Publications by authors named "Jennifer K Bender"

26 Publications

  • Page 1 of 1

Corrigendum: Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in .

Front Microbiol 2021 27;12:686197. Epub 2021 Apr 27.

Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.

[This corrects the article DOI: 10.3389/fmicb.2021.639660.].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2021.686197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111692PMC
April 2021

Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in .

Front Microbiol 2021 15;12:639660. Epub 2021 Feb 15.

Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.

Background: As next generation sequencing (NGS) technologies have experienced a rapid development over the last decade, the investigation of the bacterial genetic architecture reveals a high potential to dissect causal loci of antibiotic resistance phenotypes. Although genome-wide association studies (GWAS) have been successfully applied for investigating the basis of resistance traits, complex resistance phenotypes have been omitted so far. For this especially refers to antibiotics of last resort like daptomycin and ceftaroline. Therefore, we aimed to perform GWAS for the identification of genetic variants associated with DAP and CPT resistance in clinical isolates.

Materials/methods: To conduct microbial GWAS, we selected cases and controls according to their clonal background, date of isolation, and geographical origin. Association testing was performed with PLINK and SEER analysis. By using analysis, we also searched for rare genetic variants in candidate loci that have previously been described to be involved in the development of corresponding resistance phenotypes.

Results: GWAS revealed MprF P314L and L826F to be significantly associated with DAP resistance. These mutations were found to be homogenously distributed among clonal lineages suggesting convergent evolution. Additionally, rare and yet undescribed single nucleotide polymorphisms could be identified within and putative candidate genes. Finally, we could show that each DAP resistant isolate exhibited at least one amino acid substitution within the open reading frame of . Due to the presence of strong population stratification, no genetic variants could be associated with CPT resistance. However, the investigation of the staphylococcal cassette chromosome (SCC) revealed various SNPs to be putatively linked with CPT resistance. Additionally, some CPT resistant isolates revealed no mutations, supporting the hypothesis that further and still unknown resistance determinants are crucial for the development of CPT resistance in .

Conclusion: We hereby confirmed the potential of GWAS to identify genetic variants that are associated with antibiotic resistance traits in However, precautions need to be taken to prevent the detection of spurious associations. In addition, the implementation of different approaches is still essential to detect multiple forms of variations and mutations that occur with a low frequency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2021.639660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917082PMC
February 2021

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

Emerg Infect Dis 2021 04 18;27(4). Epub 2021 Feb 18.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2704.204576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007320PMC
April 2021

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

Antonie Van Leeuwenhoek 2021 Mar 16;114(3):235-251. Epub 2021 Feb 16.

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, 35392, Giessen, Germany.

The Gram-stain-negative, oxidase negative, catalase positive strain KPC-SM-21, 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 19606 (97.0%). Phylogenetic trees calculated based on partial rpoB and gyrB gene sequences showed a distinct clustering of strain KPC-SM-21 with Acinetobacter gerneri DSM 14967 = CIP 107464 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-21 and next related type strains supported the novel species status. The DNA G + C content of strain KPC-SM-21 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 C ω9c (44.2%), C (21.7%) and a summed feature comprising C ω7c and/or iso-C 2-OH (15.3%). Based on the obtained genotypic, phenotypic and chemotaxonomic data we concluded that strain KPC-SM-21 represents a novel species of the genus Acinetobacter, for which the name Acinetobacter stercoris sp. nov. is proposed. The type strain is KPC-SM-21 (= DSM 102168 = LMG 29413).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10482-021-01517-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902594PMC
March 2021

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

Drug Resist Updat 2020 12 27;53:100732. Epub 2020 Oct 27.

National Reference Centre for Staphylococci and Enterococci, Division Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drup.2020.100732DOI Listing
December 2020

ResFinder 4.0 for predictions of phenotypes from genotypes.

J Antimicrob Chemother 2020 12;75(12):3491-3500

National Institute of Agrarian and Veterinary Research (INIAV), National Reference Laboratory for Animal Health, Oeiras, Portugal.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkaa345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662176PMC
December 2020

Comprehensive integrated NGS-based surveillance and contact-network modeling unravels transmission dynamics of vancomycin-resistant enterococci in a high-risk population within a tertiary care hospital.

