Stephan Göttig

Rapid Detection and Quantification of RNA of Ebola and Marburg Viruses, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Rift Valley Fever Virus, Dengue Virus, and Yellow Fever Virus by Real-Time Reverse Transcription-PCR

ABSTRACT Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5′-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5′-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5′-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The ≥95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.

Detection of NDM-7 in Germany, a new variant of the New Delhi metallo-β-lactamase with increased carbapenemase activity

OBJECTIVES This study characterized a new variant of the New Delhi metallo-β-lactamase (NDM). METHODS A multidrug-resistant Escherichia coli isolate was recovered from the wounds, throat and rectum of a Yemeni patient who presented at the Frankfurt University Hospital in Germany. The presence of β-lactamase genes was analysed by PCR and sequencing. The isolate was further characterized by susceptibility testing, conjugation and transformation assays and plasmid analysis. RESULTS The E. coli isolate was resistant to all β-lactams including carbapenems. By PCR analysis, the β-lactamase genes blaCMY-2, blaCTX-M-15, blaTEM-1 and blaNDM were identified. Sequencing revealed a blaNDM gene that differed from blaNDM-1 by two point mutations at positions 388 (G→A) and 460 (A→C) corresponding to amino acid substitutions Asp130Asn and Met154Leu, respectively. This NDM variant was identified as NDM-7. The blaNDM-7 gene was located on a self-transferable IncX3 plasmid of 60 kb. E. coli TOP10 transformants harbouring NDM-7 showed higher MICs of β-lactams including carbapenems compared with transformants harbouring NDM-1. Multilocus sequence typing analysis revealed that the E. coli isolate belonged to a novel sequence type (ST599). CONCLUSIONS This study identified a novel NDM variant in E. coli, NDM-7, possessing a high ability to hydrolyse β-lactam antibiotics. Given the diversity of NDM variants located on self-transferable plasmids found in different Gram-negative species and isolated in different countries, the blaNDM gene will most likely efficiently disseminate worldwide.

Detection of pan drug-resistant Acinetobacter baumannii in Germany

blaNDM-1 gene and type IV secretion system in clinical isolates of Acinetobacter lwoffii. Antimicrob Agents Chemother 2012; 56: 1698–702. 10 Fu Y, Du X, Ji J et al. Epidemiological characteristics and genetic structure of blaNDM-1 in non-baumannii Acinetobacter spp. in China. J Antimicrob Chemother 2012; 67: 2114–22. 11 Bogaerts P, Huang T, Rezende de Castro R et al. Could Acinetobacter pittii act as an NDM-1 reservoir for Enterobacteriaceae? J Antimicrob Chemother 2013; 68: 2414–5. 12 Carattoli A, Bertini A, Villa L et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–28. 13 Yang J, Chen Y, Jia X et al. Dissemination and characterization of NDM-1-producing Acinetobacter pittii in an intensive care unit in China. Clin Microbiol Infect 2012; 18: E506–13. 14 Zhang R, Hu YY, Yang XF et al. Emergence of NDM-producing non-baumannii Acinetobacter spp. isolated from China. Eur J Clin Microbiol Infect Dis 2014; 33: 853–60.

In Vivo Horizontal Gene Transfer of the Carbapenemase OXA-48 During a Nosocomial Outbreak

BACKGROUND OXA-48 is a highly prevalent carbapenemase and has been isolated worldwide. Here, we investigate the in vivo horizontal gene transfer (HGT) of blaOXA-48 from Klebsiella pneumoniae to Escherichia coli in an infected patient. METHODS Bacterial isolates were characterized by susceptibility testing, multilocus sequence typing, DiversiLab, and plasmid analyses. Transferability of blaOXA-48 was evaluated by in vitro transconjugation using the outbreak strain and E. coli J53. In vivo transconjugation was investigated using the larvae of the greater wax moth (Galleria mellonella) and low-complexity-microbiota mice. RESULTS OXA-48-harboring K. pneumoniae isolates belonging to ST14 were isolated during a nosocomial outbreak from 6 patients. Molecular and epidemiological analyses revealed the HGT of an approximately 60-kb OXA-48-containing IncL/M-type plasmid from K. pneumoniae to E. coli belonging to the novel ST666 in a patient. In vitro conjugation experiments revealed a transconjugation frequency of 8.7 × 10(-7). HGT of OXA-48 in a newly developed in vivo model using G. mellonella larvae revealed a higher transconjugation frequency of 1.3 × 10(-4). The conjugation frequency of OXA-48 from K. pneumoniae and E. coli in the gut of low-complexity-microbiota mice was determined to be 2.9 × 10(-5). CONCLUSIONS The in vivo intergenus gene transfer of OXA-48 in the gut of an infected patient was verified in vitro and in 2 in vivo models, which both showed even higher transmission frequencies vs in vitro conditions. This implies that the current in vitro protocols might not correctly reflect the HGT of carbapenemase genes in vivo.

