Gottfried Wilharm

Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007

OBJECTIVES To investigate the genetic environment of the metallo-β-lactamase gene bla(NDM-1) in an Acinetobacter baumannii isolated in 2007 in a German hospital. METHODS Antimicrobial susceptibility testing was performed and resistance genes were characterized by PCR amplification and sequencing. Transferability of β-lactam resistance was tested by broth mating assays and transformation of plasmids. The genetic background of bla(NDM-1) was analysed by primer walking. Typing of the A. baumannii strain was performed by repetitive extragenic palindromic sequence-based PCR (rep-PCR) using the DiversiLab system. RESULTS The multidrug-resistant A. baumannii isolate harboured β-lactamase genes bla(NDM-1) and intrinsic bla(OXA-64), but without the insertion sequence ISAba1 often located upstream. Transfer of carbapenem resistance by conjugation and transformation failed. Hybridization of isolated plasmid DNA with bla(NDM) probes was not successful. Shotgun cloning of whole genomic DNA and sequence analyses revealed that bla(NDM-1) was located between two insertion elements of ISAba125. Furthermore, this bla(NDM-1)-containing transposon structure was integrated into a chromosomal gene encoding a putative A. baumannii major facilitator superfamily (MFS) metabolite/H+ symporter. CONCLUSIONS The metallo-β-lactamase gene bla(NDM-1) in this A. baumannii strain was integrated in the chromosome on a new transposon structure composed of two copies of insertion sequence ISAba125. The variability of the genetic environment of bla(NDM-1) likely facilitates the rapid dissemination of this gene within many Gram-negative bacterial species.

Yersinia enterocolitica Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB

ABSTRACT The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

DNA Uptake by the Nosocomial Pathogen Acinetobacter baumannii Occurs during Movement along Wet Surfaces

The emergence of Acinetobacter baumannii as an increasingly multidrug-resistant nosocomial pathogen largely relies on acquisition of resistance genes via horizontal gene transfer. Here, we demonstrate that many clinical isolates of A. baumannii take up DNA while they move along wet surfaces. We show that both motility and DNA uptake are abolished after inactivation of pilT, which putatively encodes the type 4 pilus (T4P) retraction ATPase, and comEC, which putatively encodes the DNA uptake channel, respectively. Inactivation of pilT correlates with an increase in the number and length of pili with an average diameter of 7.2 nm. In the Galleria mellonella infection model, the comEC mutant is significantly attenuated, whereas the pilT mutant is not, dissecting biologically distinct roles of T4P and the DNA uptake channel. Collectively, these findings promote our understanding of the mechanisms of DNA uptake and resistance development in A. baumannii, which may also apply to other important pathogens.

Surface-associated motility, a common trait of clinical isolates of Acinetobacter baumannii, depends on 1,3-diaminopropane.

While flagella-independent motility has long been described in representatives of the genus Acinetobacter, the mechanism of motility remains ambiguous. Acinetobacter baumannii, a nosocomial pathogen appearing increasingly multidrug-resistant, may profit from motility during infection or while persisting in the hospital environment. However, data on the frequency of motility skills among clinical A. baumannii isolates is scarce. We have screened a collection of 83 clinical A. baumannii isolates of different origin and found that, with the exception of one isolate, all were motile on wet surfaces albeit to varying degrees and exhibiting differing morphologies. Screening a collection of transposon mutants of strain ATCC 17978 for motility defects, we identified 2 akinetic mutants carrying transposon insertions in the dat and ddc gene, respectively. These neighbouring genes contribute to synthesis of 1,3-diaminopropane (DAP), a polyamine ubiquitously produced in Acinetobacter. Supplementing semi-solid media with DAP cured the motility defect of both mutants. HPLC analyses confirmed that DAP synthesis was abolished in ddc and dat mutants of different A. baumannii isolates and was re-established after genetic complementation. Both, the dat and ddc mutant of ATCC 17978 were attenuated in the Galleria mellonella caterpillar infection model. Taken together, surface-associated motility is a common trait of clinical A. baumannii isolates that requires DAP and may play a role in its virulence.

Yersinia enterocolitica YopQ: strain-dependent cytosolic accumulation and post-translational secretion

YopQ in Yersinia enterocolitica (YopK in Yersinia pseudotuberculosis) is a type III secreted protein required for virulence of yersiniae. In this study YopQ expression, secretion and nucleotide sequences of the corresponding yopQ gene from different yersinia strains were analysed. The cytosolic accumulation differed significantly among serotypes of Y. enterocolitica. These differences might be attributable to variations in the nucleotide sequence and their consequences on mRNA secondary structure. An mRNA signal hypothesis has been proposed for YopQ, predicting the coupling of translation and secretion via an mRNA signal. This hypothesis claims a strictly co-translational secretion of YopQ without its intracellular accumulation. The presence of YopQ in the cytosol, even with a closed secretion apparatus, is demonstrated. Moreover, post-translational secretion of YopQ could be demonstrated. These findings do not support the mRNA signal hypothesis for co-translational secretion.