PLoS One 2020 24;15(6):e0235160. Epub 2020 Jun 24.

Division of Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, Wernigerode, Germany.

Vancomycin-resistant E. faecium (VRE) are an important cause of nosocomial infections, which are rapidly transmitted in hospitals. To identify possible transmission routes, we applied combined genomics and contact-network modeling to retrospectively evaluate routine VRE screening data generated by the infection control program of a hemato-oncology unit. Over 1 year, a total of 111 VRE isolates from 111 patients were collected by anal swabs in a tertiary care hospital in Southern Germany. All isolated VRE were whole-genome sequenced, followed by different in-depth bioinformatics analyses including genotyping and determination of phylogenetic relations, aiming to evaluate a standardized workflow. Patient movement data were used to overlay sequencing data to infer transmission events and strain dynamics over time. A predominant clone harboring vanB and exhibiting genotype ST117/CT469 (n = 67) was identified. Our comprehensive combined analyses suggested intra-hospital spread, especially of clone ST117/CT469, despite of extensive screening, single room placement, and contact isolation. A new interactive tool to visualize these complex data was designed. Furthermore, a patient-contact network-modeling approach was developed, which indicates both the periodic import of the clone into the hospital and its spread within the hospital due to patient movements. The analyzed spread of VRE was most likely due to placement of patients in the same room prior to positivity of screening. We successfully demonstrated the added value for this combined strategy to extract well-founded knowledge from interdisciplinary data sources. The combination of patient-contact modeling and high-resolution typing unraveled the transmission dynamics within the hospital department and, additionally, a constant VRE influx over time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235160PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314025PMC
September 2020

A Nosocomial Cluster of Tigecycline- and Vancomycin-Resistant Isolates and the Impact of and (M) Mutations on Tigecycline Resistance.

Microb Drug Resist 2020 Jun 31;26(6):576-582. Epub 2019 Dec 31.

Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, National Reference Centre (NRC) for Staphylococci and Enterococci, Robert Koch Institute, Wernigerode, Germany.

Tigecycline-resistant enterococci are only rarely detected worldwide. In 2017, the National Reference Centre for Staphylococci and Enterococci noticed a nosocomial cluster of tigecycline- and vancomycin-resistant (TVRE) in a hospital of tertiary care in Northern Germany. Nineteen isolates were analyzed by means of antimicrobial susceptibility testing and pulsed-field gel electrophoresis. A subset of isolates was subjected to whole-genome sequencing. The genetic basis of tigecycline resistance was assessed by ResFinder and by comparative analyses to known tetracycline and tigecycline resistance genes. Phylogenetic investigations revealed the clustering of 11 TVRE that exhibited genotype ST117/CT1489. Two tigecycline-susceptible isolates were unrelated. Characterization of the genetic determinant putatively responsible for tigecycline resistance revealed two chromosomal changes in the TVRE population: (1) a deletion within the ribosomal protein gene and (2) a serine insertion in and removal of transcriptional regulation of the ribosomal protection protein Tet(M). We here report the first nosocomial cluster of TVRE in a German hospital and disclosed the resistance mechanism that was most likely causative for tigecycline insusceptibility. Clonal spread of TVRE isolates can be assumed because all isolates were highly related and harbored identical chromosomal alterations associated with tigecycline resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/mdr.2019.0346DOI Listing
June 2020

Validating a screening agar for linezolid-resistant enterococci.

BMC Infect Dis 2019 Dec 23;19(1):1078. Epub 2019 Dec 23.

Division Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, National Reference Centre for Staphylococci and Enterococci (NRC), Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12879-019-4711-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929501PMC
December 2019

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

J Antimicrob Chemother 2019 10;74(10):2926-2929

National Reference Centre for Staphylococci and Enterococci (NRC), Division of Nosocomial Pathogens and Antibiotic Resistance, Department of Infectious Diseases, Robert Koch Institut, Wernigerode Branch, Wernigerode, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkz310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753474PMC
October 2019

Development of a multiplex-PCR to simultaneously detect acquired linezolid resistance genes cfr, optrA and poxtA in enterococci of clinical origin.