Detection of the carbapenemase GIM-1 in Enterobacter cloacae in Germany

OBJECTIVES To characterize the mechanisms involved in reduced susceptibility to carbapenems in two Enterobacter cloacae clinical isolates. METHODS Two E. cloacae isolates recovered from different regions in Germany and showing reduced susceptibility to carbapenems were analysed. Susceptibility testing, conjugation, transformation assays, plasmid analysis, sequencing and molecular typing using rep-PCR were performed. RESULTS The two clinical isolates carried the bla(GIM-1) gene and showed resistance to ertapenem, with variable MIC values of imipenem and meropenem. The isolates were clonally unrelated. The bla(GIM-1) gene was located on self-transferable and non-typeable plasmids. Both isolates harboured distinct plasmids and integron structures containing the bla(GIM-1) gene cassette. Interestingly, one of the two plasmids was able to replicate in Pseudomonas aeruginosa, demonstrating its broad host range. CONCLUSIONS This is the first identification in E. cloacae of the bla(GIM-1) gene, which is responsible for reduced susceptibility to carbapenems. We showed that this gene, previously identified in P. aeruginosa, was located in a different genetic background in E. cloacae. The bla(GIM-1) gene might spread quite efficiently in Enterobacteriaceae and P. aeruginosa, as it is difficult to detect and in addition is located on conjugative plasmids.

Acinetobacter baumannii virulence is mediated by the concerted action of three phospholipases D

Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.

Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation.

To investigate the role of the monomeric guanosine triphosphatase (GTPase) Rho on migration of hematopoietic progenitor cells (HPC), we employed different clostridial toxins which inhibit the Rho family of GTPases. Pretreatment with C2I‐C3, a cell‐accessible C3 transferase fusion protein that targets Rho, increased chemokinetic migration of the factor‐dependent multipotent cell line Factor Dependent Cell Paterson with mixed lineage differentiation potential (FDCP‐mix) and of primary lineage marker‐depleted HPC in vitro. In contrast, treatment with lethal toxin (LT) from Clostridium sordellii, which predominantly inactivates Rac, and with toxin B from C. difficile, which inactivates Rho, Rac and Cdc42, decreased in vitro migration. When HPC pretreated with LT or toxin B were transplanted into mice, homing to the bone marrow was impaired, whereas C2I‐C3 treatment did not alter HPC homing. However, in a competitive hematopoietic repopulation experiment in C57BL/6 mice, pretreatment of bone marrow cells with any of the inhibitors, including the Rho inhibitor C2I‐C3, resulted in suppressed donor‐type hematopoiesis. Our data indicate that whereas Rac supports HPC cell cycling, migration, short‐term homing and hematopoietic regeneration, Rho coordinates down‐regulation of HPC migration and is required for hematopoietic regeneration.

Evaluation of the effectiveness of a pathogen inactivation technology against clinically relevant transfusion-transmitted bacterial strains

To increase blood safety, various procedures are currently implemented, including donor selection, optimized donor arm disinfection, and diversion. In addition, pathogen inactivation (PI) techniques can be used for platelets (PLTs) and plasma concentrates.

Pathogenicity of pan-drug-resistant Serratia marcescens harbouring blaNDM-1

OBJECTIVES To characterize a pan-drug-resistant Serratia marcescens clinical isolate carrying the New Delhi metallo-β-lactamase (NDM)-1. METHODS The presence of β-lactamase genes was examined by PCR and sequencing. Antibiotic susceptibility was determined by antibiotic gradient test. Transformation assays, transconjugation assays, PFGE and PCR-based replicon typing were used for plasmid analysis. Horizontal gene transfer was evaluated by liquid mating using Escherichia coli J53 as a recipient. Pathogenicity of NDM-1 expressing S. marcescens was analysed using the Galleria mellonella infection model. RESULTS S. marcescens isolate SM1890 was non-susceptible to all tested antibiotics, with minocycline retaining intermediate activity. blaNDM-1 was located on a 140 kb IncA/C-type plasmid which was transferable to E. coli and Klebsiella pneumoniae by conjugation. The LD50 of the NDM-positive, SM1890 isolate was higher than that of other, NDM-1 negative, S. marcescens strains. CONCLUSIONS The presence of a blaNDM-1-harbouring IncA/C plasmid resulted in marked resistance to β-lactam antibiotics, but had no significant effect on virulence of isogenic strains. Because of the intrinsic resistance of S. marcescens to colistin and reduced susceptibility to tigecycline, treatment options for infections by NDM-1-positive isolates are extremely limited in this species.

Impact of blaNDM-1 on fitness and pathogenicity of Escherichia coli and Klebsiella pneumoniae.

The objective of this study was to determine whether acquisition of New Delhi metallo-β-lactamase-1 (NDM-1) has an impact on the fitness and virulence of Escherichia coli and Klebsiella pneumoniae. Growth kinetics and the cost of fitness of NDM-1 plasmid carriage were assessed in isogenic E. coli J53 and K. pneumoniae PRZ in vitro by pairwise competition assays. The pathogenicity of NDM-1-expressing E. coli and K. pneumoniae strains and their isogenic controls was analysed in vivo using a Galleria mellonella infection model. The cytotoxicity of NDM-1 was assessed in A549 human lung epithelial cells using the lactate dehydrogenase (LDH) assay. No differences in growth kinetics were recorded between NDM-1-expressing strains and controls (P = 0.92). A reduction in fitness of NDM-1-carrying strains was observed both for E. coli J53 and K. pneumoniae PRZ [selection rate constant (s) = -1.27 ± 0.27 for E. coli J53 and -0.19 ± 0.14 for K. pneumoniae PRZ; P < 0.0001]. Survival of G. mellonella larvae infected with NDM-1-expressing strains and controls was similar for E. coli J53 and K. pneumoniae PRZ. Cytotoxicity in A549 cells was not affected by NDM-1 expression (P > 0.05). The presence of blaNDM-1 did not increase the virulence or cytotoxicity of isogenic strains. However, there was a considerable cost of fitness incurred by carriage of the pNDM-1 plasmid. Interestingly, the cost of fitness was significantly higher in E. coli J53 compared with K. pneumoniae PRZ.