CHROMAgar™ LIN-R as an efficient screening tool to assess the prevalence of linezolid-resistant enterococci in German hospital patients-a multicentre study approach, 2021-2022.

BACKGROUND: In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES: To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS: During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS: The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS: CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

The Mosaic mtr Locus as Major Genetic Determinant of Azithromycin Resistance of Neisseria gonorrhoeae-Germany, 2018.

Within the German Gonococcal Resistance Network's (GORENET) Neisseria gonorrhoeae (NG) sample collection, azithromycin-resistant NG isolates increased from 4.3% in 2016 to 9.2% in 2018. We aim to understand this observed increase using whole genome sequencing of NG isolates combined with epidemiological and clinical data. Reduced susceptibility to azithromycin in 2018 was predominately clonal (NG multiantigen sequence typing G12302) and could mainly be attributed to the recently described mosaic-like mtr locus. Our data suggest that, together with horizontal gene transfer of resistance determinants and well-established point mutations, international spread of resistant lineages plays a major role regarding azithromycin resistance in Germany.

Analysis of Asymptomatic and Presymptomatic Transmission in SARS-CoV-2 Outbreak, Germany, 2020.

We determined secondary attack rates (SAR) among close contacts of 59 asymptomatic and symptomatic coronavirus disease case-patients by presymptomatic and symptomatic exposure. We observed no transmission from asymptomatic case-patients and highest SAR through presymptomatic exposure. Rapid quarantine of close contacts with or without symptoms is needed to prevent presymptomatic transmission.

Factors preventing SARS-CoV-2 transmission during unintentional exposure in a GP practice: a cohort study of patient contacts; Germany, 2020.

Two general practitioners (GPs) with SARS-CoV-2 infection provided in-person patient care to patients of their joint medical practice before and after symptom onset, up until SARS-CoV-2 laboratory confirmation. Through active contact tracing, the local public health authorities recruited the cohort of patients that had contact with either GP in their putative infectious period. In this cohort of patient contacts, we assess the frequency and determinants of SARS-CoV-2-transmission from GPs to patients. We calculated incidence rate ratios (IRR) to explore the type of contact as an explanatory variable for COVID-19 cases. Among the cohort of 83 patient contacts, we identified 22 (27%) COVID-19 cases including 17 (21%) possible, three (4%) probable and two (2%) confirmed cases. All 22 cases had contact with a GP when the GP did not wear a mask, and/or when contact was ≥10 min. Importantly, patients who had contact <10 min with a GP wearing a facemask were at reduced risk (IRR 0.21; 95% CI 0.01-0.99) of COVID-19. This outbreak investigation adds to the body of evidence in supporting current guidelines on measures at preventing the transmission of SARS-CoV-2 in an outpatient setting.

Acinetobacter stercoris sp. nov. isolated from output source of a mesophilic german biogas plant with anaerobic operating conditions.

The Gram-stain-negative, oxidase negative, catalase positive strain KPC-SM-21T, isolated from a digestate of a storage tank of a mesophilic German biogas plant, was investigated by a polyphasic taxonomic approach. Phylogenetic identification based on the nearly full-length 16S rRNA gene revealed highest gene sequence similarity to Acinetobacter baumannii ATCC 19606T (97.0%). Phylogenetic trees calculated based on partial rpoB and gyrB gene sequences showed a distinct clustering of strain KPC-SM-21T with Acinetobacter gerneri DSM 14967T = CIP 107464T and not with A. baumannii, which was also supported in the five housekeeping genes multilocus sequence analysis based phylogeny. Average nucleotide identity values between whole genome sequences of strain KPC-SM-21T and next related type strains supported the novel species status. The DNA G + C content of strain KPC-SM-21T was 37.7 mol%. Whole-cell MALDI-TOF MS analysis supported the distinctness of the strain to type strains of next related Acinetobacter species. Predominant fatty acids were C18:1 ω9c (44.2%), C16:0 (21.7%) and a summed feature comprising C16:1 ω7c and/or iso-C15:0 2-OH (15.3%). Based on the obtained genotypic, phenotypic and chemotaxonomic data we concluded that strain KPC-SM-21T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter stercoris sp. nov. is proposed. The type strain is KPC-SM-21T (= DSM 102168T = LMG 29413T).

Thirty years of VRE in Germany - "expect the unexpected": The view from the National Reference Centre for Staphylococci and Enterococci.

Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

Comparative Target Analysis of Chlorinated Biphenyl Antimicrobials Highlights MenG as a Molecular Target of Triclocarban.