J Microbiol Methods 2019 05 30;160:101-103. Epub 2019 Mar 30.

National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode, Germany.

Linezolid-resistant enterococcus spp. are increasingly recognized by diagnostic laboratories. Resistance can be mediated by the expression of cfr, optrA or poxtA. We developed a multiplex-PCR to simultaneously detect all three genes. The PCR is suitable for microbiological diagnostics in order to restrict further spread of resistances in enterococci.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mimet.2019.03.025DOI Listing
May 2019

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

J Clin Microbiol 2019 03 27;57(3). Epub 2019 Feb 27.

Robert Koch Institute, Division of Nosocomial Pathogens and Antibiotic Resistances, Wernigerode, Germany.

Among enterococci, occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of 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 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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.01686-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425188PMC
March 2019

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

Drug Resist Updat 2018 09 2;40:25-39. Epub 2018 Nov 2.

Department of Infectious Diseases, Division of Nosocomial Pathogens and Antibiotic Resistances, Robert Koch Institute, Wernigerode, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drup.2018.10.002DOI Listing
September 2018

Rapid emergence of highly variable and transferable oxazolidinone and phenicol resistance gene optrA in German Enterococcus spp. clinical isolates.

Int J Antimicrob Agents 2018 Dec 17;52(6):819-827. Epub 2018 Sep 17.

National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode, Saxony-Anhalt, Germany.

The number of linezolid-resistant Enterococcus spp. isolates received by the National Reference Centre for Staphylococci and Enterococci in Germany has been increasing since 2011. Although the majority are E. faecium, clinical linezolid-resistant E. faecalis have also been isolated. With respect to the newly discovered linezolid resistance protein OptrA, the authors conducted a retrospective polymerase chain reaction screening of 698 linezolid-resistant enterococcus clinical isolates. That yielded 43 optrA-positive strains, of which a subset was analysed by whole-genome sequencing in order to infer linezolid resistance-associated mechanisms and phylogenetic relatedness, and to disclose optrA genetic environments. Multiple optrA variants were detected. The originally described variant from China (optrA) was the only variant shared between the two Enterococcus spp.; however, distinct optrA loci were detected for E. faecium and E. faecalis. Generally, optrA localized to a plethora of genetic backgrounds that differed even for identical optrA variants. This suggests transmission of a mobile genetic element harbouring the resistance locus. Additionally, identical optrA variants detected on presumably identical plasmids, that were present in unrelated strains, indicates dissemination of the entire optrA-containing plasmid. In accordance, in vitro conjugation experiments verified transfer of optrA plasmids between enterococci of the same and of different species. In conclusion, multiple optrA variants located on distinct plasmids and mobile genetic elements with the potential for conjugative transfer are supposedly causative for the emergence of optrA-positive enterococci. Hence, rapid dissemination of the resistance determinant under selective pressure imposed by extensive use of last-resort antibiotics in clinical settings could be expected.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijantimicag.2018.09.009DOI Listing
December 2018

Comparative whole genome analysis of three consecutive Salmonella diarizonae isolates.

Int J Med Microbiol 2017 Dec 5;307(8):542-551. Epub 2017 Sep 5.

Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg and University of Regensburg, Regensburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijmm.2017.09.001DOI Listing
December 2017

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

Environ Microbiol 2017 10 9;19(10):4349-4364. Epub 2017 Oct 9.

Faculty of Biological Sciences, University of Zielona Góra, Prof. Z. Szafrana Street 1, 65-561 Zielona Góra, Poland.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.13931DOI Listing
October 2017

Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium.

PLoS One 2017 17;12(2):e0172630. Epub 2017 Feb 17.

Project Group 5, Robert Koch Institute, Wernigerode, Germany.

Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give new insights into mechanisms of Salmonella host cell sensing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172630PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315404PMC
August 2017

Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp.

PLoS One 2016 28;11(11):e0167042. Epub 2016 Nov 28.

National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.

The National Reference Centre for Staphylococci and Enterococci in Germany has received an increasing number of clinical linezolid-resistant E. faecium isolates in recent years. Five isolates harbored a cfr(B) variant gene locus the product of which is capable of conferring linezolid resistance. The cfr(B)-like methyltransferase gene was also detected in Clostridium difficile. Antimicrobial susceptibility was determined for cfr(B)-positive and linezolid-resistant E. faecium isolates and two isogenic C. difficile strains. All strains were subjected to whole genome sequencing and analyzed with respect to mutations in the 23S rDNA, rplC, rplD and rplV genes and integration sites of the cfr(B) variant locus. To evaluate methyltransferase function, the cfr(B) variant of Enterococcus and Clostridium was expressed in both E. coli and Enterococcus spp. Ribosomal target site mutations were detected in E. faecium strains but absent in clostridia. Sequencing revealed 99.9% identity between cfr(B) of Enterococcus and cfr of Clostridium. The methyltransferase gene is encoded by transposon Tn6218 which was present in C. difficile Ox3196, truncated in some E. faecium and absent in C. difficile Ox3206. The latter finding explains the lack of linezolid and chloramphenicol resistance in C. difficile Ox3206 and demonstrates for the first time a direct correlation of elevated linezolid MICs in C. difficile upon cfr acquisition. Tn6218 insertion sites revealed novel target loci for integration, both within the bacterial chromosome and as an integral part of plasmids. Importantly, the very first plasmid-association of a cfr(B) variant was observed. Although we failed to measure cfr(B)-mediated resistance in transformed laboratory strains the occurrence of the multidrug resistance gene cfr on putatively highly mobile and/or extrachromosomal DNA in clinical isolates is worrisome with respect to dissemination of antibiotic resistances.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167042PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125667PMC
July 2017

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

J Bacteriol 2017 01 13;199(1). Epub 2016 Dec 13.

FG11 Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Wernigerode, Germany

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00393-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165104PMC
January 2017

Complete Genome Sequences of Two Methicillin-Sensitive Staphylococcus aureus Isolates Representing a Population Subset Highly Prevalent in Human Colonization.

Genome Announc 2016 Jul 28;4(4). Epub 2016 Jul 28.

National Reference Centre for Staphylococci and Enterococci, Division Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany

Here, we report the high-quality draft genome sequences of two methicillin-susceptible Staphylococcus aureus isolates, 08-02119 and 08-02300. Belonging to sequence type 582 (ST582) and ST7, both isolates are representatives of clonal lineages often associated with asymptomatic colonization of humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.00716-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966458PMC
July 2016

Population structure and acquisition of the vanB resistance determinant in German clinical isolates of Enterococcus faecium ST192.

Sci Rep 2016 Feb 23;6:21847. Epub 2016 Feb 23.

National Reference Centre for Staphylococci and Enterococci, Robert Koch Institute, Department of Infectious Diseases, Wernigerode, 38855, Germany.

In the context of the global action plan to reduce the dissemination of antibiotic resistances it is of utmost importance to understand the population structure of resistant endemic bacterial lineages and to elucidate how bacteria acquire certain resistance determinants. Vancomycin resistant enterococci represent one such example of a prominent nosocomial pathogen on which nation-wide population analyses on prevalent lineages are scarce and data on how the bacteria acquire resistance, especially of the vanB genotype, are still under debate. With respect to Germany, an increased prevalence of VRE was noted in recent years. Here, invasive infections caused by sequence type ST192 VRE are often associated with the vanB-type resistance determinant. Hence, we analyzed 49 vanB-positive and vanB-negative E. faecium isolates by means of whole genome sequencing. Our studies revealed a distinct population structure and that spread of the Tn1549-vanB-type resistance involves exchange of large chromosomal fragments between vanB-positive and vanB-negative enterococci rather than independent acquisition events. In vitro filter-mating experiments support the hypothesis and suggest the presence of certain target sequences as a limiting factor for dissemination of the vanB element. Thus, the present study provides a better understanding of how enterococci emerge into successful multidrug-resistant nosocomial pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep21847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763178PMC
February 2016

Complete Genome Sequence of the Gut Commensal and Laboratory Strain Enterococcus faecium 64/3.

