Characterization of a new integron containing blaVIM-1 and aac(6')-IIc in an Enterobacter cloacae clinical isolate from Greece

Irene Galani, Maria Souli, Zoi Chryssouli, Konstantina Orlandou and Helen Giamarellou*

4th Department of Internal Medicine, Molecular Biology Section, University of Athens Medical School, Athens; University General Hospital ‘Attikon’, Rimini 1, 124 62 Chaidari, Greece


* Corresponding author. Tel: +30-210-583-1000; Fax: +30-210-532-6426; Email: hgiama{at}ath.forthnet.gr

Received 15 October 2004; returned 10 November 2004; revised 24 January 2005; accepted 24 January 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production.

Materials and methods: MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of ß-lactamases. PCR assays with primers specific for the blaVIM gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-ß-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the blaVIM-1 gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the blaVIM-1 gene as a probe, amplified by PCR.

Results: The isolate was resistant to extended-spectrum ß-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 108 cfu/mL was used. Isoelectric focusing detected a ß-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the blaVIM-1 gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The blaVIM-1 probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene.

Conclusion: We describe a novel class 1 integron containing blaVIM-1 and aac(6')-IIc genes in an E. cloacae clinical isolate.

Keywords: metallo-ß-lactamases , carbapenemases , E. cloacae


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, Enterobacter cloacae has emerged as an important nosocomial pathogen. This microorganism is intrinsically resistant to ampicillin and narrow-spectrum cephalosporins. Resistance to expanded-spectrum cephalosporins and aztreonam is usually related to mutational derepression of the chromosomal Ambler class C ß-lactamase or to the production of plasmid-mediated extended-spectrum ß-lactamase. Imipenem resistance in clinical isolates of E. cloacae is unusual and has been described in strains with porin alterations combined with hyperproduction of chromosomal cephalosporinase,1 and in strains producing class A carbapenem-hydrolysing non-metallo-ß-lactamases, such as NmcA ß-lactamase2 and IMI-1 ß-lactamase.3 Metallo-ß-lactamases have been reported in E. cloacae clinical isolates from various geographical areas, IMP-8 from Taiwan,4 VIM-2 from Korea5 and VIM-4 from Italy,6 but the MICs of carbapenems against these strains cover a wide range, and do not always exceed the susceptibility breakpoint.

Since the introduction of routine use of the imipenem-EDTA-disc synergy test7 in our Infectious Diseases Research Laboratory, we have encountered several strains of Enterobacteriaceae that have shown a positive result although they were not resistant to carbapenems according to the NCCLS.8 In this report, we describe an E. cloacae clinical isolate exhibiting reduced susceptibility to imipenem and ertapenem and a positive EDTA-disc synergy test due to production of VIM-1 metallo-ß-lactamase encoded by a novel class 1 integron.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In October 2003, E. cloacae isolate 87 was isolated from the blood culture of a patient hospitalized in Attikon University Hospital in Athens, Greece. The 70-year-old patient had been admitted to a regional hospital suffering from Fournier's gangrene. Ten days later and while the patient was treated with imipenem, he developed catheter-related bacteraemia from E. cloacae intermediately susceptible to imipenem. He was transferred to our hospital with persistent bacteraemia of 25 days duration and septic pulmonary emboli, which were unresponsive to imipenem although a new catheter was inserted at another site. Endocarditis was excluded by a negative transoesophageal echocardiogram. The patient was successfully treated with intravenous colistin (3 000 000 U, three times a day) and catheter removal. Colistin was prescribed because a multiresistant Acinetobacter baumannii was also isolated from one blood culture. The E. cloacae isolate was studied for metallo-ß-lactamase production.

E. cloacae isolate 87 was identified by the ATB 32 GN System (bioMérieux SA, Marcy 1'Étoile, France). The MICs of ß-lactams and aminoglycosides were determined by the broth microdilution method according to the NCCLS,9 with the exception of ertapenem for which the Etest (AB Biodisk, Solna, Sweden) was used according to the manufacturer's instructions. The MICs of carbapenems were also determined using a higher inoculum (108 cfu/mL). Susceptibility to other antimicrobial agents was assessed by the disc diffusion method.9 To screen for metallo-ß-lactamase production, a synergy test using imipenem- and EDTA-containing discs was employed.7 Conjugation experiments were carried out by the filter mating method, with Escherichia coli RC85 R K12 strain (rifampicin MIC, > 128 mg/L) as the recipient.10 Rifampicin (128 mg/L) and ceftazidime (16 mg/L)-containing agar plates were used to select for transconjugants. The isoelectric points of ß-lactamases were determined by isoelectric focusing of cell sonicates on precast polyacrylamide gels (PhastGel IEF 3–9; Amersham Biosciences, Uppsala, Sweden) with a PhastSystem Apparatus (Pharmacia Biotech AB, Uppsala, Sweden). Enzyme bands were visualized with nitrocefin (Oxoid Ltd, Basingstoke, UK). Hydrolysis of imipenem (0.25 mg/L) by the metallo-ß-lactamase band was detected with a microbiological method, as described previously.11

