JAC Advance Access originally published online on June 18, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):410-413; doi:10.1093/jac/dkm215
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Collateral damage of flomoxef therapy: in vivo development of porin deficiency and acquisition of blaDHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 ß-lactamases
1 Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123 Ta-Pei Road, Niao-Sung, Kaohsiung 833, Taiwan 2 Department of Applied Microbiology, National Chiayi University, 300 University Road, Chiayi 600, Taiwan 3 Department of Clinical Pathology, Lin-Kou Medical Center, Chang Gung Memorial Hospital, 5 Fu-Hsin Street, Kweishan, Taoyuan 333, Taiwan 4 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, 259 Wenhua 1st Road, Kweishan, Taoyuan 333, Taiwan
* Corresponding author. Tel: +886-3-3281200, ext. 8363; Fax: +886-3-3971827; E-mail: sulh{at}adm.cgmh.org.tw
Received 10 April 2007; returned 13 April 2007; revised 22 May 2007; accepted 22 May 2007
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Abstract |
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Objectives: The study aimed to characterize the genetic basis of flomoxef and collateral ertapenem resistance in a clinical isolate of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP) after flomoxef exposure.
Methods: Four ESBL-KP isolates (Lkp11–14) were recovered sequentially from four episodes of bacteraemia in an elderly patient. Laboratory investigations included genotyping by PFGE, resistance gene analysis by PCR and sequencing, and outer membrane protein analysis by SDS–PAGE. Plasmid analysis by DNA–DNA hybridization, electroporation and conjugation was also performed.
Results: Lkp14 was recovered after 21 days of flomoxef therapy. It demonstrated an indistinguishable PFGE pattern when compared with those produced by Lkp11–13. However, resistance to both flomoxef and ertapenem emerged in Lkp14. Molecular characterization revealed that, in addition to the pre-existing ESBL production (CTX-M-3 and SHV-5) and OmpK35 deficiency found in Lkp11–13, Lkp14 had acquired an extra plasmid-mediated AmpC ß-lactamase gene (blaDHA-1) and failed to express OmpK36, because of insertional inactivation by an insertion sequence IS5. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Conjugational transfer of the plasmid-mediated blaDHA-1 gene into Lkp11 resulted in a significant increase in the MICs of cephamycins and ß-lactamase inhibitors, but not in those of carbapenems.
Conclusions: Lkp14 was apparently derived from the previously flomoxef-susceptible isolates, Lkp11–13. After flomoxef exposure, the in vivo acquisition of the plasmid-mediated blaDHA-1 gene has led to flomoxef resistance in Lkp14, and the concomitant depletion of OmpK36 expression has resulted in a collateral effect of ertapenem resistance and diminished susceptibilities to imipenem and meropenem.
Keywords: outer membrane proteins , OMPs , antimicrobial resistance mechanisms , carbapenems
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Introduction |
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Carbapenems have the most consistent in vitro activity against extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP).1 Cephamycins also have potential therapeutic effects on such organisms.1,2 However, one report described that, after the continuous use of cefotetan (a cephamycin), simultaneous resistance to cephamycins and carbapenems occurred in K. pneumoniae isolates through the acquisition of a plasmid-mediated AmpC-type ß-lactamase as well as the loss of a porin.3 Flomoxef is unique among cephamycins in having a difluoromethylthioacetamido group at position 7, giving it a better in vitro activity against ESBL-producing Enterobacteriaceae.2 Whether or not the agent will produce similar collateral damage as demonstrated by other cephamycins has not yet been reported.
Here, we report the in vivo acquisition of AmpC-type ß-lactamase activities and concomitant porin changes in a strain of ESBL-KP isolated sequentially from the blood of an elderly patient after flomoxef exposure. It resulted in full resistance to cephamycins and collaterally to ertapenem as well as diminished susceptibilities to imipenem and meropenem.
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Materials and methods |
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Case history
A 91-year-old woman was admitted to Chang Gung Memorial Hospital, Kaohsiung, Taiwan, for aspiration pneumonia on 11 March 2004. The first blood culture after admission yielded ESBL-KP Lkp11 and imipenem was prescribed. Afterwards she experienced another two episodes of bacteraemia, in which ESBL-KP isolates, Lkp12 and Lkp13, were recovered respectively, and imipenem was used in each episode. On 30 May, flomoxef was used to replace imipenem and discontinued subsequently on 18 June. The patient died of septic shock on 30 June 2004. The last blood culture grew another ESBL-KP, Lkp14, which was resistant to flomoxef and ertapenem.
Bacterial strains and antimicrobial susceptibility
Clinical isolates of K. pneumoniae, Lkp11–14, were identified and the antimicrobial susceptibility determined by standard methods. MICs of various antimicrobial agents (Table 1) were determined using Etest strips (AB Biodisk, Solna, Sweden), except that of flomoxef (Shionogi Ltd, Tokyo, Japan) which was determined by a standard broth microdilution method.
