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Vancomycin-resistant enterococci (VRE) have been isolated in increasing numbers. Hospital-adapted VRE exhibit relatively high pathogenicity by expressing factors like enterococcal surface protein (Esp), which facilitates epidemic spread. By contrast, 'community-acquired' VRE show low pathogenicity and non-epidemic features. In 2004 and 2005 an extended outbreak of VRE occurred at a university hospital in Southwestern Germany and an infection control programme was implemented to confine the outbreak. Pulsed-field gel electrophoresis (PFGE), esp PCR, multiple-locus variable number of tandem repeat analysis (MLVA), purK1 typing and multiple-locus sequence typing (MLST) were performed on representative VRE isolates. Twenty-six non-epidemic and two epidemic VRE types (MLST203, MLST280) were identified by PFGE. Seven of the non-outbreak VRE types were esp gene negative, whereas 19 non-outbreak and both epidemic VRE types were esp positive. Eight MLVA types were identified. MLVA type 1 included five PFGE types and MLVA type 159 included 16 PFGE types. Currently there is no efficient method available to identify non-epidemic VRE and avoid unnecessary isolation of patients. More than 50% non-epidemic clones were esp positive; nevertheless, esp PCR appears to be the most promising approach to identify non-epidemic VRE. Copyright © 2007 The Hospital Infection Society [ABSTRACT FROM AUTHOR]

Objectives: To determine prevalence, incidence, and factors associated with Pseudomonas aeruginosa (PA) intestinal carriage in residents of long-term care facilities (LTCFs) and to understand the population structure of this pathogen in LTCFs from two European countries., Methods: We assessed the prevalence of PA intestinal carriage and the incidence of acquisition by collecting fecal samples from 403 residents of 20 LTCFs. We collected 289 environmental samples from sinks and drinking water. Factors associated with carriage and acquisition of intestinal PA were identified. All PA isolates had their antibiotic phenotypic resistance profile determined and their genome sequenced, from which we assessed the population structure of the collection and identified resistance determinants., Results: We found a high proportion of residents with PA intestinal carriage (51.6%) over the entire study period. Over the follow-up period, 28.6% of the residents acquired intestinal PA. Older age (OR, 1.29; 95% CI, 1.09-1.52; p = 0.002), urinary incontinence (OR, 2.56; 95% CI, 1.37-4.88; p = 0.003), and male sex (OR, 2.55; 95% CI, 1.05-6.18; p = 0.039) were associated with higher probability of carriage. Wheelchair usage (OR, 4.56; 95% CI, 1.38-15.05; p = 0.013) and a body mass index >25 (OR, 3.71; 95% CI, 1.17-11.82; p = 0.026) were associated with higher risk of PA acquisition. Population structure of our isolates was mainly non-clonal with 112 different STs among the 241 isolates. Most represented STs were high risk clones ST253 (n = 26), ST17 (n = 11), ST244 (n = 11), ST309 (n = 10), and ST395 (n = 10). Most PA isolates (86.3%) were susceptible to antibiotics, with no acquired genes conferring resistance to antipseudomonal agents., Discussion: We found an unexpected high prevalence of PA intestinal carriage in LTCF residents mainly associated with individual-level factors. Our study revealed a polyclonal PA population structure suggesting that individual acquisition is more frequent than resident-to-resident transmission., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The complex interplay of a pathogen with its virulence and fitness factors, the host's immune response, and the endogenous microbiome determine the course and outcome of gastrointestinal infection. The expansion of a pathogen within the gastrointestinal tract implies an increased risk of developing severe systemic infections, especially in dysbiotic or immunocompromised individuals. We developed a mechanistic computational model that calculates and simulates such scenarios, based on an ordinary differential equation system, to explain the bacterial population dynamics during gastrointestinal infection. For implementing the model and estimating its parameters, oral mouse infection experiments with the enteropathogen, Yersinia enterocolitica (Ye), were carried out. Our model accounts for specific pathogen characteristics and is intended to reflect scenarios where colonization resistance, mediated by the endogenous microbiome, is lacking, or where the immune response is partially impaired. Fitting our data from experimental mouse infections, we can justify our model setup and deduce cues for further model improvement. The model is freely available, in SBML format, from the BioModels Database under the accession number MODEL2002070001.