Genome Announc 2015 Nov 19;3(6). Epub 2015 Nov 19.

National Reference Centre for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Germany.

The genome sequence of the commensal and widely used laboratory strain Enterococcus faecium 64/3 was resolved by means of PacificBioscience and Illumina whole-genome sequencing. The genome comprises 2,575,333 bp with 2,382 coding sequences as assigned by NCBI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.01275-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653773PMC
November 2015

Evaluation of DiversiLab®, MLST and PFGE typing for discriminating clinical Enterococcus faecium isolates.

J Microbiol Methods 2015 Nov 1;118:81-4. Epub 2015 Sep 1.

Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.

We evaluated and critically assessed the performance and discriminatory power of a rep-PCR based commercial test DiversiLab® Enterococcus kit (bioMerieux) for typing a set of 65 representative isolates of Enterococcus faecium/VRE and compared it to state-of-the-art typing techniques such as PFGE and MLST.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mimet.2015.08.019DOI Listing
November 2015

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

Infect Immun 2015 Jan 3;83(1):346-53. Epub 2014 Nov 3.

Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.02758-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288888PMC
January 2015

LPS structure and PhoQ activity are important for Salmonella Typhimurium virulence in the Galleria mellonella infection model [corrected].

PLoS One 2013 8;8(8):e73287. Epub 2013 Aug 8.

Robert Koch-Institute, Wernigerode Branch, Wernigerode, Germany.

The larvae of the wax moth, Galleria mellonella, have been used experimentally to host a range of bacterial and fungal pathogens. In this study we evaluated the suitability of G. mellonella as an alternative animal model of Salmonella infection. Using a range of inoculum doses we established that the LD₅₀ of SalmonellaTyphimurium strain NCTC 12023 was 3.6 × 10³ bacteria per larva. Further, a set of isogenic mutant strains depleted of known virulence factors was tested to identify determinants essential for S. Typhimurium pathogenesis. Mutants depleted of one or both of the type III secretion systems encoded by Salmonella Pathogenicity Islands 1 and 2 showed no virulence defect. In contrast, we observed reduced pathogenic potential of a phoQ mutant indicating an important role for the PhoPQ two-component signal transduction system. Lipopolysaccharide (LPS) structure was also shown to influence Salmonella virulence in G. mellonella. A waaL(rfaL) mutant, which lacks the entire O-antigen (OAg), was virtually avirulent, while a wzz(ST)/wzz(fepE) double mutant expressing only a very short OAg was highly attenuated for virulence. Furthermore, shortly after infection both LPS mutant strains showed decreased replication when compared to the wild type in a flow cytometry-based competitive index assay. In this study we successfully established a G. mellonella model of S. Typhimurium infection. By identifying PhoQ and LPS OAg length as key determinants of virulence in the wax moth larvae we proved that there is an overlap between this and other animal model systems, thus confirming that the G. mellonella infection model is suitable for assessing aspects of Salmonella virulence function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073287PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738532PMC
April 2014

Involvement of PatE, a prophage-encoded AraC-like regulator, in the transcriptional activation of acid resistance pathways of enterohemorrhagic Escherichia coli strain EDL933.

Appl Environ Microbiol 2012 Aug 11;78(15):5083-92. Epub 2012 May 11.

Department of Microbiology and Immunology, The University of Melbourne, Melbourne, and Murdoch Childrens Research Institute, Royal Children’s Hospital, Australia.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach. In silico analysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene, patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis in E. coli showed that the expression of patE is induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperone hdeA by binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.00617-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416394PMC
August 2012