers and some isolates were susceptible to only one antibiotic substance. In accordance with these results, qPCR of resistance genes showed that 90% of the daily load of carbapenemase genes entering the municipal wastewater treatment plant was supplied by the clinically influenced wastewater, which constituted approximately 6% of the wastewater at this sampling point. Likewise, the signature of the clinical wastewater was still visible in the resistance profiles of the bacteria isolated at the entry into the wastewater treatment plant. Carbapenemase producers were not detected in slaughterhouse wastewater, but strains harboring the colistin resistance gene mcr-1 could be isolated. Resistances against orally available antibiotics like ciprofloxacin and trimethoprim/sulfamethoxazole were widespread in strains from all three wastewaters.

WalKR (YycFG) is the only essential two-component regulator in the human pathogenStaphylococcus aureus.WalKR regulates peptidoglycan synthesis, but this function alone appears not to explain its essentiality. To understand WalKR function we investigated a suppressor mutant that arose when WalKR activity was impaired; a single histidine to tryptophan substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introduction of the WalKH271Ymutation into wild-typeS.aureusactivated the WalKR regulon. Structural analyses of the WalK PASCYTdomain revealed a hitherto unknown metal binding site, in which a zinc ion (Zn2+) was tetrahedrally-coordinated by four amino acid residues including H271. The WallkH271Ymutation abrogated metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR activity. Identification of a metal ligand sensed by the WalKR system substantially expands our understanding of this criticalS.aureusregulon.

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.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses like the cell wall stress response. However, (1) activity assays using the truncated phosphorylation domains have been shown to produce artifacts and (2) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from gram-positive bacteria that do not harbor more than two trans-membrane domains using an Escherichia coli host. This guide also presents in vitro phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases using radioactively labeled ATP and, as a novel approach, Phos-tag acrylamide gel electrophoresis to detect phosphorylated proteins by mobility shift in the polyacrylamide gel.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses like the cell wall stress response. However, (1) activity assays using the truncated phosphorylation domains have been shown to produce artifacts and (2) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from gram-positive bacteria that do not harbor more than two trans-membrane domains using an Escherichia coli host. This guide also presents in vitro phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases using radioactively labeled ATP and, as a novel approach, Phos-tag acrylamide gel electrophoresis to detect phosphorylated proteins by mobility shift in the polyacrylamide gel.

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).