Institut für Infektionsprävention und Krankenhaushygiene

Resistance Determinants by Comparative Transcriptome Analysis of Enterobacter cloacae strains  

Enterobacter cloacae is a nosocomial pathogen that can virtually infect any compartment of the body. The isolation rate of carbapenem resistant strains of E. cloacae has increased over time all over the world. Due to the emergence of carbapenem resistance, tigecycline can be the first choice for treatment. However, isolation of tigecycline resistant strains is already being reported, which could further limit therapeutic choices in the near future. In our investigations of 190 E. cloacae strains from large outbreaks, we also found tigecycline resistant strains. Interestingly, we found pairs of strains from different outbreaks that were genetically identical or very similar based on Restriction Fragment Length Polymorphism pattern (RFLP), but which displayed significant differences in Minimum Inhibitory Concentrations (MIC) against tigecycline. This unique collection of strains provides the opportunity to investigate the mechanisms of tigecycline resistance by employing an entirely novel approach. We will compare the transcriptome of genetically related pairs of outbreak-strains with significant differences in their MIC against tigecycline. Furthermore, susceptible as well as resistant strains of E. cloacae will be challenged with sub-inhibitory concentrations of tigecycline. Prior to total RNA extraction, bacterial cells will be stabilized to minimize RNA degradation. Subsequently, rRNA will be depleted to enrich the mRNA molecules. mRNA enriched RNA samples from genetically related strains with an either susceptible or resistant phenotype, challenged or unchallenged with tigecycline will be sequenced by the recently developed high-throughput (HT) sequencing technique (Hiseq illumina 2000). The mRNA obtained sequences will be aligned and analyzed with the tools implemented in Galaxy (http://galaxy.psu.edu) a web-based platform for interactive large-scale genome analysis. Comparative transcriptome analysis from resistant and susceptible pairs and tigecycline challenged vs. unchallenged strains are expected to provide us with novel candidate transcripts directly or indirectly associated with resistance against tigecycline. These findings will have to be proven by further recombination studies. The outcome of this study could possibly be used to develop novel antimicrobials against this or related resistant enteric pathogens.

Investigation of Stenotrophomonas maltophilia by Multi Locus Sequence Typing

Stenotrophomonas maltophilia is an ubiquitous environmental species, which is also found as an opportunistic pathogen colonising or infecting especially immunocompromised patients. Previously 10 different genogroups have been delineated on the base of Amplified Fragment length Polymorphism (AFLP) [Hauben et al. 1999). To obtain more information about the genetic population structure we are developing a Multi Locus Sequence Typing (MLST) scheme for this species, utilizing seven housekeeping genes. Seventy strains from all over the world are analysed including type strains, representatives of the 10 AFLP genogroups and subsequent isolates from 5 hospital outbreaks. Among the 70 S. maltophilia isolates 54 STs and 38 to 53 alleles for the single loci could be observed. We could confirm 9 of 10 genogroups and identified five new groups. Three genogroups each included exclusively ST from isolates of either clinical or environmental origin. Twelve out of 19 isolates from epidemiologically non-associated cystic fibrosis patients clustered in one particular genogroup. Considering all isolates a significant “Index of Association” indicated to a “linkage disequilibrium” as expected for clonal species. Accordingly, the Pearson-correlation and the „likelihood score“- tests demonstrated a good congruence for the single loci-trees except for two loci. On the other Hand, there was evidence for intragenic recombination as detected by the maximum chi-squared test. The unusual separation from isolates of different ecological origins into distinct genogroups requires further investigations.

Genodiversity of Resistant Pseudomonas aeruginosa in Relation to Antimicrobial Usage and Resistance Rates in Intensive Care Units

The objective of this study was to prove the assumption that resistance rates in intensive care units (ICUs) are markedly influenced by cross-transmission events, beside high antimicrobial usage.

This was a prospective ICU and laboratory-based surveillance study involving 35 German ICUs from 1999 through 2004. Five hundred and eighty-five ciprofloxacin or imipenem-resistant isolates of Pseudomonas aeruginosa were investigated, together with resistance rates (RR) and unit-based antimicrobial use (AD). Antimicrobial use was reported in terms of defined daily doses (DDDs) per 1,000 patient-days. All the strains were assigned to ICU-based genotypes. Genodiversity was calculated as the numbers of indistinguishable ICU-based genotypes found per isolates tested. Reduced ICU-based genodiversity was taken as an indirect measure of frequently occurring cross-transmission events.

The genodiversity of ciprofloxacin as well as imipenem-resistant P. aeruginosa isolates was significantly lower (Fisher P _< 0.05) in ICUs with high RR and low AD (0.50 and 0.50) than in those ICUs that featured low RR in the presence of high AD (0.90 and 0.95). In ICUs with low genodiversity, there was a stronger rise of RR with increasing AD than in ICUs with high diversity.

This study on resistant P. aeruginosa supports the assumption that high RR in the presence of low AD results from more frequent cross-transmission events. A stronger rise of RR with increasing AD in ICUs with a low genodiversity indicates that RR in ICUs might be markedly determined by cross-transmission events beside antimicrobial usage.

Plasmid-mediated quinolone-resistance (qnr) in German ICUs

The occurrence of a novel plasmid-mediated quinolone resistance has been reported in a few cases outside Europe. This transmissible quinolone resistance gene (qnr) was detected in isolates from patients in the USA, China and Egypt. Molecular analysis demonstrated the location of the qnr-gene in class I integrons. The aim of this study was to determine the prevalence of qnr-positve strains in German ICUs.

Six hundred and forty-eight fluoroquinolone or cephalosporin-resistant Enterobacteriaceae and Acinetobacter spp., obtained from 34 ICUs during the last three years were screened for the presence of integrons. One hundred and thirty-four strains containing integron cassettes as well as integrase sequences were tested for the presence of the qnr gene-locus by use of PCR.

One isolate of Enterobacter spp. from a German ICU patient turned out to be qnr-positive as well as five Citrobacter freundii strains from three patients treated in a South-German ICU during a one month period. All C. freundii strains were indistinguishable by use of XbaI macrorestriction analysis. Moreover, none of these quinolone-resistant strains were susceptible to cefoxitin, ceftazidime or cefotaxime.

This is one of the first reports of qnr-positive strains obtained from patients in Europe. Molecular epidemiology suggests that qnr-positve strains were involved in an outbreak in one ICU. The spread of transmissible quinolone-resistance might be underscored.