JAC Advance Access originally published online on July 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):857-860; doi:10.1093/jac/dkl308
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In vivo selection of OmpK35-deficient mutant after cefuroxime therapy for primary liver abscess caused by Klebsiella pneumoniae
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 Clinical Pathology, Lin-Kou Medical Center, Chang Gung Memorial Hospital 5 Fu-Hsin Street, Kweishan, Taoyuan 333, Taiwan 3 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University 259 Wenhua 1st Road, Kweishan, Taoyuan 333, Taiwan 4 Department of Applied Microbiology, National Chiayi University 300 University Road, Chiayi 600, Taiwan
*Corresponding author. Tel: +886-3-3281200 ext. 8363; Fax: +886-3-3971827; E-mail: sulh{at}adm.cgmh.org.tw
Received 18 November 2005; returned 5 April 2006; revised 8 May 2006; accepted 5 July 2006
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Abstract |
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Objectives: The aim of the study was to characterize the genetic basis of ß-lactam resistance developed in clinical isolates of Klebsiella pneumoniae after exposure to cefuroxime.
Methods: Clinical features of two episodes of liver abscess caused by K. pneumoniae in a diabetic patient were reported. Four isolates (KP1/KP2 and KP3/KP4) of K. pneumoniae were recovered from cultures of blood/pus in the first and second episodes, respectively. Laboratory investigation of the K. pneumoniae isolates included genotyping by PFGE, resistance gene analysis by PCR amplification and DNA sequencing, and outer membrane protein analysis by SDS–PAGE.
Results: KP3 and KP4 were recovered after a 21 day cefuroxime therapy and demonstrated identical genotypes to that of KP1 and KP2. However, compared with KP1 and KP2, emerging resistance to piperacillin, cefalotin, cefuroxime and cefoxitin was observed. The other antibiotics tested, except ampicillin, retained the same effectiveness against the four isolates, although increases (4- to 8-fold) in the MICs of cefotaxime, ceftriaxone, ceftazidime, cefepime, flomoxef and aztreonam were observed in KP3 and KP4. None of the isolates produced extended-spectrum ß-lactamases or plasmid-mediated AmpC ß-lactamases. Deficiency in the expression of an outer membrane protein (OmpK35) was observed in the cefuroxime-resistant isolates, KP3 and KP4.
Conclusions: The increased resistance to cephalosporins in these clinical isolates of K. pneumoniae after exposure to cefuroxime might be related to the loss of OmpK35.
Keywords: outer membrane protein deficiency , ß-lactam resistance , cephalosporins , OMPs
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Introduction |
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Primary liver abscess caused by Klebsiella pneumoniae is a problematic infection in diabetic patients in Taiwan.1 Almost all K. pneumoniae strains causing primary liver abscess in this area had a similar antimicrobial susceptibility pattern (resistant to ampicillin, ticarcillin and carbenicillin; susceptible to all cephalosporins and aminoglycosides).1 Nevertheless, in a recent report from Taiwan, one clone of K. pneumoniae causing liver abscess was unusually found to have two colony morphotypes and resistotypes, with one being resistant to first- and second-generation cephalosporins.2
Resistance to ß-lactam antibiotics in K. pneumoniae occurs by one or both of the following mechanisms: (i) production of various ß-lactamases to inactivate the associated ß-lactam antibiotics; and (ii) altered outer membrane proteins (OMPs) resulting in a decreased diffusion of antibiotics to the target site through the membrane.3 Loss of both OmpK35 and OmpK36 may lead to antimicrobial resistance in K. pneumoniae.4
We report herein the finding that in one patient with recurrent liver abscess antimicrobial susceptibilities of the presumed causative pathogen, K. pneumoniae, shifted from being susceptible towards being resistant to some ß-lactam antibiotics after cefuroxime therapy. Laboratory investigation was performed to clarify the genetic basis for the development of such resistance.
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Case report
A 44-year-old man was admitted to the Chang Gung Memorial Hospital, Kaohsiung, Taiwan, on 1 December 2004 because of a 1 week fever. He had a 6 year history of diabetes mellitus and his blood sugar had not been regularly controlled. Abdominal echography showed an abscess (3.9 cm x 3.7 cm x 4.5 cm) over the right lobe of his liver. Two isolates of K. pneumoniae were recovered from his blood (KP1) and abscess (KP2) cultures. The morphology of both isolates was mucoid and opaque. They were susceptible to all tested ß-lactam antibiotics except ampicillin (Table 1). Parenteral cefuroxime (1500 mg per 8 h) was administered. A draining catheter was inserted on the second hospital day, and fever subsided 2 days later. Cefuroxime was continued for a total of 21 days. A follow-up echography showed resolution of the abscess. The patient was discharged on 24 December 2004.