Background: Asymptomatic C. difficile colonization is believed to predispose to subsequent C. difficile infection (CDI). While emerging insights into the role of the commensal microbiota in mediating colonization resistance against C. difficile have associated CDI with specific microbial components, corresponding prospectively collected data on colonization with C. difficile are largely unavailable., Methods: C. difficile status was assessed by GDH EIA and real-time PCR targeting the toxin A (tcdA) and B (tcdB) genes. 16S V3 and V4 gene sequencing results from fecal samples of patients tested positive for C. difficile were analyzed by assessing alpha and beta diversity, LefSe, and the Piphillin functional inference approach to estimate functional capacity., Results: 1506 patients were recruited into a prospective observational study (DRKS00005335) upon admission into one of five academic hospitals. 936 of them provided fecal samples on admission and at discharge and were thus available for longitudinal analysis. Upon hospital admission, 5.5% (83/1506) and 3.7% (56/1506) of patients were colonized with toxigenic (TCD) and non-toxigenic C. difficile (NTCD), respectively. During hospitalization, 1.7% (16/936) acquired TCD. Risk factors for acquisition of TCD included pre-existing lung diseases, lower GI endoscopy and antibiotics. Species protecting against hospital-related C. difficile acquisition included Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococcus spp. Metagenomic pathway analysis identified steroid biosynthesis as the most underrepresented metabolic pathway in patients who later acquire C. difficile colonization., Conclusions: Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococci were associated with a decreased risk of C. difficile acquisition., Clinical Trials Registration: DRKS00005335., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC, and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface., Importance: The secretion of trimeric autotransporters (TAA's) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rooke, Icke, Wells, Rossiter, Browning, Morris, Leo, Schütz, Autenrieth, Cunningham, Linke and Henderson.)

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal β-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in β-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa ., (Copyright © 2020 Sonnabend et al.)

Members of the Enterobacter ( E. ) cloacae complex have emerged as important pathogens frequently encountered in nosocomial infections. Several outbreaks with E. cloacae complex have been reported in recent years, especially in neonatal units. Fast and reliable strain typing methods are crucial for real-time surveillance and outbreak analysis to detect pathogen reservoirs and transmission routes. The aim of this study was to evaluate the performance of Fourier-transform infrared (FTIR) spectroscopy as a fast method for typing of clinical E. cloacae complex isolates, when whole genome sequencing (WGS) analysis was used as reference. First, the technique was used retrospectively on 24 first isolates of E. cloacae complex strains from neonatal patients and showed good concordance with SNP-based clustering [adjusted rand index (ARI) = 0.818] and with the sequence type (ST) (ARI = 0.801). 29 consecutive isolates from the same patients were shown by WGS analysis to almost always belong to the same SNP cluster as the first isolates, which was only inconsistently recognized by FTIR spectroscopy. Training of an artificial neural network (ANN) with all FTIR spectra from sequenced strains markedly improved the recognition of related and unrelated isolate spectra. In a second step, FTIR spectroscopy was applied on 14 strains during an outbreak with E. cloacae complex and provided fast typing results that were confirmed by WGS analysis. In conclusion, FTIR spectroscopy is a promising tool for strain typing of clinical E. cloacae complex strains. Discriminatory power can be improved by implementing an ANN for spectrum analysis. Due to its low costs and fast turnaround times, the method presents a valuable tool for real-time surveillance as well as outbreak analysis., (Copyright © 2019 Vogt, Löffler, Dinkelacker, Bader, Autenrieth, Peter and Liese.)

Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c + cells. Thus, weak agonistic LPS represents a promising agent to treat diseases involving pathological overactivation of the intestinal immune system, e.g., in inflammatory bowel diseases., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The selection pressure exercised by antibiotic drugs is an important consideration for the wise stewardship of antimicrobial treatment programs. Treatment decisions are currently based on crude assumptions, and there is an urgent need to develop a more quantitative knowledge base that can enable predictions of the impact of individual antibiotics on the human gut microbiome and resistome., Results: Using shotgun metagenomics, we quantified changes in the gut microbiome in two cohorts of hematological patients receiving prophylactic antibiotics; one cohort was treated with ciprofloxacin in a hospital in Tübingen and the other with cotrimoxazole in a hospital in Cologne. Analyzing this rich longitudinal dataset, we found that gut microbiome diversity was reduced in both treatment cohorts to a similar extent, while effects on the gut resistome differed. We observed a sharp increase in the relative abundance of sulfonamide antibiotic resistance genes (ARGs) by 148.1% per cumulative defined daily dose of cotrimoxazole in the Cologne cohort, but not in the Tübingen cohort treated with ciprofloxacin. Through multivariate modeling, we found that factors such as individual baseline microbiome, resistome, and plasmid diversity; liver/kidney function; and concurrent medication, especially virostatic agents, influence resistome alterations. Strikingly, we observed different effects on the plasmidome in the two treatment groups. There was a substantial increase in the abundance of ARG-carrying plasmids in the cohort treated with cotrimoxazole, but not in the cohort treated with ciprofloxacin, indicating that cotrimoxazole might contribute more efficiently to the spread of resistance., Conclusions: Our study represents a step forward in developing the capability to predict the effect of individual antimicrobials on the human microbiome and resistome. Our results indicate that to achieve this, integration of the individual baseline microbiome, resistome, and mobilome status as well as additional individual patient factors will be required. Such personalized predictions may in the future increase patient safety and reduce the spread of resistance., Trial Registration: ClinicalTrials.gov, NCT02058888 . Registered February 10 2014.