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Genotyping
Genetic relationship of the isolates was analysed by PFGE of the XbaI-digested macrofragments of the bacterial DNA.4 PFGE patterns that varied by more than four bands were considered to be of different genotypes.4
Analysis of outer membrane protein profiles and PCR–sequencing of specific porin and ß-lactamase genes
Outer membrane protein (OMP) profiles of the isolates were examined.4 To specifically define the DNA sequences of OMP genes, two primer pairs were designed. OmpK36-F (5'-ACAGAGGGTTAATAACATGAA) and OmpK36-R (5'-TAGAACTGGAAACCAGGC) were used to amplify a 1107 bp fragment from the ompK36 gene (GenBank accession no. Z33506), whereas OmpK35-F (5'-TGATCCCTGCCCTGCTGGT) and OmpK35-R (5'-CCGGAGTCATGTTGTAAGTCT) were used to amplify a 738 bp fragment from the ompK35 gene (GenBank accession no. AJ303057).
PCR detection of blaSHV, blaTEM and blaCTX-M genes and a multiplex PCR for plasmid-mediated AmpC ß-lactamase genes were performed.4 PCR amplification was also used to detect K. pneumoniae carbapenemase (KPC)-associated genes.5 Primers OXA48F (5'-CAAAGGAATGGCAAGAAAACAAAA) and OXA48R (5'-GCGCAGCCCTAAACCATCC) were designed to amplify a 686 bp fragment from the blaOXA-48 gene (GenBank accession no. AY236073). All PCR products were purified, sequenced and analysed as described elsewhere.4
Plasmid profile analysis, DNA–DNA hybridization, electroporation and conjugation
Plasmid profiles and DNA–DNA hybridization were performed, as described previously.6 To examine the transferability of plasmids harbouring the ß-lactamase genes, plasmid DNA of Lkp14 was extracted and transferred into competent Escherichia coli DH5
by electroporation.6 Specific transformants were used as the donor in the subsequent conjugation experiment using K. pneumoniae Lkp11 as the recipient. Bacterial conjugation was performed as described previously,7 and the transconjugants were selected on Luria–Bertani agar media containing 16 mg/L flomoxef.
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Results |
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Indistinguishable PFGE patterns were found among Lkp11–14, suggesting that they were of the same clone. Results of antimicrobial susceptibility testing indicated that Lkp11–13 were all ESBL producers (Table 1). MIC reduction in the presence of clavulanate was not determinable for Lkp14 because the MICs all exceeded the detection limits; however, PCR investigation did suggest that Lkp14 was an ESBL producer, too. Compared with the other three isolates, Lkp14 demonstrated a higher resistance to cephamycins and carbapenems. Although Lkp14 showed an intermediate resistance to imipenem (MIC = 8 mg/L), the strain did not seem to produce metallo-ß-lactamases because the MIC of imipenem was not reduced by more than 8-fold in the presence of EDTA (Table 1).8 MICs of minocycline were not different among the four isolates, indicating that up-regulation of chromosomal efflux may not be involved in the antimicrobial resistance of Lkp14.9 PCR and sequencing analysis indicated that Lkp11–14 all produced TEM-1, SHV-5 and CTX-M-3 ß-lactamases. Lkp14 also produced an additional plasmid-mediated DHA-1 ß-lactamase. Neither KPC- nor OXA-associated carbapenem-hydrolysing ß-lactamase genes were found among the isolates.
Lkp11–14 all carried multiple large plasmids. The three plasmids (70, 80 and 90 kb) of Lkp14 were transformed into E. coli DH5 and harboured, respectively, by three transformants A, B and C (Figure 1). DNA–DNA hybridization revealed that the 80 kb plasmid was common to the four isolates and accommodated both the blaTEM-1 and blaCTX-M-3 genes. Another two plasmids (70 and 90 kb) were unique to Lkp14, and the plasmid-mediated DHA-1 ß-lactamase gene was harboured on the 90 kb plasmid. The 70 kb plasmid was apparently not associated with any known antimicrobial resistance gene. Antimicrobial susceptibility testing and PCR sequencing analysis of the transformants confirmed the findings from DNA–DNA hybridization (Table 1). The remaining blaSHV-5 gene was located on plasmids of >100 kb and was not transformed successfully.
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OMP analysis revealed that the Lkp11–13 produced only OmpK36 and had lost OmpK35. For Lkp14, both OmpK35 and OmpK36 were not expressed (Table 1). Sequencing analysis did not find any mutation in the ompK35 coding region. For ompK36, an intact open reading frame of 1113 bp was found among isolates Lkp11–13, whereas a much larger DNA fragment was amplified from Lkp14. Sequencing analysis revealed that an additional insertion sequence, IS5 (1196 bp), was inserted after nucleotide position 97 of the ompK36 gene of Lkp14.
To elucidate the respective contributions of blaDHA-1 and loss of OmpK36 to the antimicrobial resistance phenotype observed in Lkp14, the blaDHA-1-carrying plasmid was transferred from transformant C to Lkp11 by conjugation. A flomoxef-resistant transconjugant was obtained and results of antimicrobial susceptibility testing indicated that addition of the blaDHA-1-carrying plasmid in Lkp11 only increased significantly the MICs of cephamycins and ß-lactamase inhibitors. Among the carbapenems, however, only the MIC of ertapenem showed a slight 4-fold increase. Thus, in Lkp14, the various levels of carbapenem resistance were apparently due to the concomitant loss of OmpK36.