PCR amplification was carried out as described previously with primers VIM-B (5'-ATGGTGTTTGGTCGCATATC-3') and VIM-F (5'-TGGGCCATTCAGCCAGATC-3') amplifying a 510 bp (nucleotides 152–661) internal fragment of the blaVIM-1 gene (EMBL/GenBank accession no. AF 191564).12 Detection and mapping of a class 1 integron were carried out using primers specific for the conserved segments 5'-CS (5'-CTTCTAGAAAACCGAGGATGC-3') and 3'-CS (5'-CTCTCAAGATTTTAATGCGGATG-3')13 amplifying the variable region containing the resistance gene cassettes. The PCR amplicon (In87) was ligated to pCR2.1 vector and the ligation product pCR2.1(In87) was used to transform E. coli TOP10F' (TA Cloning Kit; Invitrogen, Carlsbad, CA, USA), as described by the manufacturer. Nucleotide sequences of the cloned PCR product were determined on both strands with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequence analysis and database similarity searches for nucleotide and deduced amino acid sequences were carried out at the NCBI website (http://www.ncbi.nlm.nih.gov/). Alignment of protein sequences was carried out by the CLUSTAL W multiple sequence alignment program available over the Internet (http://www.clustalw.genome.ip/).

Extraction of plasmid DNA was carried out with the QIAprep Miniprep (Qiagen GmbH, Hilden, Germany), as described by the manufacturer and by an alkaline lysis method.14 In order to identify the location of the blaVIM-1 gene, genomic DNA in the form of a plug was digested with SpeI (New England BioLabs (UK) Ltd, Hitchin, Hertfordshire, UK), electrophoresed in Gene Navigator apparatus (Pharmacia Biotech AB, Uppsala, Sweden Dassell, Germany), transferred onto a nylon membrane (Schleicher & Schuell, Dassell, Germany) and hybridized with a PCR-obtained 510 bp internal fragment of the blaVIM-1 gene labelled with a digoxigenin DNA labelling and detection kit (Roche Diagnostics, Mannheim, Germany).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
E. cloacae isolate 87 was resistant to ampicillin, ampicillin/sulbactam, co-amoxiclav, piperacillin, piperacillin/tazobactam, ticarcillin, ticarcillin/clavulanic acid, cephalosporins and aztreonam. The MICs of imipenem, ertapenem and meropenem were 1, 3 and 0.25 mg/L, respectively. The isolate was susceptible to the aminoglycosides (Table 1) and ciprofloxacin. The MICs of imipenem, ertapenem and meropenem rose to 512, > 32 and 125 mg/L, respectively, when an inoculum of 108 cfu/mL was used. The EDTA-disc synergy test was positive. Isoelectric focusing showed two ß-lactamase bands, one with a pI value of approximately 9.0, probably corresponding to the chromosomal AmpC cephalosporinase of E. cloacae, and the second with a pI of approximately 6.1. The microbiological assay showed that this pI 6.1 band inactivated imipenem, allowing the growth of the indicator strain.


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Table 1. MICs of various antimicrobial agents for E. cloacae isolate 87, E. coli TOP10F'/pCR2.1 and E. coli TOP10F'/pCR2.1(In87)

 
Neither imipenem nor ceftazidime resistance were transferred by conjugation. Repeated attempts to isolate plasmid DNA were unsuccessful. PCR amplification and nucleotide sequence analysis of the cloned PCR product identified blaVIM-1. We proceeded with sequence analysis of the 2130 bp PCR amplicon which revealed the structure of a class 1 integron, designated In87 (Figure 1). The 5'-CS element included an IntI1 integrase gene with its own promoter region, and an attI1 recombination site whereas the 3'-CS element contained the disinfectant resistance determinant qacE{Delta}1. The integron contained two inserted gene cassettes: blaVIM-1 and aac(6')-IIc. The blaVIM-1 gene was inserted in the attI1 recombination site and had a core site (GTTATGC) and an inverse core site (GCATAAC) at its boundaries. The initiation codon of the blaVIM-1 gene was preceded by a strong P1 promoter (TTGACAN17TAAACT) and the inactive form of P2 (TTGTTAN14TACAGT) located at the 5' end of the integrase 1 gene. The blaVIM-1 cassette (including the 81 nucleotides of the 59-base element) was identical to that originally described in Pseudomonas aeruginosa15 and in an E. coli isolated in Greece.16 The aac(6')-IIc gene cassette was located downstream of the blaVIM-1 gene cassette and it was identical to that previously sequenced from a P. aeruginosa isolate (GenBank accession no. AF 162771). The AAC(6')-IIc shows 31–80% homology with a number of previously reported N-aminoglycoside acetyltransferases (Figure 2). The phylogenetic tree (Figure 3), which is based on a CLUSTAL W multiple alignment of the putative amino acid sequences demonstrates that it clusters to the largest AAC(6') subfamily which is composed of AAC(6')-Ib, AAC(6')-IIa, AAC(6')-IIb and the amino-terminal portion of the AAC(6') + APH(2'') bifunctional enzyme [AAC(6')-Ie] and closer to AAC(6')-IIa with which it shares only 65% nucleotide sequence identity but 80% similarity in the predicted protein sequence. AAC(6')-II enzymes modify gentamicin, tobramycin and netilmicin but not amikacin, in contrast to AAC(6')-I enzymes, which also modify amikacin. Although the typical AAC(6')-II pattern of resistance was not observed, there was an increase in the MICs of gentamicin, tobramycin and netilmicin but not of amikacin against E. coli TOP10F'/pCR2.1(In87) compared with E. coli TOP10F'/pCR2.1 (Table 1).