Triclocarban (TCC), a formerly used disinfectant, kills bacteria via an unknown mechanism of action. A structural hallmark is its N,N'-diaryl urea motif, which is also present in other antibiotics, including the recently reported small molecule PK150. We show here that, like PK150, TCC exhibits an inhibitory effect on Staphylococcus aureus menaquinone metabolism via inhibition of the biosynthesis protein demethylmenaquinone methyltransferase (MenG). However, the activity spectrum (MIC90) of TCC across a broad range of multidrug-resistant staphylococcus and enterococcus strains was much narrower than that of PK150. Accordingly, TCC did not cause an overactivation of signal peptidase SpsB, a hallmark of the PK150 mode of action. Furthermore, we were able to rule out inhibition of FabI, a confirmed target of the diaryl ether antibiotic triclosan (TCS). Differences in the target profiles of TCC and TCS were further investigated by proteomic analysis, showing complex but rather distinct changes in the protein expression profile of S. aureus Downregulation of the arginine deiminase pathway provided additional evidence for an effect on bacterial energy metabolism by TCC.IMPORTANCE TCC's widespread use as an antimicrobial agent has made it a ubiquitous environmental pollutant despite its withdrawal due to ecological and toxicological concerns. With its antibacterial mechanism of action still being unknown, we undertook a comparative target analysis between TCC, PK150 (a recently discovered antibacterial compound with structural resemblance to TCC), and TCS (another widely employed chlorinated biphenyl antimicrobial) in the bacterium Staphylococcus aureus We show that there are distinct differences in each compound's mode of action, but also identify a shared target between TCC and PK150, the interference with menaquinone metabolism by inhibition of MenG. The prevailing differences, however, which also manifest in a remarkably better broad-spectrum activity of PK150, suggest that even high levels of TCC or TCS resistance observed by continuous environmental exposure may not affect the potential of PK150 or related N,N'-diaryl urea compounds as new antibiotic drug candidates against multidrug-resistant infections.

The status of the genus Prolinoborus (Pot et al. 1992) and the species Prolinoborus fasciculus (Pot et al. 1992).

On the basis of two other publications (Yarza et al. 2013; Nemec et al. 2019) and on the basis of resequencing of the 16S rRNA gene of Prolinoborus fasciculus CIP 103579T it is concluded that Prolinoborus fasciculus CIP 103579T, which is the only available strain of the species from culture collections, does not conform to the original description given by Pot et al. (1992). The strain investigated is a member of the genus Acinetobacter within the Moraxellaceae, a family of the Gammaproteobacteria and not a member of the Betaproteobacteria as originally proposed. Prolinoborus fasciculus CIP 103579T shared 99.8 % 16S rRNA gene sequence similarity with Acinetobacter lwoffii DSM 2403T. The two strains clustered together by rpoB- and core genome-based phylogenetic analyses and shared an average nucleotide identity of 96.47% (reciprocal, 96.56 %) and a digital genome distance calculation (GGDC) value of 66.9 %. Furthermore, the two strains shared matrix-assisted laser desorption/ionization time of flight MS profiles to a high extent and showed highly similar cellular fatty acid profiles and physiological substrate utilization patterns. It is proposed that the Judicial commission consider (1) that the strain currently deposited as CIP 103579 be recognized as a member of Acinetobacter lwoffii; (2) placing Prolinoborus fasciculus (Pot et al. 1992) on the list of rejected names if a suitable replacement strain, or a neotype strain cannot be found within 2 years of publication of this request; and (3) place the genus name Prolinoborus (Pot et al. 1992) on the list of rejected names [Recommendation 20D (3) of the Code].

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Comparison of VITEK® 2, three different gradient strip tests and broth microdilution for detecting vanB-positive Enterococcus faecium isolates with low vancomycin MICs.

OBJECTIVES: In 2018, EUCAST issued a warning regarding unreliable results of gradient strip tests for confirming vancomycin resistance in enterococci. We compared the performance of various diagnostic standard and confirmatory tests to identify and determine vanB-type vancomycin resistance. METHODS: We analysed a collection of vanB-positive Enterococcus faecium isolates (n = 68) with low vancomycin MICs and compared the performance of VITEK® 2 (bioMérieux), broth microdilution and three gradient strip tests from different providers (Oxoid, Liofilchem and bioMérieux). For the latter we compared the standard procedure with a protocol with increased inoculum, a rich agar medium and a longer incubation time ('macromethod'). RESULTS: The sensitivity of VITEK® 2 was 81% compared with 72% for broth microdilution and 61%-63% for the three gradient strip tests using standard conditions. The macromethod substantially improved the performance of all strip tests resulting in a sensitivity of 89%-96% after 48 h of incubation. CONCLUSIONS: We recommend that EUCAST changes the present warning against the general use of MIC strips. When MIC strips are used to either exclude or confirm suspected vancomycin resistance in E. faecium, and a PCR is not available, the macromethod should be employed. For clinically relevant enterococci, where a rapid therapeutic decision is needed, a molecular test (e.g. PCR) should be favoured in order to save time and to further increase sensitivity.

