JAC Advance Access published online on December 6, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm471
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Bloodstream infections caused by multidrug-resistant Klebsiella pneumoniae producing the carbapenem-hydrolysing VIM-1 metallo-β-lactamase: first Italian outbreak
1 Di.S.C.A.T., Microbiology Unit, University of Genoa, Genoa, Italy 2 Department of Molecular Biology, Microbiology Unit, University of Siena, Siena, Italy 3 Department of Infectious Diseases, S. Martino Hospital, Genoa, Italy 4 Department of Anaesthesiology—Critical Care, S. Martino Hospital, Genoa, Italy
* Corresponding author. Tel: +39-010-3537502; Fax: +39-010-3537655; E-mail: anna.marchese{at}unige.it
Received 28 August 2007; returned 23 September 2007; revised 9 November 2007; accepted 13 November 2007
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Abstract |
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Objectives: To investigate the first Italian outbreak of bloodstream infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae producing metallo-β-lactamase (MBL), which occurred in three wards of one large tertiary-care hospital in Genoa, Italy, from September 2004 to March 2005.
Methods: MBL production was screened by an imipenem–EDTA disc synergy test and confirmed by a conventional hydrolysis test. Antibiotic susceptibility was determined by broth microdilution or disc diffusion. PFGE was used to study the genetic relatedness of isolates. PCR and sequencing were carried out to identify the β-lactamase genes and to analyse the genetic context of the MBL gene. Outer membrane protein (OMP) profiles were analysed by SDS–PAGE.
Results: Nine cases of bloodstream infections caused by an MDR strain of K. pneumoniae producing the VIM-1 MBL and the SHV-5 extended-spectrum β-lactamase (ESBL) were identified. The isolates exhibited various carbapenem resistance levels (imipenem MICs ranged from 4 to 64 mg/L) and were resistant to other β-lactams, fluoroquinolones, trimethoprim/sulfamethoxazole and chloramphenicol. The isolate with the highest imipenem MIC also lacked the k36 OMP. The blaVIM-1 gene cassette was part of the variable region of a class 1 integron that also included an aac(6')-IIc cassette. The ESBL and MBL genes were transferable by conjugation.
Conclusions: This is the first report on the emergence of an MDR strain of K. pneumoniae producing the VIM-1 MBL, causing an outbreak of bloodstream infections in an Italian hospital. The strain evolved through OMP alterations generating a mutant with increased carbapenem resistance.
Key Words: carbapenem resistance , carbapenemases , extended-spectrum β-lactamases , class 1 integrons
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Introduction |
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Carbapenems currently represent the drugs of choice for treatment of serious infections caused by multidrug-resistant (MDR) strains of Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs). Recently, however, the emergence of carbapenem resistance mediated by the production of acquired carbapenemases has been increasingly reported among Enterobacteriaceae and is a matter of major clinical concern.1
Two major classes of acquired carbapenemases have been reported in Enterobacteriaceae, namely, the molecular class A serine-β-lactamases of the IMI-, SME- and KPC-types and the molecular class B metallo-β-lactamases (MBLs) of the IMP- and VIM-types.1 Enterobacteriaceae producing acquired MBLs still remain uncommon worldwide, except in Greece.2 Recently, sporadic isolates of Enterobacter cloacae and Klebsiella pneumoniae producing VIM-type MBLs have been described in Italy.3,4
In this report, we describe the first Italian outbreak of bloodstream infections caused by an MDR strain of K. pneumoniae producing the VIM-1 MBL and the SHV-5 ESBL.
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Materials and methods |
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Bacterial strains
Nine K. pneumoniae isolated from blood samples of nine inpatients at the S. Martino University Hospital (Genoa, Italy) from September 2004 to March 2005 were investigated in this work.
Antibiotic susceptibility testing and phenotypic screening for MBL production
Broth dilution and disc diffusion tests were performed according to the recommendations of CLSI.5,6 To detect MBL production, a synergy test using imipenem and EDTA discs was used.7
Isoelectric focusing (IEF) was performed in polyacrylamide gels (pH range 3.5–9.5, GE Healthcare Bio-Science AB, Sweden), with crude cell extracts prepared by sonic disruption. MBL activity was determined by spectrophotometry essentially as described previously.8
Pulsed-field gel electrophoresis
Genomic DNA was prepared, digested with XbaI (New England Biolabs Inc., MA, USA) and subjected to PFGE with the CHEF DRII device (Bio-Rad, Milan, Italy), as described previously.9
Transfer of β-lactam resistance
Conjugation experiments were carried out between each test isolate and Escherichia coli K12 J53 RifR in broth medium. Transconjugants were selected on rifampicin (256 mg/L) and either imipenem (1, 2 or 4 mg/L) or aztreonam (1, 2 or 4 mg/L).
