Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 β-lactamases co-mediated by porin loss

doi:10.1016/j.ijantimicag.2006.09.006

International Journal of Antimicrobial Agents

Volume 29, Issue 2, February 2007, Pages 201-206

https://doi.org/10.1016/j.ijantimicag.2006.09.006 Get rights and content

Abstract

We investigated the resistance mechanisms and clonality among 42 imipenem-non-susceptible Klebsiella pneumoniae isolated at a tertiary care hospital in Korea. Two isolates had bla_VIM-2 alleles, whereas bla_CMY-2- and bla_DHA-1-like alleles were detected in 24 and 16 isolates, respectively, with these enzymes confirmed by sequencing for representative isolates. Transfer of bla_CMY-2 and bla_DHA-1 was achieved by conjugation. Addition of 300 mg/L 3-aminophenylboronic acid (APB) reduced the minimum inhibitory concentration for 90% of the organisms (MIC₉₀) of imipenem and meropenem eight- and four-fold, respectively, for the bla_CMY-2- and bla_DHA-1-positive isolates, confirming the role of these enzymes in resistance. SDS-PAGE of outer membrane proteins for representative isolates showed lack or greatly diminished expression of OmpK35 and OmpK36 porins. Pulsed-field gel electrophoresis of XbaI-restricted genomic DNA revealed two closely related clusters among 23 bla_CMY-2-positive isolates, whereas those with bla_DHA-1 were more heterogeneous. In conclusion, reduced imipenem susceptibility among K. pneumoniae at this Korean hospital was largely co-mediated by production of plasmid-mediated AmpC β-lactamases along with lack or greatly diminished expression of OmpK35 and OmpK36 porins.

Introduction

Klebsiella pneumoniae is frequently isolated from serious nosocomial infections and is increasingly resistant to expanded-spectrum cephalosporins via production of various extended-spectrum β-lactamases (ESBLs) [1] and plasmid-mediated AmpC enzymes, with the latter also conferring resistance to cephamycin [2]. A plasmidic AmpC enzyme, CMY-1, was detected in Korea in 1988, with CMY-2 and other AmpC types subsequently becoming widespread in Escherichia coli and K. pneumoniae[3].

Klebsiella pneumoniae isolates producing ESBLs and plasmid-mediated AmpC enzymes mostly remain susceptible to carbapenems and, whilst carbapenem resistance is increasingly prevalent among Acinetobacter spp. and Pseudomonas spp. in Korea, it remains very rare among Enterobacteriaceae, as is other countries. Nevertheless, carbapenem resistance can arise in Enterobacteriaceae, mediated by class A, D or B carbapenemases [4] or via combination of AmpC or ESBLs together with impermeability [5].

The aim of this study was to investigate the mechanism of resistance and clonality among 42 imipenem-non-susceptible K. pneumoniae isolates collected between 2002 and 2004 at one tertiary care hospital in Korea.

Section snippets

Isolates and susceptibility testing

Forty-two non-duplicate isolates of K. pneumoniae isolated between 2002 and 2004 from patients at one tertiary care hospital in Korea were investigated. Isolates were identified by conventional phenotypic tests [6] and the imipenem susceptibility was determined by a disk diffusion method [7] using commercial disks and Mueller–Hinton agar (Becton Dickinson and Co., Sparks, MD). ESBLs were determined by double-disk tests with amoxicillin/clavulanic acid (20/10 μg), cefepime (30 μg), cefotaxime (30

Antimicrobial susceptibility and detection of plasmid-mediated AmpC genes

Among the 42 imipenem-non-susceptible isolates of K. pneumoniae, 2 were positive by the imipenem disk Hodge test and the EDTA-SMA double-disk synergy test, indicating the presence of MBL. bla_VIM-2 alleles were detected in these isolates by PCR. All the remaining 40 isolates were negative for these tests and lacked genes for known MBLs. However, they were resistant to cefoxitin and 33 gave positive results in the cefoxitin disk Hodge test, suggesting production of AmpC β-lactamases, although it

Acknowledgments

We are grateful to Ms. Myungsook Kim for collecting the isolates and to Ms. Younghee Seo and Ms. Chasoon Lee for technical assistance.

