Skip Navigation



JAC Advance Access published online on February 20, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn049
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
61/5/1020    most recent
dkn049v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Liu, Y.-F.
Right arrow Articles by Wu, J.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Y.-F.
Right arrow Articles by Wu, J.-J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Characterization of carbapenem-non-susceptible Escherichia coli isolates from a university hospital in Taiwan

Yi-Fang Liu1,{dagger}, Jing-Jou Yan2,{dagger}, Wen-Chien Ko3, Shu-Huei Tsai2 and Jiunn-Jong Wu1,4,*

1 Institute of Basic Medical Sciences, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 2 Department of Pathology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 3 Department of Internal Medicine, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 4 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan

* Correspondence address. Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng-Kung University, No. 1 University Road, Tainan 70101, Taiwan. Tel: +886-6-2353535, ext. 5775; Fax: +886-6-2363956; E-mail: jjwu{at}mail.ncku.edu.tw

Received 5 November 2007; returned 21 January 2008; revised 28 November 2007; accepted 22 January 2008


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Funding
 Transparency declarations
 References
 
Objectives: To investigate characteristics of nine carbapenem-non-susceptible (CP-NS) Escherichia coli isolates collected between 1999 and 2005 at a Taiwanese university hospital.

Methods: Genetic relatedness was analysed by PFGE. β-Lactamases were characterized by PCR and isoelectric focusing. Outer membrane proteins and transcripts were investigated by SDS–PAGE and northern blotting. Cloning experiments were performed to investigate the role of membrane permeability in carbapenem non-susceptibility.

Results: The nine CP-NS isolates were found to produce the CMY-2 AmpC enzyme (n = 8), the CTX-M-14-type extended-spectrum β-lactamase (ESBL) (n = 1), the SHV-12 ESBL (n = 1) and the IMP-8-type metallo-β-lactamase (n = 1) alone or in combination. All CP-NS isolates revealed a decrease in the transcription and protein expression of ompC, and susceptibility to carbapenems was restored in one isolate by introducing the cloned ompC gene. PFGE revealed genetic diversity among the nine isolates. All patients with the CP-NS isolates had been treated with carbapenems (six patients) and/or extended-spectrum cephalosporins (five patients) before isolation.

Conclusions: Our study suggests that the decreased susceptibility to carbapenems in E. coli in the hospital might arise by the stepwise accumulations of multiple drug-resistance determinants in different clones.

Key Words: outer membrane proteins , ompC , β-lactamases


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Funding
 Transparency declarations
 References
 
The emergence of carbapenem resistance in Enterobacteriaceae has become a matter of great concern.1 In Escherichia coli, carbapenem resistance remains extremely rare and is mostly attributable to an outer membrane porin deficiency in combination with the production of CMY-2-related AmpC enzymes or CTX-M-type extended-spectrum β-lactamases (ESBLs).2–4 The present study was conducted to characterize carbapenem-non-susceptible (CP-NS) E. coli isolates collected between 1999 and 2005 at a university hospital in Taiwan.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Funding
 Transparency declarations
 References
 
Clinical isolates and patients

A total of 18 345 E. coli isolates were consecutively collected between 1999 and 2005 at the Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan. Among these isolates, 17 (0.09%) isolates from 10 patients were non-susceptible (resistant or intermediately susceptible) to imipenem, meropenem or both based on the CLSI (formerly NCCLS) criteria for the standard disc diffusion method.5 The annual rates of carbapenem non-susceptibility ranged from 0% to 0.08% between 1999 and 2003, and were 0.13% and 0.27% in 2004 and 2005, respectively.

Nine CP-NS isolates from different patients were selected for further examination. Among them, seven isolates were associated with nosocomial infections and five isolates might be acquired at intensive care units. Before isolation, six patients had been exposed to imipenem or meropenem and five patients had been treated with third- or fourth-generation cephalosporins. Five patients died of infections caused by CP-NS isolates. A carbapenem-susceptible isolate (2837-2/05) recovered with a carbapenem-resistant isolate (2837-1/05) from a wound sample was also analysed for comparison. E. coli strain K12 was used as the positive control in analysis of outer membrane proteins (OMPs).