In this study, we aimed to elucidate a prolonged outbreak of extensively drug-resistant (XDR) Pseudomonas aeruginosa , at two adjacent hospitals over a time course of 4 years. Since all strains exhibited a similar antibiotic susceptibility pattern and carried the carbapenemase gene bla VIM , a monoclonal outbreak was assumed. To shed light on the intra-hospital evolution of these strains over time, whole genome sequence (WGS) analysis of 100 clinical and environmental outbreak strains was employed. Phylogenetic analysis of the core genome revealed the outbreak to be polyclonal, rather than monoclonal as initially suggested. The vast majority of strains fell into one of two major clusters, composed of 27 and 59 strains, and their accessory genome each revealed over 400 and 600 accessory genes, respectively, thus indicating an unexpectedly high structural diversity among phylogenetically clustered strains. Further analyses focused on the cluster with 59 strains, representing the hospital from which both clinical and environmental strains were available. Our investigation clearly shows both accumulation and loss of genes occur very frequently over time, as reflected by analysis of protein enrichment as well as functional enrichment. In addition, we investigated adaptation through single nucleotide polymorphisms (SNPs). Among the genes affected by SNPs, there are a multidrug efflux pump ( mexZ ) and a mercury detoxification operon ( merR ) with deleterious mutations, potentially leading to loss of repression with resistance against antibiotics and disinfectants. Our results not only confirm WGS to be a powerful tool for epidemiologic analyses, but also provide insights into molecular evolution during an XDR P. aeruginosa hospital outbreak. Genome mutation unveiled a striking genetic plasticity on an unexpectedly high level, mostly driven by horizontal gene transfer. Our study adds valuable information to the molecular understanding of "real-world" Intra-hospital P. aeruginosa evolution and is a step forward toward more personalized medicine in infection control.

Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-β-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22-mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow-chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients., Competing Interests: The authors have declared that no competing interests exist.

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the β-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC , of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.

Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae , K. variicola , and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS., (Copyright © 2018 American Society for Microbiology.)

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.

The metallo-beta-lactamase GIM-1 has been found in various bacterial host species nearly exclusively in western Germany. However, not much is known about the epidemiology of GIM-1-positive Serratia marcescens Here we report on a surprisingly protracted regional dissemination. In-hospital transmission was investigated by using conventional epidemiological tools to identify spatiotemporal links. Strain typing was performed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Bayesian phylogeny was used to infer the time axis of the observed occurrence. Thirteen S. marcescens strains from 10 patients from 6 different German hospitals were investigated. Suspected in-hospital transmissions were confirmed by molecular typing at a higher resolution by WGS than by PFGE. A detailed sequence analysis demonstrated the spread of one predominant strain variant but also provided evidence for transfer of the bla GIM-1 gene cassette between different strains. A Bayesian phylogenetic analysis showed that the most recent common ancestor of the identified clonal cluster could be dated back to April 1993 (95% highest posterior density interval, January 1973 to March 2003) and that this strain might have already harbored the bla GIM-1 at that time and, therewith, years before the first detection of this resistance gene in clinical specimens. This study shows a long-standing clonal and plasmid-mediated expansion of GIM-1-producing S. marcescens that might have gone unnoticed in the absence of a standardized and effective molecular screening for carbapenemases. The systematic and early detection of resistance is thus highly advisable, especially for the prevention of potentially long-term dissemination that may progress beyond control., (Copyright © 2017 American Society for Microbiology.)

Competing Interests: Compliance with ethical standards Funding German Research Foundation (DFG) Priority Program 1656; German Center for Infection Research, DZIF.

Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis., (© 2016 S. Karger AG, Basel.)

β-Barrel proteins are found in the outer membrane (OM) of Gram-negative bacteria, chloroplasts and mitochondria. The assembly of these proteins into the corresponding OM is facilitated by a dedicated protein complex that contains a central conserved β-barrel protein termed BamA in bacteria and Tob55/Sam50 in mitochondria. BamA and Tob55 consist of a membrane-integral C-terminal domain that forms a β-barrel pore and a soluble N-terminal portion comprised of one (in Tob55) or five (in BamA) polypeptide transport-associated (POTRA) domains. Currently the functional significance of this difference and whether the homology between BamA and Tob55 can allow them to replace each other are unclear. To address these issues we constructed hybrid Tob55/BamA proteins with differently configured N-terminal POTRA domains. We observed that constructs harboring a heterologous C-terminal domain could not functionally replace the bacterial BamA or the mitochondrial Tob55 demonstrating species-specific requirements. Interestingly, the various hybrid proteins in combination with the bacterial chaperones Skp or SurA supported to a variable extent the assembly of bacterial β-barrel proteins into the mitochondrial OM. Collectively, our findings suggest that the membrane assembly of various β-barrel proteins depends to a different extent on POTRA domains and periplasmic chaperones.

Cathepsin S (CTSS) is a lysosomal protease whose activity regulation is important for MHC-II signaling and subsequent activation of CD4 + T cell mediated immune responses. Dysregulation of its enzymatic activity or enhanced secretion into extracellular environments is associated with the induction or progression of several autoimmune diseases. Here we demonstrate that commensal intestinal bacteria influence secretion rates and intracellular activity of host CTSS and that symbiotic bacteria, i.e. Bacteroides vulgatus mpk, may actively regulate this process and help to maintain physiological levels of CTSS activities in order to prevent from induction of pathological inflammation. The symbiont-controlled regulation of CTSS activity is mediated by anticipating reactive oxygen species induction in dendritic cells which, in turn, maintains cystatin C (CysC) monomer binding to CTSS. CysC monomers are potent endogenous CTSS inhibitors. This Bacteroides vulgatus caused and CysC dependent CTSS activity regulation is involved in the generation of tolerant intestinal dendritic cells contributing to prevention of T-cell mediated induction of colonic inflammation. Taken together, we demonstrate that symbionts of the intestinal microbiota regulate host CTSS activity and secretion and might therefore be an attractive approach to deal with CTSS associated autoimmune diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Most frequently, gram-negative bacterial infections in humans are caused by Enterobacteriaceae and remain a major challenge in medical diagnostics. We non-invasively imaged moderate and severe systemic Yersinia enterocolitica infections in mice using the positron emission tomography (PET) tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), which is a marker of proliferation, and compared the in vivo results to the ex vivo biodistributions, bacterial loads, and histologies of the corresponding organs. Y. enterocolitica infection is detectable with histology using H&E staining and immunohistochemistry for Ki 67. [18F]FLT revealed only background uptake in the spleen, which is the main manifestation site of systemic Y. enterocolitica-infected mice. The uptake was independent of the infection dose. Antibody-based thymidine kinase 1 (Tk-1) staining confirmed the negative [18F]FLT-PET data. Histological alterations of spleen tissue, observed via Ki 67-antibody-based staining, can not be detected by [18F]FLT-PET in this model. Thus, the proliferation marker [18F]FLT is not a suitable tracer for the diagnosis of systemic Y. enterocolitica infection in the C57BL/6 animal model of yersiniosis., Competing Interests: BJP received grant/research support from AstraZeneca, Bayer Healthcare, Boehringer-Ingelheim, Bruker, Oncodesign, Merck and Siemens. This did not alter our adherence to PLOS ONE policies on sharing data and materials.