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An Unusual Carbapenem Resistant Escherichia coli Carrying Plasmid-mediated AmpC and Mutated ompC in A Patient with Recurrent Urinary Tract Infections
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This highly mutated ompC is predicted to result in porin loss, which in conjunction with the plasmid-mediated blaCMY-2, explains the carbapenem resistance in our case. Notably this resistance mechanism (i.e., pAmpC + porin loss) has already been identified in both K. pneumoniae and E. coli previously [3,21]. However, the current prevalence of carbapenem resistant E. coli with this mechanism in the U.S. is still unknown.
We describe a case of carbapenem resistant E. coli isolated from urine in an 87-year-old woman with recurrent urinary tract infections. Using whole genome sequencing (WGS), we identified the carbapenem resistance mechanism to be a combination of ompC porin loss and plasmid-mediated AmpC gene bla_CMY-2, which was not detected by routine molecular and phenotypic carbapenemase assays. Our case raises a concern for the limitation of current CRE screening tools for emerging resistance mechanisms and demonstrates the utility of WGS as a better tool for characterization of CRE in the clinical setting.
Multi-drug carbapenem-resistant Klebsiella pneumoniae infection carrying the OXA-48 gene and showing variations in outer membrane protein 36 causing an outbreak in a tertiary care hospital in Riyadh, Saudi Arabia
2014, International Journal of Infectious Diseases
To investigate the genes of antibiotic resistance among isolates from the first reported carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak in a tertiary care hospital, Riyadh, Saudi Arabia.
Antimicrobial susceptibility testing was performed on bacterial isolates using the Microscan Walkaway system (Siemens, Germany) and was confirmed by Etest (AB Biodisk, Sweden). bla_-CTX-M, _-SHV, _-TEM, _-OXA-48, _OXA-A,B,C,D, _-KPC, _-NDM, _-VIM, _-IMP, integron 1, and outer membrane proteins(Omp)-35 and Omp-36 were investigated by PCR amplification and direct sequencing of PCR products. Isolates were sequence-typed by multilocus sequence typing (MLST).
All isolates were resistant to cefotaxime, ceftazidime, cefepime, ciprofloxacin, and piperacillin–tazobactam, and 91% (21 out of 23) were resistant to amikacin and gentamicin. All isolates except two from a single patient were resistant to one of the carbapenems. CTX-M and SHV genes were detected in all isolates, CTX-M-15 and SHV-1 types being predominant among these extended-spectrum beta-lactamases (ESBLs). TEM-1 was found in all except one isolate (isolate 3). Significantly, the OXA-48 gene was also found in all isolates. OXA-D-gene was found in three out of 23 isolates. KPC, NDM, OXA-A, -B, -C, VIM, and IMP genes were absent in all isolates. Disruption of the Omp-36 gene due to insertion of transposon IS903 and/or IS4 was detected in four out of 23 isolates, and some unique variations were also observed in this gene, including an insertion of two amino acids in the L3 region of Omp-36 in one isolate (isolate 3) and a mutation resulting in a premature stop codon in another isolate (isolate 25). MLST revealed ST29 to be the predominant sequence type (17 out of 23 isolates, 74%). Three were ST709 and one each was ST37 and ST111; one isolate had an unknown ST.
This is probably the first reported outbreak of multidrug/carbapenem-resistant Klebsiella infection involving the OXA-48 gene from Saudi Arabia. Although the presence of ESBLs such as OXA, CTX-M, TEM, and SHV are predictable reasons for resistance, variations in the Omp-36 gene might also have precipitated this phenomenon. Disruption of the Omp-36 sequence by large insertional elements, the insertion of two amino acids in a very crucial part of this protein, and the presence of a premature stop codon in one isolate might have rendered this protein incomplete and non-functional. The study also demonstrated that more than one type of clone was responsible for this reported apparent outbreak and that ST29, a clone not reported from this region before, was the major clone responsible.
Successful containment of carbapenem-resistant Enterobacteriaceae by strict contact precautions without active surveillance
2014, American Journal of Infection Control
Citation Excerpt :
Noncarbapenemase-producing CRE are generally regarded as less significant in terms of public health than carbapenemase producers, and the importance of infection control measures is also less emphasized. In principle, however, the horizontal spread of plasmid-mediated β-lactamases remains a possibility, and a clonal outbreak of plasmid-mediated AmpC β-lactamases producing KPN has indeed been reported.21 Our isolates were not clonally related, and we implemented several infection control strategies at once; therefore, we cannot estimate the impact of each separately.
Carbapenem-resistant Enterobacteriaceae (CRE) are a growing problem worldwide. Guidelines focus on carbapenemase-producing organisms, and little is known about whether strict adherence to infection control measures is effective for CRE without carbapenemase. During 2009, CRE increased markedly in a tertiary hospital, and enhanced infection control measures without active surveillance were adopted.
