Yvonne Pfeifer

Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens

During the past 15 years, emergence and dissemination of beta-lactam resistance in nosocomial Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, became a serious problem worldwide. Especially the increasing resistance to 3rd and 4th generation cephalosporins and carbapenems is of particular concern. Gram-negative bacteria pursue various molecular strategies for development of resistance to these antibiotics: (a) generation of extended-spectrum beta-lactamases (ESBL) according to the original definition due to extension of the spectrum of already widely disseminated plasmid-encoded beta-lactamases by amino acid substitution; (b) acquisition of genes encoding ESBL from environmental bacteria as, for instance the CTX-M-type beta-lactamases from Kluyvera spp.; (c) high-level expression of chromosome-encoded beta-lactamase (bla) genes as bla(OXA) or bla(ampC) genes due to modifications in regulatory genes, mutations of the beta-lactamase promoter sequence as well as integration of insertion sequences containing an efficient promoter for intrinsic bla genes; (d) mobilization of bla genes by incorporation in integrons and horizontal transfer into other Gram-negative species such as the transfer of the ampC gene from Citrobacter freundii to Klebsiella spp.; (e) dissemination of plasmid-mediated carbapenemases as KPC and metallo-beta-lactamases, e.g. VIM and IMP; (f) non-expression of porin genes and/or efflux pump-based antibiotic resistance. This mini-review summarizes the historical emergence of beta-lactam resistance and beta-lactamases as major resistance mechanism in enteric bacteria, and also highlights recent developments such as multidrug- and carbapenem resistance.

Detection of NDM-1, VIM-1, KPC, OXA-48, and OXA-162 carbapenemases by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a potentially useful tool for the detection of antimicrobial resistance, especially that conferred by β-lactamases. Here we describe a modification of a previously reported MALDI-TOF MS meropenem hydrolysis assay. The modified method was validated on 108 carbapenemase-producing members of the Enterobacteriaceae, two NDM-1-producing Acinetobacter baumannii isolates, and 35 carbapenem-resistant enterobacteria producing no carbapenemase. The detection of carbapenemases by MALDI-TOF MS seems to be a powerful, quick, and cost-effective method for microbiological laboratories.

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.

Emergence of carbapenem-non-susceptible extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tübingen, Germany.

The spread of Gram-negative bacteria with plasmid-borne extended-spectrum beta-lactamases (ESBLs) has become a worldwide problem. This study analysed a total of 366 ESBL-producing Enterobacteriaceae strains isolated from non-selected patient specimens at the university hospital of Tübingen in the period January 2003 to December 2007. Although the overall ESBL rate was comparatively low (1.6 %), the percentages of ESBL-producing Enterobacter spp. and Escherichia coli increased from 0.8 and 0.5 %, respectively, in 2003 to 4.6 and 3.8 % in 2007. In particular, the emergence was observed of one carbapenem-resistant ESBL-producing E. coli isolate and five carbapenem-non-susceptible ESBL-positive Klebsiella pneumoniae isolates, in two of which carbapenem resistance development was documented in vivo under a meropenem-containing antibiotic regime. The possible underlying mechanism for this carbapenem resistance in three of the K. pneumoniae isolates was loss of the Klebsiella porin channel protein OmpK36 as shown by PCR analysis. The remaining two K. pneumoniae isolates exhibited increased expression of a tripartite AcrAB-TolC efflux pump as demonstrated by SDS-PAGE and mass spectrometry analysis of bacterial outer-membrane extracts, which, in addition to other unknown mechanisms, may contribute towards increasing the carbapenem MIC values further. Carbapenem-non-susceptible ESBL isolates may pose a new problem in the future due to possible outbreak situations and limited antibiotic treatment options. Therefore, a systematic exploration of intestinal colonization with ESBL isolates should be reconsidered, at least for haemato-oncological departments from where four of the five carbapenem-non-susceptible ESBL isolates originated.

High prevalence of extended-spectrum-β-lactamase-producing Enterobacteriaceae in organic and conventional retail chicken meat, Germany

OBJECTIVES To determine the prevalence of extended-spectrum β-lactamase (ESBL) production in Enterobacteriaceae in retail chicken meat in Germany. METHODS A total of 399 chicken meat samples from nine supermarket chains, four organic food stores and one butchers shop in two geographically distinct regions (Berlin and Greifswald) were screened for ESBL production using selective agar. Phenotypic ESBL isolates were tested for bla(TEM), bla(CTX-M) and bla(SHV) genes using PCR and DNA sequencing. Antibiotic coresistances were determined and strain typing was performed using PCR-based phylogenetic grouping and XbaI-PFGE. RESULTS A total of 185 confirmed ESBL isolates were obtained from 175 samples (43.9%) from all tested sources. The majority of isolates were Escherichia coli producing ESBL types SHV-12 (n = 82), CTX-M-1 (n = 77) and TEM-52 (n = 16). No differences could be observed in the prevalence of ESBL producers between organic and conventional samples. 73.0% of the ESBL producers showed coresistance to tetracycline, 35.7% to co-trimoxazole and 7.6% to ciprofloxacin. Strain typing of selected E. coli isolates from Berlin revealed identical macrorestriction patterns for several isolates from samples taken from the same stores. CONCLUSIONS This is the first comprehensive study from Germany showing a high prevalence of TEM-, CTX-M- and SHV-type ESBLs in Enterobacteriaceae isolated from retail chicken meat. The high rate of coresistance to different classes of antibiotics in the ESBL producers might reflect the common veterinary usage of these and related substances. There is an urgent need to further evaluate the role of poultry in the transmission of highly resistant ESBL-producing bacteria in humans.