Enteropathogenic Yersinia enterocolitica (Ye) enters the host via contaminated food. After colonisation of the small intestine Ye invades the Peyer's patches (PPs) via M cells and disseminates to the mesenteric lymph nodes (MLNs), spleen and liver. Whether Ye uses other invasion routes and which pathogenicity factors are required remains elusive. Oral infection of lymphotoxin-β-receptor deficient mice lacking PPs and MLNs with Ye revealed similar bacterial load in the spleen 1h post infection as wild-type mice, demonstrating a PP-independent dissemination route for Ye. Immunohistological analysis of the small intestine revealed Ye in close contact with mononuclear phagocytes (MPs), specifically CX3CR1(+) monocyte-derived cells (MCs) as well as CD103(+) dendritic cells (DCs). This finding was confirmed by flow cytometry and imaging flow cytometry analysis of lamina propria (LP) leukocytes showing CD103(+) DCs and MCs with intracellular Ye. Uptake of Ye by LP CD103(+) DCs and MCs was dependent on the pathogenicity factor invasin, whereas the adhesin YadA was dispensable as demonstrated by Ye deletion mutants. Furthermore, Ye were found exclusively associated with CD103(+) DCs in the MLNs from wild-type mice, but not from CCR7(-/-) mice, demonstrating a CCR7 dependent transport of Ye by CD103(+) DCs from LP to the MLNs. In contrast, dissemination of Ye to the spleen was dependent on MCs as significantly less Ye could be recovered from the spleen of CX3CR1(GFP/GFP) mice compared to wild-type mice. Altogether, MCs and CD103(+) DCs contribute to immediate invasion and dissemination of Ye. This together with data from other bacteria suggests MPs as general pathogenic entry site in the intestine., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Lipopolysaccharides (LPS) of Gram negative bacteria are one of the most potent stimulators of the host innate immune system and LPS recognition is essential for the host organism to clear infections of invading bacterial pathogens. Here we review on the latest research on how LPS is sensed by host cells and how distinct LPS structures differentially modulate the strength of the host immune response. Much is known about host immunological reactions towards pathogens via recognition of their LPS, as well as strategies of pathogens to modulate their LPS structure in order to evade the immune system. However, less is known about differential sensing of lipopolysaccharides of commensal bacteria in the intestine and how this contributes to manifestation or destruction of the intestinal homeostasis. LPS sensing is necessary to fight pathogens. However, sensing of LPS of gut commensal bacteria can simultaneously be disadvantageous for the genetically predisposed host, since this might lead to damage of the intestinal homeostasis and therefore to chronic intestinal inflammation. However, less immunogenic LPS could also serve as therapeutics to antagonize an overreacting innate immune system. Therefore, commensal gut bacteria-derived LPS could prevent from uncontrolled intestinal immune response in the intestine which makes LPS an attractive therapeutical approach to treat e.g. IBD., (Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.)

A strain of obligately anaerobic, Gram-stain-negative and non-spore-forming rod-shaped bacterium was isolated from a human wound and characterized both phenotypically and genotypically. The strain was moderately saccharolytic and proteolytic. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to represent a member of the genus Prevotella, but to be different from the described species, with the closest relationship to Prevotella bergensis and Prevotella multisaccharivorax. The genomic DNA G+C content was 43.2 mol%. The most abundant cellular long-chain fatty acids were 3-OH iso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. In view of phenotypical and biochemical characteristics as well as gene sequencing, strain A1336T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella colorans sp. nov. is proposed. The type strain is A1336T (=DSM 100333T =CCUG 67421T =CCOS 902T).

Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory., (Copyright © 2016. Published by Elsevier B.V.)

Like many other Bacteroides species, Bacteroides vulgatus strain mpk, a mouse fecal isolate which was shown to promote intestinal homeostasis, utilizes a variety of mobile elements for genome evolution. Based on sequences collected by Pacific Biosciences SMRT sequencing technology, we discuss the challenges of assembling and studying a bacterial genome of high plasticity. Additionally, we conducted comparative genomics comparing this commensal strain with the B. vulgatus type strain ATCC 8482 as well as multiple other Bacteroides and Parabacteroides strains to reveal the most important differences and identify the unique features of B. vulgatus mpk. The genome of B. vulgatus mpk harbors a large and diverse set of mobile element proteins compared with other sequenced Bacteroides strains. We found evidence of a number of different horizontal gene transfer events and a genome landscape that has been extensively altered by different mobilization events. A CRISPR/Cas system could be identified that provides a possible mechanism for preventing the integration of invading external DNA. We propose that the high genome plasticity and the introduced genome instabilities of B. vulgatus mpk arising from the various mobilization events might play an important role not only in its adaptation to the challenging intestinal environment in general, but also in its ability to interact with the gut microbiota., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.

Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors., (Copyright © 2015. Published by Elsevier GmbH.)

The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed- and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (-2.66 hits per million sample reads/defined daily dose; P = 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy., (© 2015 John Wiley & Sons Ltd.)