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Figure 1. Schematic structure of In87. The 5'-CS contains the IntI1 integrase gene and the 3'-CS includes the resistance determinant qacE{Delta}1. Inserted gene cassettes and their transcriptional orientation are indicated by arrows. The attI1 recombination site is represented by an open circle, and 59 bp elements are represented by filled circles.

 


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Figure 2. Alignment of 6'-N-aminoglycoside acetyltransferase amino acid sequences which can be found in gene banks by the indicated accession numbers and in the published references: AAC(6')-IIc, AY648125 (this study); AAC(6')-IIa, M29695;17 AAC(6')-Ib, M55547;18 AAC(6')-IIb, L06163 (Shaw, K. J. & Ramirez, F., unpublished data); AAC(6')-APH(2'), M13771.19 Asterisks (*) indicate positions which have a single, fully conserved residue. Colons (:) indicate that one of the following ‘strong’ groups is fully conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. Full stops (.) indicate that one of the following ‘weaker’ groups is fully conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, HFY.20

 


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Figure 3. Phylogenetic tree of the AAC(6')-II subfamily of enzymes.

 
The labelled blaVIM-1-probe hybridized with an approximately 130 kb SpeI fragment of genomic DNA, suggesting that the In87 integron was located on the chromosome.

The nucleotide sequence of integron In87 has been assigned the EMBL/GenBank accession no. AY648125.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MICs of carbapenems against E. cloacae isolate 87 were below the resistance breakpoint. Nevertheless, imipenem failed to treat the catheter-related bacteraemia of our patient despite prompt replacement of the catheter. It is possible that, in vivo in the presence of a high inoculum, high-level resistance arises, as was suggested by our in vitro experiments. Little clinical information is available on the outcome of patients infected with carbapenem-susceptible metallo-ß-lactamase-producing Enterobacteriaceae. The emergence and spread of this new threat necessitates a consensus guideline on the interpretation and reporting of the antibiogram. Furthermore, in vivo data from relevant animal models are warranted for the evaluation of the efficacy of carbapenems for the treatment of these infections.

The other question that arises upon isolation of a carbapenem-susceptible metallo-ß-lactamase-producing strain is whether infection control measures are required. Phenotypically, E. cloacae isolate 87 did not exhibit multiple resistances and the same is true for all the Enterobacteriaceae carrying blaVIM genes that we have isolated thus far.8 Nevertheless, an outbreak of VIM-1-producing Klebsiella pneumoniae causing fatal infections in many tertiary care hospitals in Athens has already been described.21 These strains are now endemic in many Intensive Care Units in Athens. The mobile nature of metallo-ß-lactamase genes, the broad hydrolysis profile of the enzymes, and their frequent association with other resistance genes such as those coding for aminoglycoside-modifying enzymes, make them an important potential threat for hospital epidemiology. For these reasons, we advocate barrier precautions (private room, if available, gloves and gowns) for the infected or colonized patient, although consensus guidelines on this issue are also lacking. We have described the presence of the blaVIM-1 gene in an E. cloacae clinical isolate and the association of blaVIM-1 and aac(6')-IIc in the same integron for the first time. The identity of these gene cassettes with those previously described in P. aeruginosa15 and E. coli16 suggests extensive spread of resistance genes between various Gram-negative species.


    References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lee, E. H., Nicolas, M. H., Kitzis, M. D. et al. (1994). Association of two resistance mechanisms in a high level imipenem-resistant clinical isolate of Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 35, 1093–8.

2 . Naas, T. & Nordmann, P. (1994). Analysis of a carbapenem-hydrolyzing class A ß-lactamase from Enterobacter cloacae and its LysR-type regulatory protein. Proceedings of the National Academy of Sciences, USA 91, 7693–7.[Abstract/Free Full Text]

3 . Rasmussen, B. A., Bush, K., Keeney, D. et al. (1996). Characterization of IMI-1 ß-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 40, 2080–6.[Abstract]

4 . Yan, J. J., Ko, W. W., Chuang, C. L. et al. (2002). Metallo-ß-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. Journal of Antimicrobial Chemotherapy 50, 503–11.[Abstract/Free Full Text]

5 . Jeong, S. H., Lee, K., Chong, Y. et al. (2003). Characterization of a new integron containing VIM-2, a metallo-ß-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae. Journal of Antimicrobial Chemotherapy 51, 397–400.[Abstract/Free Full Text]

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21 . Giakkoupi, P., Xanthaki, A., Kanelopoulou, M. et al. (2003). VIM-1 metallo-ß-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals. Journal of Clinical Microbiology 41, 3893–6.[Abstract/Free Full Text]