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Discussion |
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It is known that prolonged use of cephamycins is associated with in vivo development of porin-deficient mutants and acquisition of plasmids encoding class C ß-lactamases in ESBL-producing organisms.3 As a collateral damage, these plasmid-mediated class C ß-lactamases not only provide a broader spectrum of resistance than ESBLs through their hydrolysing activities against cephamycins and resistance to ß-lactamase inhibitors, but in conjunction with porin losses, they can also confer resistance to carbapenems.3 The present study demonstrated that flomoxef, having better in vitro activity against ESBL-producing Enterobacteriaceae, could produce similar collateral effects.
Genotyping analysis indicated that the flomoxef-resistant isolate, Lkp14, was apparently derived from the former flomoxef-susceptible K. pneumoniae Lkp11–13. Molecular investigation, however, revealed two major differences in Lkp14: loss of OmpK36 in addition to the original OmpK35 deficiency and in vivo acquisition of an extra plasmid-encoded blaDHA-1 gene. Both changes occurred after prolonged flomoxef exposure and resulted in the collateral effect of carbapenem resistance observed in Lkp14. Further transformation and conjugation experiments suggested that, without the loss of OmpK36, acquisition of the blaDHA-1 gene by the OmpK35-deficient ESBL-producing isolate, Lkp11, may only result in resistance to cephalosporins (Table 1). Carbapenem resistance occurred only when OmpK36 was also lost, as was observed in Lkp14 (Table 1). Although cephamycins are theoretically effective against ESBL-KP, clinical use of these agents to treat such infections, especially when prolonged use is expected, should take into account their potential to induce porin deficiency. Once this has occurred, the impaired membrane permeability may not only lead to treatment failure, but also collaterally compromise the effectiveness of carbapenems, which are to be considered as the gold standard therapeutic choices for infections caused by ESBL-producing pathogens.
The OmpK36 deficiency in Lkp14 was due to disruption of the coding sequence via the presence of an insertion sequence, IS5. Varied mechanisms, including point mutations, deletions and insertions, have been shown to be associated with OmpK36 deficiency, but insertional interruptions appear to be the most frequent type of changes.10 Different insertion sequences, IS1, IS5, IS26, IS102 and IS903, have been reported, and similar to the finding in the present study, the insertion positions are usually within the first 100 bp from the ompK36 start codon.10 Previous reports also indicated that various insertion sequences could be present in multiple copies in the chromosome of K. pneumoniae.10 It is possible that, under antimicrobial selection pressure, transposition of these insertion sequences occurs in response to environment changes to increase survival.
In conclusion, the present study demonstrated the development of flomoxef and collateral ertapenem resistance in an OmpK35-deficient ESBL-KP strain after prolonged flomoxef exposure. It is likely that the resistance was related to the loss of OmpK36, although in vivo acquisition of a new DHA-1-encoding plasmid was also noted.
Nucleotide sequence accession number
The DNA sequence of ompK36 from Lkp14 has been deposited in GenBank and EMBL under accession no. EF061849.
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The study was supported by grant CMRPG33125 from Chang Gung Memorial Hospital and grant NSC94-2320-B-182A-015 from the National Science Council, Executive Yuan, Taipei, Taiwan.
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None to declare.
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References |
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2
Lee CH, Su LH, Tang YF, et al. Treatment of ESBL-producing Klebsiella pneumoniae bacteraemia with carbapenems or flomoxef: a retrospective study and laboratory analysis of the isolates. J Antimicrob Chemother (2006) 58:1074–7.
3 Bradford PA, Urban C, Mariano N, et al. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother (1997) 41:563–9.[Abstract]
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Lee CH, Chia JH, Chu C, et al. In vivo selection of OmpK35-deficient mutant after cefuroxime therapy for primary liver abscess caused by Klebsiella pneumoniae. J Antimicrob Chemother (2006) 58:857–60.
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Chu C, Chiu CH, Chu CH, et al. Nucleotide and amino acid sequences of oriT-traM-traJ-traY-traA-traL regions and mobilization of virulence plasmids of Salmonella enterica serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium. J Bacteriol (2002) 184:2857–62.
7 Chiu CH, Lin TY, Ou JT. Lack of evidence of an association between the carriage of virulence plasmid and the bacteremia of Salmonella typhimurium in humans. Microbiol Immunol (2000) 44:741–8.[ISI][Medline]
8
Walsh TR, Bolmstrom A, Qwärnström A, et al. Evaluation of a new Etest for detecting metallo-ß-lactamases in routine clinical testing. J Clin Microbiol (2002) 40:2755–9.
9 Elliott E, Brink AJ, van Greune J, et al. In vivo development of ertapenem resistance in a patient with pneumonia caused by Klebsiella pneumoniae with an extended-spectrum ß-lactamase. Clin Infect Dis (2006) 42:e95–8.[CrossRef][ISI][Medline]
10
Hernández-Allés S, Benedi VJ, Martinez-Martinez L, et al. Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes. Antimicrob Agents Chemother (1999) 43:937–9.
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