Beginning in April 2010, enhanced antimicrobial stewardship, strict contact precautions, and cohort isolation were adopted. After September 2010, hand hygiene performance was prospectively monitored by active surveillance, and results were monthly fed back to medical personnel. Available carbapenem-resistant Escherichia coli (ECO) and carbapenem-resistant Klebsiella pneumoniae (KPN) isolated during 2008-2010 were characterized. Imipenem and meropenem minimal inhibitory concentrations were confirmed by E-test (AB biodisk, Solna, Sweden). Phenotypic screening assays and polymerase chain reaction (PCR) amplification of known β-lactamase and carbapenemase genes were performed.
From 3,511 ECO and 2,279 KPN, 44 (0.76%) were CRE (3 ECO, 41 KPN). CRE incidence rates rose from 1.61 in 2008 to 5.49 in 2009; they rose further to 9.81 per 100,000 patient days in early 2010. After adoption of strict infection control measures, CRE frequency fell back in 2011 and remained at baseline afterward. Phenotypic screening and PCR showed AmpC β-lactamase and extended spectrum β-lactamases with or without loss of porins; carbapenemases were not detected.
Enhanced infection control measures, even without active surveillance, seem effective to prevent further spread of CRE in a low-prevalence setting with mainly carbapenemase-nonproducing CRE.
Underlying mechanisms of carbapenem resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: Role of OXA-48 and NDM-1 carbapenemases
2013, International Journal of Antimicrobial Agents
A recent increase in carbapenem resistance among extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli isolates at a major tertiary care centre in Lebanon prompted the initiation of this study. Consecutive ESBL-producing isolates were tested for resistance to carbapenems, with initial screening by disk diffusion and Etest using ertapenem. The modified Hodge test was also performed. PCR of β-lactamase-encoding genes, including bla_NDM-1, bla_KPC, bla_OXA-48, bla_CTX-M, bla_TEM, bla_SHV, bla_CMY-2 and bla_OXA-1, as well as outer membrane porin genes (ompC and ompF) was performed. Sequencing, efflux pump inhibitor tests and random amplified polymorphic DNA (RAPD) analysis were performed. In total, 14 (2.45%) of 572 K. pneumoniae and 24 (1.07%) of 2243 E. coli were ertapenem-non-susceptible [minimum inhibitory concentration (MIC) ≥0.25 μg/mL]. Resistance to other carbapenems was variable. PCR and sequencing analysis revealed that isolates harboured different β-lactamase genes, including bla_OXA-1, bla_CTX-M-15, bla_TEM-1, bla_CMY-2, bla_OXA-48 and bla_NDM-1. In addition, K. pneumoniae lacked the outer membrane porin-encoding genes, whilst E. coli harboured them with detected mutations. CTX-M-15 was carried on a 90 kb plasmid, whilst OXA-48 was carried on a 70 kb plasmid. Efflux pump inhibition significantly decreased MICs in E. coli. RAPD analysis demonstrated genomic variability. In conclusion, carbapenem resistance in ESBL-producing K. pneumoniae and E. coli is due to the combined effect of β-lactamases with porin impermeability and/or efflux pump activity observed in these organisms, and in a number of isolates is due to the production of the carbapenemase-encoding genes bla_OXA-48 and the newly emerging bla_NDM-1.
Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae
2012, Indian Journal of Medical Microbiology
Purpose: Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR). Materials and Methods: Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25) and K. pneumoniae (n = 52) were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL). Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test). Results: Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii), DHA (Dhahran Hospital, Saudi Arabia), ACC (Ambler class C), EBC (Amp C origin - Enterobacter cloacae) groups. ESBL was co-produced in 54 isolates. Conclusions: Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.
Carbapenem resistance in Enterobacteriaceae: Here is the storm!
2012, Trends in Molecular Medicine
Citation Excerpt :
Plasmid-mediated cephalosporinase genes are often associated with coresistance to other antibiotics (e.g. aminoglycosides, tetracycline, sulfonamides) as a consequence of the colocalization of antibiotic resistance genes on the same plasmids [10]. The selection of isolates with carbapenem resistance or decreased carbapenem susceptibility has been sometimes observed during a carbapenem-based treatment [10,17,19]. Similarly, production of an ESBL in combination with outer membrane permeability defects can be responsible for carbapenem resistance in Enterobacteriaceae [8,19,20].
The current worldwide emergence of resistance to the powerful antibiotic carbapenem in Enterobacteriaceae constitutes an important growing public health threat. Sporadic outbreaks or endemic situations with enterobacterial isolates not susceptible to carbapenems are now reported not only in hospital settings but also in the community. Acquired class A (KPC), class B (IMP, VIM, NDM), or class D (OXA-48, OXA-181) carbapenemases, are the most important determinants sustaining resistance to carbapenems. The corresponding genes are mostly plasmid-located and associated with various mobile genetic structures (insertion sequences, integrons, transposons), further enhancing their spread. This review summarizes the current knowledge on carbapenem resistance in Enterobacteriaceae, including activity, distribution, clinical impact, and possible novel antibiotic pathways.