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On 2 February 2005, he was admitted again with the chief complaint of fever and chills for 4 days. Abdominal echography revealed an abscess (3.2 cm x 4.3 cm x 5 cm) over the right lobe of his liver. Cefuroxime was empirically used again, and immediate drainage was carried out. K. pneumoniae isolates were found from blood (KP3) and abscess (KP4) cultures, which were resistant to both first- and second-generation cephalosporins, including the administered cefuroxime (Table 1). Cefuroxime was replaced by ceftriaxone (1 g per 12 h) and fever disappeared 2 days later. Parenteral ceftriaxone was administered for a total of 28 days, followed by oral cefixime (200 mg per 12 h) for 4 weeks. Follow-up echography showed that the abscess had disappeared.
Bacterial strains and antimicrobial susceptibility
Clinical isolates of K. pneumoniae were identified by standard methods. To elucidate the associated mechanisms of the emerging resistance, the four isolates of K. pneumoniae, KP1–KP4, were subjected to further laboratory investigation. K. pneumoniae ATCC 13883 and two epidemiologically non-related clinical isolates of K. pneumoniae were included in some of the experiments for comparison.
Susceptibilities of the isolates to antimicrobial agents were determined by the Etest in accordance with the recommendations of the manufacturer (AB BIODISK, Solna, Sweden). The MICs of cefotaxime, ceftazidime, cefepime and flomoxef were determined by a standard broth microdilution method. The interpretation criteria were in accordance with those suggested by the CLSI.5
Genotyping
To determine the genetic relationship between the K. pneumoniae isolates, XbaI-digested macro-fragments of the bacterial DNA were analysed by PFGE as described previously.6 PFGE patterns that varied by more than four bands were considered of different genotypes.6
PCR amplification and DNA sequencing
To clarify whether any extended-spectrum ß-lactamases were associated with the development of cephalosporin resistance in the K. pneumoniae isolates, PCR detection of the associated genes was conducted. Four primer sets previously described for the detection of blaSHV, blaTEM and blaCTX-M genes were used.7 A multiplex PCR described previously for the detection of plasmid-mediated AmpC ß-lactamase genes was also performed.8 The PCR products were purified, sequenced and analysed as described previously.8
Analysis of OMPS
The production of OMPs by the K. pneumoniae isolates was examined by a method described previously.9 Briefly, OMPs of the K. pneumoniae isolates were collected after sonication of overnight broth cultures of the bacterial strains. The OMPs were separated by SDS–PAGE and visualized after Coomassie Blue staining (Bio-Rad, Hercules, CA, USA). To accurately locate the position of OmpK35, two broth cultures were grown simultaneously for each isolate. In one of the paired cultures, 20% sorbitol was added to create a high osmolarity to purposely inhibit the expression of OmpK35. The result was compared with another broth culture in which intact OmpK35 could be produced efficiently.
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Results |
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PFGE demonstrated indistinguishable patterns among the four isolates obtained from the patient and completely different patterns for the two epidemiologically non-related isolates, indicating that the four isolates might be of the same clone (Figure 1a) and thus the second episode of liver abscess may be a relapse by the same strain of K. pneumoniae that caused the first episode.
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Antibiotic susceptibility profiles of the four isolates are shown in Table 1. MICs of all ß-lactams, except imipenem, increased at least 2-fold for KP3 and KP4. The MICs of piperacillin, cefalotin, cefuroxime and cefoxitin show that KP1 and KP2 were susceptible to these antibiotics, but that KP3 and KP4 (isolated after cefuroxime therapy) were resistant to these antibiotics.
Molecular analysis indicated that none of the isolates produced TEM, CTX-M or plasmid-mediated AmpC-type ß-lactamases. The gene blaSHV-1 was identified in all four isolates. KP1, KP2 and K. pneumoniae ATCC 13883 produced OmpK34, OmpK35 and OmpK36, while KP3 and KP4 produced OmpK34 and OmpK36, but not OmpK35 (Figure 1b).
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Discussion |
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Most K. pneumoniae isolates causing liver abscess in Taiwan have remained susceptible to cefazolin over the past two decades.1 In the present report, PFGE demonstrated that a strain of K. pneumoniae might be responsible for the two consecutive episodes of liver abscess in the patient studied. Molecular investigation of the four K. pneumoniae isolates demonstrated that the resistance to cephalosporins developed without the production of extended-spectrum or AmpC ß-lactamases. This finding coupled with the missing OmpK35 in the two resistant isolates suggested that porin deficiency might play an important role in the resistance to cephalosporins in the K. pneumoniae strain.