Analysis of the outer membrane proteins (OMPs)
OMP preparations were obtained after sonic disruption of bacterial cells grown in nutrient broth, followed by selective solubilization of cytoplasmic material with sodium N-lauroyl sarcosinate (2% w/v) and ultracentrifugation. The preparations were run on SDS–polyacrylamide gels (11%) and stained with Coomassie Blue.
PCR was used for the detection of β-lactamase determinants (blaTEM, blaSHV, blaCTX-M, blaIMP and blaVIM) and for mapping of the variable region of class 1 integrons.8,10 DNA sequencing was carried out on both strands of PCR amplification products, as described previously.10
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Description of the outbreak and characterization of the isolates
Between September 2004 and March 2005, a total of nine bloodstream infection episodes caused by MDR isolates of K. pneumoniae showing a putative MBL-positive phenotype according to an imipenem–EDTA synergy test were observed in nine inpatients at three wards [two intensive care units (ICUs) and the transplant surgery unit] of the S. Martino University Hospital (Genoa, Italy).
All isolates exhibited the same PFGE macrorestriction profile (data not shown), revealing the clonal nature of the outbreak.
Molecular analysis revealed the presence of the following β-lactamase genes in all isolates: blaTEM-1, blaSHV-5 and blaVIM-1. These findings were overall consistent with the IEF profiles obtained with crude extracts, which showed the presence of β-lactamase bands of both acidic pI (around 5.3, accountable for by TEM-1 and VIM-1) and alkaline (around 8.2, accountable for by SHV-5 and, possibly, by the resident SHV-type enzyme). Conventional spectrophotometric assays confirmed the presence of MBL activity in the crude extracts of all isolates (imipenemase specific activity, 27–33 nmol/min/mg protein; in all cases inhibitable by >80% after incubation with 1 mM EDTA).
From all patients, isolation of the K. pneumoniae occurred >72 h after hospital admission, indicating nosocomial acquisition. For eight patients, isolation of the K. pneumoniae occurred >72 h after ICU admission, suggesting that acquisition occurred in the ICU (Table 1). The majority of the patients had strong epidemiological links. Eight of nine were in the same ICU (ICU p4), although temporal links were evident for only seven of them. The patient in the transplantation surgery unit (E) was previously hospitalized in ICU p4, where they probably acquired the VIM-1-producing K. pneumoniae. A single patient (G) was infected with the same microorganism and in the same period of time but in a different ICU (ICU er). Contact between the patients in the two ICUs through the same medical equipment or medical staff was excluded. One patient (C), who was moved from ICU p4 to ICU er for 2 days and then back to ICU p4, possibly played a role in the dissemination.
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All patients had underlying disease prior to admission: five had undergone recent liver or kidney transplantation, one had colon cancer, one had lung cancer and one had serious obesity.
Antimicrobial therapy prior to and after isolation of the MBL-producing K. pneumoniae is reported in Table 1. Four of the nine patients died, but only for two of them (Patients D and F), could the K. pneumoniae infection be considered as causative or contributory to death. In the other two patients, the K. pneumoniae infection was apparently eradicated following treatment with meropenem plus amikacin (Patient I) or treatment with piperacillin/tazobactam plus amikacin (Patient C), but the patients died because of a subsequent infection caused by an MDR Pseudomonas aeruginosa (septicaemia and pneumonia, respectively). The P. aeruginosa isolates from these patients were carbapenem-resistant and positive for the blaVIM MBL gene by PCR.
All K. pneumoniae isolates exhibited the same multiresistance pattern and were susceptible or intermediate to carbapenems, with the exception of that from the last patient included in the study (Patient I), which was resistant to imipenem and meropenem (Table 2). Imipenem hydrolytic activity of the extract from this isolate did not reveal significant differences from those of the extracts from other isolates (data not shown). Synergy between clavulanate and carbapenems or other β-lactams was weak or absent, except for aztreonam, in agreement with the β-lactamase profile of these isolates which included both an ESBL and an MBL.
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Analysis of OMPs
Electrophoresis of OMPs showed two major OMPs of 36 and 35 kDa in all K. pneumoniae isolates except that from Patient I (showing high carbapenem MICs), which lacked the k36 OMP.
The genetic context of blaVIM-1 was investigated in the index isolate 6885. The MBL gene was carried on a mobile gene cassette inserted into the variable region of a class 1 integron. The integron variable region contained two cassettes: the blaVIM-1 cassette in the first position and an aac(6')-IIc cassette in the second position.