Susceptibility testing

MICs of various antimicrobial agents were determined by the standard agar dilution method and Etest strips (AB Biodisk, Solna, Sweden).5 Isolates were tested for ESBL production by the CLSI-recommended disc-diffusion confirmatory test and the double-disc synergy test. The 2-mercaptopropionic acid double-disc potentiation method was used to screen for metallo-β-lactamases (MBLs).6

β-Lactamase analyses

Analytical isoelectric focusing was performed on polyacrylamide gels. Hydrolysis of 100 µM imipenem and meropenem by crude preparations of β-lactamases was monitored using a Beckman DU-800 ultraviolet spectrophotometer (Palo Alto, CA, USA) at 297 nm.4

PCR experiments

The presence of bla genes related to blaSHV, blaTEM, blaDHA-1, blaCMY-2, blaCTX-M-9, blaCTX-M-13 and blaIMP-2 was detected by PCR and nucleotide sequencing with primers used for PCR.7–9 The omp genes were amplified by PCR using primer pairs as follows: h_ECompA-1 (5'-CTGGGTAAGGAATAACTG-3') and h_ECompA-2 (5'-ATTTTGGATGATAACGAG-3') for ompA; com_ompC-1 (HindIII) (5'-CCCAAGCTTGCGGGTTGTGGTTTTTGATC-3') and com_ompC-2 (HindIII) (5'-CCCAAGCTTCAACAGTAAAAAGAAGCGGG-3') for ompC; and OmpF-1 (5'-ATCGTCCCTGCTCTGTTAG-3') and OmpF-2 (5'- TAAACGATACCCACAGCAA-3') for ompF. The sequences of the amplified ompC genes were determined with primers ‘com_ompC-1 (HindIII)’ and ‘seq_ompC’ (5'- GCAAATAAAGGCATATAACA-3'). Nucleotide sequences were analysed by use of BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).

SDS–PAGE of OMPs

Bacteria were cultured in medium A (7 g of nutrient broth, 1 g of yeast extract, 3.7 g of K2HPO4, 1.3 g of KH2PO4 and 2 g of glycerol per 1 L) at 37°C overnight. OMPs were prepared as described previously4 and separated by 10% SDS–PAGE with 4 M urea. The gel was stained with 0.1% Coomassie Blue. OMPs suspected of being OmpC, OmpF or OmpA on SDS–PAGE gels from selected isolates were submitted to the National Cheng Kung University Proteomics Research Core Laboratory for nanoelectrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The data were analysed by the database search software on the Mascot web site (http://matrixscience.com).

PFGE

PFGE analysis of XbaI-digested genomic DNA was carried out with a CHEF Mapper XA apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the instruction manual. PFGE patterns were interpreted in accordance with the criteria of Tenover et al.10

Cloning experiment

The amplified ompC and ompF genes including their promoter regions and downstream sequences from isolate 2837-2/05 were inserted into pACYC184 (New England Biolabs, Beverly, MA, USA) and cloned into E. coli DH5{alpha}. Isolates 2837-1/05 and 2837-2/05 were then transformed with the OmpC or OmpF expression vector and selected on Luria–Bertani plates containing chloramphenicol (25 mg/L).

Northern blot assays

Bacteria were cultured in 40 mL of medium A for 7 h to late log phase. RNA samples were prepared and northern blotting was performed as described previously.11 The probes used for hybridization were prepared by PCR with primers OmpA-1 (5'-GGAATTCCCGTCTGGAATACCAGTG-3') and OmpA-2 (5'-CCAAGCTTGTTGCCAGTAACCGGGTT-3') for ompA; OmpC_conserve-1 (5'-TCCAGCTCCAAACGTACC-3') and OmpC_conserve-2 (5'-GGTACTGAGCAACAGCTT-3') for ompC; and OmpF-1 and OmpF-2 for ompF.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Funding
 Transparency declarations
 References
 