Extended-Spectrum-β-Lactamase-Producing Escherichia coli as Intestinal Colonizers in the German Community

ABSTRACT We determined the presence of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli among 3,344 study participants from the German community. Intestinal colonization was detected in 211 persons (6.3%), without significant differences among the different age groups. The majority (95.2%) of isolates harbored CTX-M-type ESBL, with CTX-M-15 (46%) and CTX-M-1 (24.2%) as the most common types. The finding of ESBL producers and one isolate additionally producing carbapenemase OXA-244 indicates a risk of dissemination of resistant bacteria outside the hospitals.

Emergence of OXA-48 carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in dogs

OBJECTIVES To evaluate the possible occurrence of carbapenemase-producing Escherichia coli and Klebsiella spp. strains in domestic animals. METHODS Veterinary clinical E. coli (n = 1175) and Klebsiella spp. (n = 136) isolates consecutively collected from livestock and companion animals in Germany from June 2012 to October 2012 were screened for their susceptibility to carbapenems using the agar disc diffusion test. Carbapenemase genes were characterized by PCR and sequencing; conjugation assays were performed. Carbapenemase-positive isolates were assigned to phylogenetic lineages by multilocus sequence typing and the clonal relatedness was determined using macrorestriction analysis and subsequent PFGE. RESULTS Carbapenem non-susceptible isolates of Klebsiella pneumoniae (n = 5) and E. coli (n = 3) were obtained from six dogs hospitalized in a single veterinary clinic in Hessia, Germany, partly at the same time and consecutively over the study period. All isolates harboured carbapenemase gene blaOXA-48 located within Tn1999.2 transposons on conjugative ~60 kb plasmids. The K. pneumoniae isolates belonged to sequence type ST15, pulsotype 1, and coexpressed CTX-M-15, SHV-28, OXA-1 and TEM-1. Two E. coli isolates were assigned to ST1196 and pulsotype 2 and coproduced CMY-2, SHV-12 and TEM-1, while the third E. coli isolate was of ST1431 (pulsotype 3), and possessed blaCTX-M-1, blaOXA-2 and blaTEM-1. CONCLUSIONS This is the first known report of OXA-48-producing bacteria from companion animals. The clonal nature of the K. pneumoniae and two E. coli isolates suggests a nosocomial dissemination rather than repeated introduction by individual patients into the clinic.

A novel IS26 structure surrounds blaCTX-M genes in different plasmids from German clinical Escherichia coli isolates.

This report focuses on the molecular characterization of 22 extended-spectrum beta-lactamase-producing Escherichia coli isolates collected in a German university hospital during a period of 9 months in 2006. Relationship analysis of clinical isolates was done via PFGE, multilocus sequence typing, plasmid profiling and additionally PCR for bla(ESBL) detection and determination of phylogroups. After conjugal transfer, plasmid isolation and subsequent PCR for bla(ESBL) detection and determination of incompatibility groups were performed. Using one-primer walking, up to 3600 bp upstream and downstream of different bla(CTX-M) genes could be sequenced. beta-Lactamases found were TEM-1 (n=14), SHV-5 (n=1) and a wide variety of CTX-M types (n=21), i.e. CTX-M-15 (n=12), CTX-M-1 (n=4), CTX-M-14 (n=2), CTX-M-9 (n=1), CTX-M-3 (n=1) and one new type, CTX-M-65 (n=1). In 18 isolates, bla(ESBL) genes were located on conjugative plasmids of sizes between 40 and 180 kbp belonging to incompatibility groups FII (n=9), N (n=5) and I1 (n=4). bla(CTX-M) was found to be associated with the common elements ISEcp1, IS26 and IS903-D, but with unusual spacer sequences for ISEcp1 in two isolates. These insertion sequences, connected to bla(CTX-M) as well as other genes, were located between two IS26 elements in a configuration that has not yet been described. The results reveal the emergence of bla(ESBL), predominantly bla(CTX-M), located on different plasmids harboured by genotypically different E. coli strains. The identical gene arrangement in the bla(CTX-M) neighbourhood in plasmids of different incompatibility groups indicates a main role of IS26 in distribution of mobile resistance elements between different plasmids.

NDM-1-Producing Escherichia coli in Germany

The bla NDM-1 carbapenemase gene has now been identified among many different enterobacterial species from many countries ([1][1], [7][2], [10][3]-[14][4]). Its current spread has often been associated with importations from the Indian subcontinent. Here we report the case of a 70-year-old man

Emergence of OXA-48-Type Carbapenemase-Producing Enterobacteriaceae in German Hospitals

ABSTRACT Nine carbapenem-resistant Enterobacteriaceae isolates collected from eight patients in five German hospitals were investigated. Six isolates produced the OXA-48 carbapenemase, and three isolates produced OXA-162, which is a point mutant form of OXA-48. Both carbapenemase genes were located on IncL/M-type conjugative plasmids. Insertion sequence IS1999 (truncated or not by IS1R) was located upstream of the blaOXA-48 and blaOXA-162 genes in all of the isolates. Pulsed-field gel electrophoresis typing indicated the clonal transmission of an OXA-48-producing Klebsiella pneumoniae strain in two hospitals.