Transfer of β-lactam resistance
Conjugal transfer of resistance traits to imipenem and aztreonam was assayed with all isolates. E. coli transconjugants selected with aztreonam exhibited both an ESBL and an MBL phenotype and carried both β-lactamase determinants, whereas those selected with imipenem showed only an MBL phenotype. These results showed that both the ESBL and the MBL determinants were transferable by conjugation and that they were likely carried on different conjugative plasmids. Further plasmid characterization was not carried out in this work.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
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In this report, we have described the first outbreak of bloodstream infections caused by MBL-producing K. pneumoniae in Italy. The outbreak was caused by a single strain producing VIM-1 MBL and also an SHV-type ESBL. Interestingly, the structure of the variable region of the VIM-1-encoding integron was different from that carried by a VIM-1-producing E. cloacae previously detected in Genoa,4 although it was identical to that previously described in a VIM-1-producing E. cloacae from Greece.11 This suggests the possibility of a common origin of those integrons, although epidemiological links between the first patient and Greece or Greek nationals could not be ascertained.
Although the outbreak involved three different wards, the epidemiological links among patients revealed a crucial role of one of the ICUs in the dissemination of the MBL-producing strain. Because in this case the VIM-1 integron was carried on a transferable plasmid, the containment of this resistance is expected to be more difficult.
Most VIM-1-producing K. pneumoniae isolates included in this study did not show a frank carbapenem resistance phenotype, and their carbapenem MICs were still in the susceptible range. This fact complicates the detection of MBL-producing Enterobacteriaceae and may delay the enforcement of infection control measures, including isolation of these patients. Only one isolate was resistant to carbapenems because of the concomitant loss of the k36 OMP. This finding underscores the notion that carbapenem resistance can easily emerge by mutation in this OMP gene in MBL-producing klebsiellae, even if carbapenem MICs remain initially in the susceptible range.
Our data confirm the need for a systematic screening to detect MBL-producing strains among isolates circulating in high-risk wards such as ICUs.
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This work was supported by internal funds from Di.S.C.A.T., Microbiology Unit, University of Genoa and Department of Molecular Biology, Microbiology Unit, University of Siena.
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None to declare.
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Acknowledgements |
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We would like to thank O. Varnier, Head of the Diagnostic Microbiology Unit and U. Valente, Head of the Transplantation Unit (University of Genoa), who have facilitated our work. We gratefully acknowledge E. Maioli (Microbiology Unit, University of Genoa) for her technical assistance.
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References |
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1 . Queenan AM, Bush K. Carbapenemases: the versatile β-lactamases. Clin Microbiol Rev (2007) 20:440–58.
2
.
Giakkoupi P, Xanthaki A, Kanelopoulou M, et al. VIM-1 metallo-β-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals. J Clin Microbiol (2003) 41:3893–6.
3
.
Luzzaro F, Docquier JD, Colinon C, et al. Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-β-lactamase encoded by a conjugative plasmid. Antimicrob Agents Chemother (2004) 48:648–50.
4 . Castanheira M, Sader HS, Jones RN, et al. In71, an Enterobacter cloacae blaVIM-1-carrying integron related to In70.2 from Italian Pseudomonas aeruginosa isolates: a SENTRY Antimicrobial Surveillance Program report. Microb Drug Resist (2007) 13:130–4.[CrossRef][Web of Science][Medline]
5 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests—Ninth Edition: Approved Standard M2-A9 (2006) Wayne, PA, USA: CLSI.
6 . Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Seventh Edition: Approved Standard M7-A7 (2006) Wayne, PA, USA: CLSI.
7
.
Arakawa Y, Shibata N, Shibayama K, et al. Convenient test for screening metallo-β-lactamase-producing Gram-negative bacteria by using thiol compounds. J Clin Microbiol (2000) 38:40–3.
8 . Luzzaro F, Endimiani A, Docquier JD, et al. Prevalence and characterization of metallo-β-lactamases in clinical isolates of Pseudomonas aeruginosa. Diagn Microbiol Infect Dis (2004) 48:131–5.[CrossRef][Web of Science][Medline]
9 . Marchese A, Arlet G, Schito GC, et al. Detection of SHV-5 extended-spectrum β-lactamase in Klebsiella pneumoniae strains isolated in Italy. Eur J Clin Microbiol Infect Dis (1996) 15:245–8.[CrossRef][Web of Science][Medline]
10
.
Pallecchi L, Bartoloni A, Fiorelli C, et al. Rapid dissemination and diversity of CTX-M extended-spectrum β-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America. Antimicrob Agents Chemother (2007) 51:2720–5.
11
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Galani I, Souli M, Chryssouli Z, et al. Characterization of a new integron containing blaVIM-1 and aac(6')-IIc in an Enterobacter cloacae clinical isolate from Greece. J Antimicrob Chemother (2005) 55:634–8.
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