By the phenotypic detection methods for ESBLs and MBLs, isoelectric focusing and PCR experiments, eight of the nine CP-NS isolates were found to produce the CMY-2 AmpC enzyme (pI = 9.0), and the CTX-M-14-type ESBL (pI = 8.1) was detected in one CMY-2 producer (Table 1). The remaining one isolate was found to co-produce the SHV-12 ESBL (pI = 8.2) and the IMP-8 MBL (pI = 8.2). Moreover, the TEM-1 narrow-spectrum β-lactamase (pI = 5.4) was detected in eight isolates. Isolates 2837-2/05 and 2837-1/05 had the same β-lactamases. Among the nine isolates, only the IMP-8 producer showed marked carbapenemase activity against imipenem in the hydrolysis analysis (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 1. Results of β-lactamase characterization and susceptibility testing

 
The results of susceptibility testing are shown in Table 1. Among the nine CP-NS isolates, high-level resistance to imipenem and ertapenem (MIC, ≥128 mg/L) was detected in four and five isolates, respectively. All nine isolates showed high-level resistance (MIC, ≥128 mg/L) to cefotaxime, ceftazidime and aztreonam. Seven isolates were resistant to cefepime (MIC, ≥32 mg/L). Isolates 2837-2/05 and 2837-1/05 revealed discernible differences only in susceptibilities to carbapenems and cefepime.

PFGE revealed eight distinct patterns among eight of the nine CP-NS E. coli isolates (Table 1). One isolate could not be typed probably due to lysis of its genomic DNA. Isolates 2837-1/05 and 2837-2/05 differed only by three bands, suggesting that they were from the same clone.

The results of SDS–PAGE are shown in Figure 1. The expressions of OmpC and OmpF were decreased in all CP-NS isolates. For isolate 2837-2/05, only OmpA and OmpC were obtained.


Figure 1
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. OMP profiles of the carbapenem-non-susceptible and carbapenem-susceptible E. coli isolates. Lanes 1–12, E. coli 784/99, 2272/02, 464/03, 474-2/04, 430/04, 2881/04, 983/05, 773-2/05, 2837-1/05, 2837-2/05, 2837-1/05+ompC (isolate 2837-1/05 transformed by the OmpC expression vector) and K12. Lane M, molecular weight marker.

 
The presence of the three omp genes was detected by PCR in all study isolates (data not shown). Sequence analysis of ompC by ‘Needle global alignment’ (http://www.ebi.ac.uk/emboss/align/) revealed that the similarities between K12 and the clinical isolates ranged from 53% to 94%. Sequence analysis by ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) revealed that 2837-1/05 and 2837-2/05 differed by only one C to A change at nucleotide position 310, resulting in an early stop codon in the ompC gene of 2837-1/05. Six of the nine CP-NS isolates were predicted to have truncated OmpC (Table 1).

In the northern blot assay, transcripts of ompC were markedly lower in six of the nine CP-NS isolates and slightly decreased in the remaining three CP-NS isolates when compared with E. coli K12 and isolate 2937-02/05. Moreover, all CP-NS isolates except 784/99 showed decreased transcription of ompF. Transcription of ompA appeared to increase slightly in all CP-NS isolates (data not shown).

The ompC gene from 2837-02/05 was cloned and used to transform isolate 2837-1/05. The transformant revealed increased transcription and expression of ompC (Figure 1) and became susceptible to carbapenems (Table 1), indicating the importance of OmpC in carbapenem susceptibility. The transformation by ompF had no significant effects in the susceptibility to any carbapenems (Table 1), suggesting that the loss of OmpF has little role in carbapenem resistance in our CP-NS E. coli isolates.

The present study revealed that carbapenem resistance in E. coli in this Taiwanese hospital was mainly attributed to the combination of the loss of OmpC and the presence of CMY-2. MICs of imipenem and meropenem varied remarkably among the CP-NS isolates, suggesting the presence of other resistance mechanisms. The IMP-8 MBL was identified in one CP-NS isolate. Although IMP-8 has hydrolytic activity against carbapenems, the acquisition of a blaIMP-8 plasmid did not confer substantive resistance to carbapenems in E. coli.8,12 Thus, the loss of OmpC may also play an important role in carbapenem resistance in the IMP-8 producer.