Impact of Acinetobacter baumannii Superoxide Dismutase on Motility, Virulence, Oxidative Stress Resistance and Susceptibility to Antibiotics

Acinetobacter baumannii is a Gram-negative bacterium appearing as an opportunistic pathogen in hospital settings. Superoxide dismutase (SOD) contributes to virulence in several pathogenic bacteria by detoxifying reactive oxygen species released in the course of host defense reactions. However, the biological role of SODs in A. baumannii has not yet been elucidated. Here, we inactivated in A. baumannii ATCC 17978 gene A1S_2343, encoding a putative SOD of the Fe-Mn type by transposon insertion, resulting in mutant ATCC 17978 sod2343::Km. The mutation was also introduced in two naturally competent A. baumannii isolates by transformation with chromosomal DNA derived from mutant ATCC 17978 sod2343::Km. We demonstrate that inactivation of sod2343 leads to significant motility defects in all three A. baumannii strains. The mutant strains were more susceptible to oxidative stress compared to their parental strains. Susceptibility to colistin and tetracycline was increased in all mutant strains while susceptibility of the mutants to gentamicin, levofloxacin and imipenem was strain-dependent. In the Galleria mellonella infection model the mutant strains were significantly attenuated. In conclusion, sod2343 plays an important role in motility, resistance to oxidative stress, susceptibility to antibiotics and virulence in A. baumannii.

Structure and Biosynthesis of Fimsbactins A–F, Siderophores from Acinetobacter baumannii and Acinetobacter baylyi

Novel chatechol/hydroxamate siderophores (named “fimsbactins”) were identified in Acinetobacter baumannii ATCC 17978 and Acinetobacter baylyi ADP1. The major compound, fimsbactin A, was isolated from low‐iron cultures of A. baylyi ADP1, and its chemical structure was elucidated by mass spectrometry, and detailed 1H, 13C and 15N NMR spectroscopy. From inverse feeding experiments following HPLC‐MS analysis, the structures of five additional derivatives were elucidated. The gene cluster encoding the fimsbactin synthetase (fbs) was identified in both genomes, and mutants in fbs genes in A. baylyi were analyzed, thus allowing prediction of the fimsbactin biosynthesis pathway.

Yersinia enterocolitica type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

BackgroundPathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a t ype t hree s ecretion s ystem (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of s pecific Y op c haperones (called Sycs).ResultsIn this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model.ConclusionsWe present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion.

A simple and rapid method of bacterial transformation

Recently, a unique method for bacterial transformation using nanofibers to inoculate DNA has been developed by Naoto Yoshida and colleagues. We have verified the principle, transforming Escherichia coli, Yersinia enterocolitica and Acinetobacter baumannii, and have established a user-friendly protocol. A buffered suspension of sepiolite-an inexpensive, fibrous yet inoffensive mineral-is mixed with bacteria and transforming DNA and the mixture directly spread on selective agar.

Chryseobacterium gallinarum sp. nov., isolated from a chicken, and Chryseobacterium contaminans sp. nov., isolated as a contaminant from a rhizosphere sample

Two yellow-pigmented bacterial strains (100(T) and C26(T)), showing 98.4 % 16S rRNA gene sequence similarity to each other and isolated from a chicken in Germany and as a contaminant from an agar plate of a rhizosphere sample in Alabama, were studied by using a polyphasic taxonomic approach. Cells of both isolates were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequences of the two organisms with the sequences of the type strains of the most closely related species of the genus Chryseobacterium showed the highest sequence similarities of strains 100(T) and C26(T) to the type strains of Chryseobacterium joostei (respectively 97.5 and 98.2 %), C. viscerum (96.6, 97.8 %), C. gleum (97.1, 97.7 %), C. arthrosphaerae (97.3%, 97.7 %), C. indologenes (97.2, 97.7 %), C. tructae (96.6, 97.6 %), C. jejuense (97.0, 97.6 %) and C. oncorhynchi (96.3, 97.5 %); 16S rRNA gene sequence similarities to members of all other species of the genus Chryseobacterium were below 97.5 %. The fatty acid profiles of both strains consisted of the major fatty acids iso-C15 : 0, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C17 : 1ω9c and iso-C17 : 0 3-OH, but also showed slight differences (absence or presence of C16 : 0 3-OH and iso-C15 : 1 F). DNA-DNA hybridizations between the two strains and between the novel strains and the type strains of C. joostei, C. indologenes, C. jejuense, C. tructae and C. viscerum resulted in relatedness values clearly below 70 %. These DNA-DNA hybridization results and the differentiating biochemical and chemotaxonomic properties showed that both strains 100(T) and C26(T) represent novel species, for which the names Chryseobacterium gallinarum sp. nov. (type strain 100(T) = LMG 27808(T) = CCM 8493(T)) and Chryseobacterium contaminans sp. nov. (type strain C26(T) = LMG 27810(T) = CCM 8492(T)) are proposed.