Development Of Carbapenem Resistance Research Articles

Emergence of Carbapenem-Resistant Escherichia Coli in Taiwan: Resistance Due to Combined CMY-2 Production and Porin Deficiency

Eight pairs of Escherichia Coli isolates with various carbapenem susceptibilities from 8 patients were prospectively collected to study the development of resistance. All carbapenem-resistant E. coli isolates were resistant to all tested β-lactams antibiotics except tigecycline. identical pulsed-field gel electrophoresis (PFGE) patterns were found in carbapenem-susceptible and -resistant isolates but different PFGE patterns occurred among patients. A CMY-2 β-lactamase was found in all E. coli isolates. No previously reported carbapenemase genes were detected. Examination of outer membrane protein (OMP) profiles revealed that OmpA was not found in all isolates, while OmpC and Ompf were lost in carbapenem-resistant isolates. loss of both OmpC and Ompf represents the major mechanism of the development of carbapenem resistance in those patients with CMY-2-producing E. coli infections.

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.

Emergence of imipenem resistance in clinical Escherichia coli during therapy

The molecular epidemiology and the mechanisms of resistance of Escherichia coli isolated from two patients infected by imipenem-resistant strains are reported in this study. From one patient, three closely related consecutive isolates of E. coli were recovered; the first was carbapenem-susceptible but acquired imipenem resistance after treatment with ertapenem, and the third isolate was again imipenem-susceptible. An additional imipenem-resistant isolate was recovered from another patient who received imipenem. The genetic relatedness of the E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE) after digestion with XbaI. Standard polymerase chain reaction (PCR) conditions were used to amplify several β-lactamase genes coding for carbapenemases, extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC; the E. coli ampC gene promoter was also amplified and sequenced. Primers OmpF-F/OmpF-R and OmpC-F/OmpC-R were used to amplify the ompF and ompC genes. The outer membrane protein (OMP) profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Imipenem-resistant E. coli isolates did not produce carbapenemases but lacked the two major OMPs OmpF and OmpC and had ampC promoter mutations; in addition, one of the imipenem-resistant isolates produced the CMY-2 cephalosporinase, whilst the other produced the new CTX-M-67 ESBL. Carbapenem resistance in this study was associated with lack of expression of OmpF and OmpC porins. Additional mechanisms of β-lactam resistance, such as plasmid-mediated AmpC and ESBL production, were also found. Development of carbapenem resistance in a CTX-M-67-producing E. coli is first described in this study.

In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple β-lactamases

Four clinical strains of extended-spectrum β-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C 1 and C 2 were isolated prior to carbapenem therapy, whilst isolates C 3 and C 4 were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). β-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C 1 in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple β-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C 1 and C 2, isolates C 3 and C 4 failed to express OmpE36 owing to insertional inactivation by an IS 903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene ( kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C 3 and C 4 were apparently derived from the previously imipenem-susceptible isolates C 1 and C 2. Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.

Acquisition of a Plasmid-Borne bla OXA-58 Gene with an Upstream IS 1008 Insertion Conferring a High Level of Carbapenem Resistance to Acinetobacter baumannii

The oxacillinase gene was reported to confer limited resistance to carbapenem in Acinetobacter baumannii. In this study, we have demonstrated that an A. baumannii clinical isolate harboring a plasmid, pTVICU53, has 11,037 bp encoding 13 open reading frames. A bla(OXA-58) gene with an upstream insertion of truncated ISAba3 (DeltaISAba3) and IS1008 was found in this plasmid. DeltaISAba3and IS1008 provided two independent promoters for the transcription control of the bla(OXA-58) gene. The transformation of pTVICU53 or a shuttle vector bearing IS1008-DeltaISAba3-bla(OXA-58) to different A. baumannii recipients can increase their MICs of carbapenem 64- to 256-fold. The deletion of promoters provided by IS1008 resulted in dramatic decreases in bla(OXA-58) transcription and a 32- to 64-fold reduction in the carbapenem MIC. These findings highlight that A. baumannii might develop carbapenem resistance with a single transformation step, taking up a plasmid containing a genetic construct with a potentially high level of transcription of the bla(OXA-58) gene.

Carbapenem Resistance in Clinical Isolates of Pseudomonas aeruginosa

The objectives of this study were to identify the carbapenem resistance mechanisms of clinical Pseudomonas aeruginosa isolates. The strains resistant to imipenem had lost only the OprD protein, the isolates resistant to imipenem and meropenem had both loss of the OprD porin and reduced minimum inhibitory concentrations (MICs) in the presence of efflux pump inhibitors. In the isolates in which efflux had been identified (n=2) only 1 isolate had a mutation in the mexR gene corresponding to a glutamine to a stop codon change at amino acid 106. This has not been previously identified. There were no significant changes in the mexT genes. No mutations previously associated with the upregulation of the carbapenem efflux pumps in in vitro generated resistant isolates were identified in any of the clinical isolates. Therefore, the resistance mechanisms identified by development of carbapenem resistance in vitro are not sufficient to understand carbapenem resistance development in clinical isolates.

Characterization of a Large Outbreak by CTX-M-1-Producing Klebsiella pneumoniae and Mechanisms Leading to In Vivo Carbapenem Resistance Development

All extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from patients admitted to and adult intensive care unit were prospectively documented from 2002 to 2005, when a large outbreak (51 patients affected) of multiresistant ESBL-producing Klebsiella pneumoniae infection was detected. The involvement of a single K. pneumoniae clone was demonstrated by pulsed-field gel electrophoresis. In addition to the ESBL-mediated resistance, the epidemic strain uniformly showed cross-resistance to ciprofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, and tetracycline, whereas resistance to the beta-lactam-beta-lactamase inhibitor combinations was variable. The ESBL involved was CTX-M-1, as demonstrated by isoelectric focusing, PCR amplification, and sequencing. CTX-M-1 as well as the aminoglycoside resistance determinants were encoded in a 50-kb plasmid that could be transferred to Escherichia coli only by transformation. In two of the infected patients, carbapenem resistance development (MICs of 8 to 12, 16, and >32 microg/ml for imipenem, meropenem, and ertapenem, respectively) was documented, both in clinical samples and in intestinal colonization studies. The analysis of the outer membrane proteins of the carbapenem-susceptible and -resistant isolates revealed that the former expressed only one of the two major porins, OmpK36, whereas the latter did not express either of them. In one of the cases, the lack of expression of OmpK36 was demonstrated to be mediated by the interruption of the coding sequence by the insertion sequence IS26. This is the first report of a large outbreak of CTX-M-1-producing Enterobacteriaceae and, curiously, the first documented description in the literature of CTX-M-1 in K. pneumoniae, despite the fact that this enzyme has been found in multiple species. Furthermore, we document and characterize for the first time carbapenem resistance development in CTX-M-1-producing Enterobacteriaceae.

Interplay of Efflux System, ampC , and oprD Expression in Carbapenem Resistance of Pseudomonas aeruginosa Clinical Isolates

Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal beta-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. beta-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.