Outbreak Caused by a Multidrug-Resistant Klebsiella pneumoniae Clone Carrying blaVIM-12 in a University Hospital

From November 2006 to April 2007, nine nonrepetitive isolatesof Klebsiella pneumoniae with reduced susceptibility or resistanceto carbapenems were recovered from clinical specimens from separatepatients hospitalized in a tertiary care hospital. The imipenem-EDTAsynergy test was positive for all isolates. PCR, sequencing,and transferability experiments revealed the novel bla_VIM-12metallo-β-lactamase gene, which was plasmid mediated andlocated in a class 1 integron. Pulsed-field gel electrophoresisdemonstrated a single macrorestriction pattern, indicating theclonal spread of VIM-12-producing K. pneumoniae.

INTRODUCTION

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INTRODUCTION

Acquired metallo-β-lactamases (MBLs) constitute a growingclass of β-lactamases that readily hydrolyze most β-lactamantibiotics, including imipenem and meropenem (20). They areusually embedded in class 1 integron structures carrying variousgene cassettes and belong to the group B β-lactamases accordingto the classification scheme proposed by Ambler (1). Five differentgroups of acquired MBLs have been described (IMP, VIM, SPM,GIM, and SIM enzymes), but the first two of these groups havepredominated in gram-negative organisms (8, 9, 13, 20). VIM-typeMBLs are commonly encountered among nonfermenters in the FarEast and southern Europe (14, 15, 18, 21, 22), but during thelast few years, the respective bla genes have also spread inmembers of the family Enterobacteriaceae (11, 20, 23). Reportsof VIM-type MBL-producing K. pneumoniae isolates from Greekhospitals are increasing (5, 7). Recently, a novel VIM-typeMBL variant, VIM-12, was identified from a clinical isolateof Klebsiella pneumoniae in Greece (16). In this report, wedescribe an outbreak caused by K. pneumoniae harboring the bla_VIM-12gene.

MATERIALS AND METHODS

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MATERIALS AND METHODS

From November 2006 to April 2007, nine nonrepetitive K. pneumoniaeisolates exhibiting reduced susceptibility or resistance toimipenem were isolated from clinical specimens collected fromseparate patients hospitalized in different medical and surgicalwards at Hippokration General Hospital, Thessaloniki, Greece.All patients had a long-term hospitalization and had been treatedwith multiple courses of antimicrobials, including carbapenems.Identification of the isolates to the species level and initialantibiotic susceptibility testing were performed by the Vitek-2automated system (BioMérieux, Marcy l'Étoile,France), by which they were characterized as imipenem intermediateor resistant.

Susceptibilities to a range of antimicrobials (amikacin, amoxicillin-clavulanate,ampicillin, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime,ciprofloxacin, cotrimoxazole, ertapenem, imipenem, meropenem,and tetracycline) were also determined by disk diffusion (3).MICs of imipenem, meropenem, ertapenem, and tigecycline wereadditionally determined by Etest (AB Biodisk, Solna, Sweden)and the broth microdilution method (3). Susceptibilities wereinterpreted according to CLSI guidelines (3), when available.For tigecycline, the U.S. FDA (Food and Drug Administration)recommendation was used (susceptible, $≤$ 2 µg/ml; resistant, $≥$ 8 µg/ml). Possible MBL production was tested by usingthe imipenem-EDTA double-disk synergy test (2, 14). Escherichiacoli strains ATCC 25922 and 35218 and known VIM-12-producingE. coli strain 28 (6) were used as controls in all susceptibilityassays.

The isolates were tested by PCR for the bla_VIM gene with a setof primers that amplify an internal fragment of the gene (18).For sequencing, primers amplifying the entire bla_VIM gene werealso used (21). Additionally, PCRs were performed with all ofthe isolates for the bla_IMP MBL gene (18), as well as for theextended-spectrum β-lactamase-encoding genes bla_TEM, bla_CTX-M,and bla_SHV-5-type and the AmpC type-encoding gene bla_CMY (12,19). Detection and mapping of a class 1 integron were carriedout with primers specific for 5' and 3' conserved segments amplifyingthe variable region containing the resistance gene cassette(10). Nucleotide sequencing of both strands of the PCR productsderived with primers amplifying the whole bla_VIM gene and theclass 1 integron was performed with an ABI Prism 377 DNA sequencer(Applied Biosystems Division, Perkin-Elmer, Foster City, CA).

The K. pneumoniae clinical isolates studied were subjected toconjugation experiments with E. coli 26R793 (lac Rif^r) as therecipient (17). Selection of transconjugants was done on MacConkeyagar plates containing rifampin at 100 µg/ml and ceftazidimeat 2 µg/ml. Transconjugants were analyzed for plasmidsby an alkaline lysis procedure. The plasmid DNA band was extractedfrom the agarose gel with the QIAquick gel extraction kit (QiagenGmbH, Hilden, Germany) and was used as template DNA in a PCRfor the detection of bla_VIM. The expression of the bla_VIM genewas tested by reverse transcriptase PCR-specific amplificationwith DNase-treated RNA extracts obtained with an RNeasy minikit (Qiagen) and 16S rRNA as a control as described previously(7), with the same RNA extracts for amplification of both the16S rRNA and bla_VIM genes.