Objectives: Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany., Methods: Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities., Results: By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-β-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak., Conclusions: Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Toll-like receptor (TLR) expression in patients with inflammatory bowel disease is increased when compared with healthy controls. However, the impact of TLR signaling during inflammatory bowel disease is not fully understood., Methods: In this study, we used a murine model of acute phase inflammation in bone marrow chimeric mice to investigate in which cell type TLR2/4 signal induction is important in preventing intestinal inflammation and how intestinal dendritic cells are influenced. Mice were either fed with wild-type bacteria, able to initiate the TLR2/4 signaling cascade, or with mutant strains with impaired signal induction capacity., Results: The induction of the TLR2/4 signal cascade in epithelial cells resulted in inflammation in bone marrow chimeric mice, whereas induction in hematopoietic cells had an opposed function. Furthermore, feeding of wild-type bacteria prevented disease; however, differing signal induction of bacteria had no effect on lamina propria dendritic cell activation. In contrast, functional TLR2/4 signals resulted in increased frequencies of CD103-expressing lamina propria and mesenteric lymph node dendritic cells, which were able to ameliorate disease., Conclusions: The TLR-mediated amelioration of disease, the increase in CD103-expressing cells, and the beneficial function of TLR signal induction in hematopoietic cells indicate that the increased expression of TLRs in patients with inflammatory bowel disease might result in counterregulation of the host and serve in preventing disease.

Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a β-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract., (© 2014 Max Planck Society. Molecular Microbiology published by John Wiley & Sons Ltd.)

Beta-barrel proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The evolutionary conservation in the biogenesis of these proteins allows mitochondria to assemble bacterial β-barrel proteins in their functional form. In this chapter, we describe exemplarily how the capacity of yeast mitochondria to process the trimeric autotransporter YadA can be used to study the role of bacterial periplasmic chaperones in this process.

Background: This study aimed to investigate risk factors for colonisation with extensively drug-resistant P. aeruginosa (XDR-PA) in immunocompromised patients and to build a clinical risk score (CRS) based on these results., Methods: We conducted a matched case-control study with 31 cases and 93 controls (1:3). Cases were colonised with XDR-PA during hospitalisation. Independent risk factors were determined using a three step conditional logistic regression procedure. A CRS was built with respect to the corresponding risk fraction of each risk factor, and its discriminatory power was estimated by receiver operating characteristic (ROC) analysis., Results: The presence of a central venous catheter (OR 7.41, P = 0.0008), the presence of a urinary catheter (OR 21.04, P < 0.0001), CRP > 10 mg/dl (OR 7.36, P = 0.0015), and ciprofloxacin administration (OR 5.53, P = 0.025) were independent risk factors. The CRS exhibited a high discriminatory power, defining a high risk population with an approximately fourteen times greater risk for XDR-PA colonisation., Conclusions: Unnecessary use of antibiotics, particularly ciprofloxacin should be avoided, and a high standard of infection control measures must be achieved when using medical devices. A CRS can be used for adaptation of the active screening culture policy to the local setting.

Unlabelled: Severe pneumonia remains an important cause of morbidity and mortality. Polymerase chain reaction (PCR) has been shown to be more sensitive than current standard microbiological methods--particularly in patients with prior antibiotic treatment--and therefore, may improve the accuracy of microbiological diagnosis for hospitalized patients with pneumonia. Conventional detection techniques and multiplex PCR for 14 typical bacterial pneumonia-associated pathogens were performed on respiratory samples collected from adult hospitalized patients enrolled in a prospective multi-center study. Patients were enrolled from March until September 2012. A total of 739 fresh, native samples were eligible for analysis, of which 75 were sputa, 421 aspirates, and 234 bronchial lavages. 276 pathogens were detected by microbiology for which a valid PCR result was generated (positive or negative detection result by Curetis prototype system). Among these, 120 were identified by the prototype assay, 50 pathogens were not detected. Overall performance of the prototype for pathogen identification was 70.6% sensitivity (95% confidence interval (CI) lower bound: 63.3%, upper bound: 76.9%) and 95.2% specificity (95% CI lower bound: 94.6%, upper bound: 95.7%). Based on the study results, device cut-off settings were adjusted for future series production. The overall performance with the settings of the CE series production devices was 78.7% sensitivity (95% CI lower bound: 72.1%) and 96.6% specificity (95% CI lower bound: 96.1%). Time to result was 5.2 hours (median) for the prototype test and 43.5 h for standard-of-care. The Pneumonia Application provides a rapid and moderately sensitive assay for the detection of pneumonia-causing pathogens with minimal hands-on time., Trial Registration: Deutsches Register Klinischer Studien (DRKS) DRKS00005684.