All patients with the CP-NS E. coli isolates had been exposed to carbapenems and/or extended-spectrum cephalosporins before isolation. Genetic diversity was demonstrated by PFGE among the CP-NS E. coli isolates. These findings suggest that the emergence of carbapenem resistance in E. coli in this hospital might be caused by stepwise accumulation of multiple drug-resistance determinants in different clones under the selective pressure created by the use of extended-spectrum cephalosporins or carbapenems. The majority of the CP-NS isolates were resistant to nearly all of the antimicrobials tested, and a high mortality rate was observed among our patients infected by the CP-NS isolates. The spread of such multidrug-resistant strains may become a serious problem in Taiwan.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
Transparency declarations
 References
 
This work was partially supported by grants DOH95-DC-1038 from the Center for Disease Control, the Department of Health, the Executive Yuan, and NSC94-2320-B-006-085 from the National Science Council, Taiwan.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
 Transparency declarations
References
 
None to declare.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgements
 
We thank Proteomics Research Core Laboratory of National Cheng Kung University for the assistance in mass spectrometry analysis.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
 Transparency declarations
 References
 
1 . Paterson DL. Resistance in Gram-negative bacteria: Enterobacteriaceae. Am J Med (2006) 119:S20–8. discussion S62–70.[Medline]

2 . Lartigue MF, Poirel L, Poyart C, et al. Ertapenem resistance of Escherichia coli. Emerg Infect Dis (2007) 13:315–7.[Web of Science][Medline]

3 . Poirel L, Heritier C, Spicq C, et al. In vivo acquisition of high-level resistance to imipenem in Escherichia coli. J Clin Microbiol (2004) 42:3831–3.[Abstract/Free Full Text]

4 . Stapleton PD, Shannon KP, French GL. Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 β-lactamase production and loss of an outer membrane protein. Antimicrob Agents Chemother (1999) 43:1206–10.[Abstract/Free Full Text]

5 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16. (2006) Wayne, PA, USA: CLSI.

6 . Yan JJ, Wu JJ, Tsai SH, et al. Comparison of the double-disk, combined disk, and Etest methods for detecting metallo-β-lactamases in Gram-negative bacilli. Diagn Microbiol Infect Dis (2004) 49:5–11.[CrossRef][Web of Science][Medline]

7 . Saladin M, Cao VT, Lambert T, et al. Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett (2002) 209:161–8.[Web of Science][Medline]

8 . Yan JJ, Ko WC, Tsai SH, et al. Outbreak of infection with multidrug-resistant Klebsiella pneumoniae carrying blaIMP-8 in a university medical center in Taiwan. J Clin Microbiol (2001) 39:4433–9.[Abstract/Free Full Text]

9 . Yan JJ, Hsueh PR, Lu JJ, et al. Extended-spectrum β-lactamases and plasmid-mediated AmpC enzymes among clinical isolates of Escherichia coli and Klebsiella pneumoniae from seven medical centers in Taiwan. Antimicrob Agents Chemother (2006) 50:1861–4.[Abstract/Free Full Text]

10 . Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol (1995) 33:2233–9.[Web of Science][Medline]

11 . Wang CH, Lin CY, Luo YH, et al. Effects of oligopeptide permease in group A streptococcal infection. Infect Immun (2005) 73:2881–90.[Abstract/Free Full Text]

12 . Yan JJ, Ko WC, Chuang CL, et al. Metallo-β-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii. J Antimicrob Chemother (2002) 50:503–11.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J Med MicrobiolHome page
S. Grobner, D. Linke, W. Schutz, C. Fladerer, J. Madlung, I. B. Autenrieth, W. Witte, and Y. Pfeifer
Emergence of carbapenem-non-susceptible extended-spectrum {beta}-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tubingen, Germany
J. Med. Microbiol., July 1, 2009; 58(7): 912 - 922.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
G. A. Jacoby
AmpC {beta}-Lactamases
Clin. Microbiol. Rev., January 1, 2009; 22(1): 161 - 182.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
M. Pavez, P. Neves, M. Dropa, M. H. Matte, R. S. Grinbaum, M. R. Elmor de Araujo, E. M. Mamizuka, and N. Lincopan
Emergence of carbapenem-resistant Escherichia coli producing CMY-2-type AmpC {beta}-lactamase in Brazil
J. Med. Microbiol., December 1, 2008; 57(12): 1590 - 1592.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
61/5/1020    most recent
dkn049v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Liu, Y.-F.
Right arrow Articles by Wu, J.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Y.-F.
Right arrow Articles by Wu, J.-J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?