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International Journal of Antimicrobial Agents

Abstract

Introduction

Section snippets

Isolates and susceptibility testing

Antimicrobial susceptibility and detection of plasmid-mediated AmpC genes

Acknowledgments

References (15)

Evaluation of phenotypic screening methods for detecting plasmid-mediated AmpC β-lactamases-producing isolates of Escherichia coli and Klebsiella pneumoniae

Diagn Microbiol Infect Dis

Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors

Clin Microbiol Rev

Plasmid-determined AmpC-type β-lactamases

Antimicrob Agents Chemother

Prevalence of plasmid-mediated AmpC β-lactamases in Escherichia coli and Klebsiella pneumoniae in Korea

Microb Drug Resist

Metallo-β-lactamases: the quiet before the storm?

Clin Microbiol Rev

Roles of β-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae

Antimicrob Agents Chemother

Enterobacteriaceae: introduction and identification

Cited by (54)

An Unusual Carbapenem Resistant Escherichia coli Carrying Plasmid-mediated AmpC and Mutated ompC in A Patient with Recurrent Urinary Tract Infections

Multi-drug carbapenem-resistant Klebsiella pneumoniae infection carrying the OXA-48 gene and showing variations in outer membrane protein 36 causing an outbreak in a tertiary care hospital in Riyadh, Saudi Arabia

Successful containment of carbapenem-resistant Enterobacteriaceae by strict contact precautions without active surveillance

Underlying mechanisms of carbapenem resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: Role of OXA-48 and NDM-1 carbapenemases

Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae

Carbapenem resistance in Enterobacteriaceae: Here is the storm!

Short communicationReduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 β-lactamases co-mediated by porin loss

Abstract

Introduction

Section snippets

Isolates and susceptibility testing

Antimicrobial susceptibility and detection of plasmid-mediated AmpC genes

Acknowledgments

Diagn Microbiol Infect Dis

Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors

Clin Microbiol Rev

Plasmid-determined AmpC-type β-lactamases

Antimicrob Agents Chemother

Prevalence of plasmid-mediated AmpC β-lactamases in Escherichia coli and Klebsiella pneumoniae in Korea

Microb Drug Resist

Metallo-β-lactamases: the quiet before the storm?

Clin Microbiol Rev

Roles of β-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae

Antimicrob Agents Chemother

Enterobacteriaceae: introduction and identification

Short communication
Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 β-lactamases co-mediated by porin loss