Validating a screening agar for linezolid-resistant enterococci.

BACKGROUND: Linezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci. Some countries report an increasing number of isolates with resistance to linezolid. The recent publication of the Commission for Hospital Hygiene in Germany on enterococci/VRE recommends screening for linezolid-resistant enterococci (LRE). However, a suitable selective medium or a genetic test is not available. Our aim was to establish a selective screening agar for LRE detection and validate its application with a comprehensive collection of clinical LRE and linezolid-susceptible enterococci. METHODS: We decided to combine the selective power of an enterococcal screening agar with a supplementation of linezolid. Several rounds of analyses with reference, control and test strains and under varying linezolid concentrations of a wider and a smaller range were investigated and assessed. The collection of linezolid-resistant enterococcal control strains included isolates with different resistance mechanisms (23S rDNA mutations, cfr(B), optrA, poxtA). Finally, we validated our LRE screening agar with 400 samples sent to our National Reference Centre in 2019. RESULTS: Several rounds of pre-tests and confirmatory analyses favored Enterococcosel® Agar supplemented with a concentration of 2 mg/L linezolid. A 48 h incubation period was essential for accurate identification of LRE strains. Performance of the LRE screening agar revealed a sensitivity of 96.6% and a specificity of 94.4%. CONCLUSIONS: Here we describe preparation of a suitable screening agar and a procedure to identify LRE isolates with high accuracy.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Emergence and control of linezolid-resistant Staphylococcus epidermidis in an ICU of a German hospital.

Objectives: To investigate an outbreak of linezolid-resistant Staphylococcus epidermidis (LRSE) in an interdisciplinary ICU, linezolid consumption and infection control measures taken. Methods: Routine surveillance of nosocomial infections revealed colonization and infection with LRSE affecting 14 patients during a 15 month period. LRSE isolates were analysed with respect to their clonal relatedness, antimicrobial susceptibility, the presence of cfr and/or mutations in the 23S rRNA, rplC, rplD and rplV genes. cfr plasmids were characterized by Illumina sequencing. Medical records were reviewed and antibiotic consumption was determined. Results: Molecular typing identified the presence of three different LRSE clusters: PFGE type I/ST168 (n = 5), PFGE type II/ST5 (n = 10) and PFGE type III/ST2 (n = 1). Ten strains harboured the cfr gene; we also detected mutations in the respective ribosomal protein genes. WGS revealed an almost identical 39 kb cfr plasmid obtained from strains of different genetic background (ST2, ST5, ST168) that shows high similarity to the recently published LRSE plasmid p12-02300. Due to an increase in the number of patients treated for infections with MRSA, a significant increase in linezolid usage was noted from January to July 2014 (from 5.55 to 20.41 DDDs/100 patient-days). Conclusions: Here, we report the molecular epidemiology of LRSE in an ICU. Our results suggest the selection of resistant mutants under linezolid treatment as well as the spread of cfr-carrying plasmids. The reduction of linezolid usage and the strengthening of contact precautions proved to be effective infection control measures.

Evaluation of WGS based approaches for investigating a food-borne outbreak caused by Salmonella enterica serovar Derby in Germany.

In Germany salmonellosis still represents the 2nd most common bacterial foodborne disease. The majority of infections are caused by Salmonella (S.) Typhimurium and S. Enteritidis followed by a variety of other broad host-range serovars. Salmonella Derby is one of the five top-ranked serovars isolated from humans and it represents one of the most prevalent serovars in pigs, thus bearing the potential risk for transmission to humans upon consumption of pig meat and products thereof. From November 2013 to January 2014 S. Derby caused a large outbreak that affected 145 primarily elderly people. Epidemiological investigations identified raw pork sausage as the probable source of infection, which was confirmed by microbiological evidence. During the outbreak isolates from patients, food specimen and asymptomatic carriers were investigated by conventional typing methods. However, the quantity and quality of available microbiological and epidemiological data made this outbreak highly suitable for retrospective investigation by Whole Genome Sequencing (WGS) and subsequent evaluation of different bioinformatics approaches for cluster definition. Overall the WGS-based methods confirmed the results of the conventional typing but were of significant higher discriminatory power. That was particularly beneficial for strains with incomplete epidemiological data. For our data set both, single nucleotide polymorphism (SNP)- and core genome multilocus sequence typing (cgMLST)-based methods proved to be appropriate tools for cluster definition.