Porin-deficient mutants are not uncommon in members of the Enterobacteriaceae.4 In general, K. pneumoniae synthesizes two major porins, OmpK35 and OmpK36.4 The loss of the 40 kDa porin (OmpK35) was shown to be associated with the emergence of cephalosporin resistance during cefamandole therapy in a patient with suppurative thrombophlebitis caused by K. pneumoniae.10 In a previous report from Taiwan, low-level expression of OmpK35 was found in an outbreak strain of K. pneumoniae that also produced SHV-1 and TEM-1. The combination of antibiotic resistance mechanisms resulted in the false designation of the isolates as ESBL producers.9 While the cause of the OmpK35 deficiency was not indicated in that study, our report suggests that such alterations in OMPs could occur after exposure to cefuroxime. In an experimental model, a study also demonstrated that the reduced expression of OmpK36 could be affected by the presence of cefuroxime.11 However, a previous report demonstrated unaltered drug susceptibility in a laboratory-derived OmpK35 mutant of K. pneumoniae.12 It remains possible that other unidentified genetic factors may also contribute to the altered antimicrobial susceptibility observed in the present study. More investigations are required to elucidate all the associated resistance mechanisms.
The rates of penetration through the OmpF porin of E. coli and the OmpK35 porin of K. pneumoniae differ for various ß-lactams.13 Cefuroxime, a monoanionic ß-lactam carrying a positive electric charge at neutral pH, has a relatively lower diffusion rate through the OmpK35 channel.13 OmpF (homologous to OmpK35) was previously reported to have a wider channel than OmpC (homologous to OmpK36), and OmpF theoretically allows larger molecules to pass through its channel. The present study showed that in the absence of OmpK35 and unaltered OmpK36, the MICs of extended-spectrum cephalosporins are moderately (4- to 8-fold) increased. This may result from the relatively lower efficiency in penetration of these cephalosporins through the OmpK36.
Unlike previous reports that used molecular cloning or genetic manipulation methods to produce and study the function of OmpK35,12 the uniqueness of our present study lies in that the resistant K. pneumoniae isolates occurred spontaneously in a clinical setting and thus represented the most likely in vivo situation that may take place after K. pneumoniae is exposed to antibiotics, i.e. cefuroxime in this study. Our report demonstrated that the development of cefuroxime resistance in a K. pneumoniae strain after a 3 week exposure to this antibiotic might be associated with the depletion of OmpK35.
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None to declare.
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Acknowledgements |
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This work was supported in part by grants CMRPG33125 (to L.-H. S.) and CMRPG 33124 (to T.-L. W.) from Chang Gung Memorial Hospital, Taoyuan, Taiwan.
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References |
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1 Lee CH, Leu HS, Wu TS, et al. (2005) Risk factors for spontaneous liver abscess caused by Klebsiella pneumoniae. Diag Microbiol Infect Dis 52:79–84.[CrossRef][ISI][Medline]
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Nikaido H. (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656.
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6 Su LH, Leu HS, Chiu YP, et al. (2000) Molecular investigation of two clusters of nosocomial bacteraemia caused by multiresistant Klebsiella pneumoniae using pulsed-field gel electrophoresis and infrequent-restriction-site PCR. J Hosp Infect 46:110–7.[CrossRef][ISI][Medline]
7 Lee CH, Su LH, Chia JH, et al. (2004) Recurrent Klebsiella pneumoniae mycotic aneurysm in a diabetic patient and emergence of an extended-spectrum ß-lactamase (CTX-M-24)-containing Klebsiella pneumoniae strain after prolonged treatment with first-generation cephalosporins for mycotic aneurysm. Microb Drug Resist 10:359–63.[CrossRef][ISI][Medline]
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Pérez-Pérez FJ and Hanson ND. (2002) Detection of plasmid-mediated AmpC ß-lactamase genes in clinical isolates by using multiple PCR. J Clin Microbiol 40:2153–62.
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Wu TL, Siu LK, Su LH, et al. (2001) Outer membrane protein change combined with co-existing TEM-1 and SHV-1 ß-lactamases lead to false identification of ESBL-producing Klebsiella pneumoniae. J Antimicrob Chemother 47:755–61.
10 van de Klundert JA, van Gestel MH, Meerdink G, et al. (1988) Emergence of bacterial resistance to cefamandole in vivo due to outer membrane protein deficiency. Eur J Clin Microbiol Infect Dis 7:776–8.[CrossRef][ISI][Medline]
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Nelson EC, Segal H, Elisha BG. (2003) Outer membrane protein alterations and blaTEM-1 variants: their role in ß-lactam resistance in Klebsiella pneumoniae. J Antimicrob Chemother 52:899–903.
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Hernandez-Alles S, Conejo M, Pascual A, et al. (2000) Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae. J Antimicrob Chemother 46:273–7.
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