Suppressor Mutations Linking gpsB with the First Committed Step of Peptidoglycan Biosynthesis in Listeria monocytogenes.

The cell division protein GpsB is a regulator of the penicillin binding protein A1 (PBP A1) in the Gram-positive human pathogen Listeria monocytogenes Penicillin binding proteins mediate the last two steps of peptidoglycan biosynthesis as they polymerize and cross-link peptidoglycan strands, the main components of the bacterial cell wall. It is not known what other processes are controlled by GpsB. L. monocytogenes gpsB mutants are unable to grow at 42°C, but we observed that spontaneous suppressors correcting this defect arise on agar plates with high frequency. We here describe a first set of gpsB suppressors that mapped to the clpC and murZ genes. While ClpC is the ATPase component of the Clp protease, MurZ is a paralogue of the listerial UDP-N-acetylglucosamine (UDP-GlcNAc) 1-carboxyvinyltransferase MurA. Both enzymes catalyze the enolpyruvyl transfer from phosphoenolpyruvate to UDP-GlcNAc, representing the first committed step of peptidoglycan biosynthesis. We confirmed that clean deletion of the clpC or murZ gene suppressed the ΔgpsB phenotype. It turned out that the absence of either gene leads to accumulation of MurA, and we show that artificial overexpression of MurA alone was sufficient for suppression. Inactivation of other UDP-GlcNAc-consuming pathways also suppressed the heat-sensitive growth of the ΔgpsB mutant, suggesting that an increased influx of precursor molecules into peptidoglycan biosynthesis can compensate for the lack of GpsB. Our results support a model according to which PBP A1 becomes misregulated and thus toxic in the absence of GpsB due to unproductive consumption of cell wall precursor molecules. IMPORTANCE: The late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.

Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide.

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock.

Comparative whole genome analysis of three consecutive Salmonella diarizonae isolates.

Infections of very young children or immunocompromised people with Salmonella of higher subspecies are a well-known phenomenon often associated with contact to cold-blooded animals. We describe the molecular characterization of three S. enterica subsp. diarizonae strains, isolated consecutively over a period of several months from a hospital patient suffering from diarrhea and sepsis with fatal outcome. With the initial isolate the first complete genome sequence of a member of subsp. diarizonae is provided and based on this reference we revealed the genomic differences between the three isolates by use of next-generation sequencing and confirmed by phenotypical tests. Genome comparisons revealed mutations within gpt, hfq and purK in the first isolate as a sign of clonal variation rather than host-directed evolution. Furthermore, our work demonstrates that S. enterica subsp. diarizonae possess, besides a conserved set of known Salmonella Pathogenicity Islands, a variable portfolio of additional genomic islands of unknown function.

Control of acid resistance pathways of enterohemorrhagic Escherichia coli strain EDL933 by PsrB, a prophage-encoded AraC-like regulator.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes bloody diarrhea and hemolytic-uremic syndrome (HUS) and is the most prevalent E. coli serotype associated with food-borne illness worldwide. This pathogen is transmitted via the fecal-oral route and has a low infectious dose that has been estimated to be between 10 and 100 cells. We and others have previously identified three prophage-encoded AraC-like transcriptional regulators, PatE, PsrA, and PsrB in the EHEC O157:H7 EDL933 strain. Our analysis showed that PatE plays an important role in facilitating survival of EHEC under a number of acidic conditions, but the contribution of PsrA and PsrB to acid resistance (AR) was unknown. Here, we investigated the involvement of PsrA and PsrB in the survival of E. coli O157:H7 in acid. Our results showed that PsrB, but not PsrA, enhanced the survival of strain EDL933 under various acidic conditions. Transcriptional analysis using promoter-lacZ reporters and electrophoretic mobility shift assays demonstrated that PsrB activates transcription of the hdeA operon, which encodes a major acid stress chaperone, by interacting with its promoter region. Furthermore, using a mouse model, we showed that expression of PsrB significantly enhanced the ability of strain EDL933 to overcome the acidic barrier of the mouse stomach. Taken together, our results indicate that EDL933 acquired enhanced acid tolerance via horizontally acquired regulatory genes encoding transcriptional regulators that activate its AR machinery.