The relatedness of the K. pneumoniae isolates was analyzed bypulsed-field gel electrophoresis (PFGE) with XbaI-digested genomicDNA and a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UnitedKingdom) as described elsewhere (18). XbaI macrorestrictionpatterns were compared visually.

RESULTS

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RESULTS

The nine K. pneumoniae isolates studied were recovered fromblood cultures, wounds, and bronchial secretions (two caseseach) and from urine, sputum, and drainage fluid (one case each).The ages of the patients ranged from 23 to 90 years; they wereadmitted for several reasons and were hospitalized in six differentwards of the hospital. Four of them had received carbapenemsprior to the isolation of the bla_VIM-12-producing K. pneumoniae.Table 1 summarizes the characteristics of the patients and theorigins of the isolates.

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TABLE 1. Origins of bla_VIM-12-containing K. pneumoniae isolates and clinical characteristics of the patients

By the automated system, all of the isolates were initiallycharacterized as intermediately resistant or resistant to imipenemand meropenem (Table 2). By disk diffusion testing, five ofthe isolates were susceptible to all carbapenems, three weresusceptible to imipenem and meropenem but intermediately resistantto ertapenem, and one was resistant to all three carbapenems.In accordance with disk diffusion, the Etest and broth microdilutionMICs for all of the isolates but one were in the susceptiblerange for imipenem and meropenem (1 to 4 µg/ml for imipenemand 0.5 to 4 µg/ml for meropenem) and ranged from 0.5to 16 µg/ml for ertapenem, while the remaining isolatehad MICs of >32 µg/ml for all three carbapenems (Table2). The isolates were also susceptible to tigecycline, whoseMICs ranged from 0.032 to 2 mg/liter. All of the isolates exhibitedsynergy with EDTA in the imipenem-EDTA double-disk synergy test,indicating MBL production. The susceptibilities of the isolatesto other antimicrobial agents by the disc diffusion method arereported in Table 3. All of the isolates were found resistantor intermediately resistant to ampicillin, amoxicillin-clavulanate,aztreonam, expanded-spectrum cephalosporins, ciprofloxacin,amikacin, cotrimoxazole, and tetracycline.

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TABLE 2. Susceptibilities of the nine K. pneumoniae isolates studied to carbapenems by different assays

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TABLE 3. Antibiotic susceptibilities of the nine K. pneumoniae isolates studied to various antimicrobials by the disk diffusion method^a

The nine K. pneumoniae isolates were PCR positive for the bla_VIMgene with both sets of consensus primers. DNA sequencing ofthe entire bla_VIM amplicons showed that all of the isolatescarried the novel bla_VIM-12 MBL gene. The structure of the bla_VIM-12-containingintegron was characterized in three representative K. pneumoniaeisolates. The bla_VIM-12 gene was inserted as a gene cassetteinto a class 1 integron. This gene cassette, with its truncated59-base element, was part of a 5'-to-3' region and was flankedby two copies of an aacA7 gene cassette which confers resistanceto aminoglycosides. A strong P1 promoter and an activated P2promoter were the promoters of the 5' conserved segment sequence.Reverse transcriptase-PCR confirmed that, irrespectively ofthe carbapenem MICs, the bla_VIM-12 gene was expressed in allnine isolates. PCR was negative for the bla_IMP, bla_SHV-5-type,bla_CTX-M, and bla_TEM β-lactamase genes but positive forbla_CMY in all of the isolates.

Conjugation experiments showed that the ceftazidime resistancedeterminant was successfully transferred to E. coli 26R793 fromall nine isolates at transfer frequencies ranging from 1.7 x10^–2 to 3.4 x 10^–5 per recipient cell. Susceptibilitytesting confirmed the transfer of resistance to β-lactams,as well as resistance to amikacin and cotrimoxazole. The molecularsizes of the plasmids transferred from each isolate to E. coli26R793 were approximately 70 kb. When the plasmid bands of thetransconjugants were extracted from the gel and used as templatesfor the amplification of the bla_VIM and bla_CMY genes, the specificproducts were detected, suggesting that both resistance determinantsresided in this transferable plasmid. Sequencing of both strandsof the entire bla_VIM gene amplicons derived from the extractedplasmidic DNA confirmed the presence of bla_VIM-12 in the transconjugants.PFGE analysis showed that all of the clinical isolates belongedto a single clone producing the same macrorestriction pattern(Fig. 1).

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FIG. 1. PFGE of XbaI-digested genomic DNA from the nine nonrepetitive bla_VIM-12-positive K. pneumoniae isolates.

DISCUSSION

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DISCUSSION

This report describes an outbreak caused by a single clone ofK. pneumoniae carrying the novel plasmid-mediated bla_VIM-12carbapenem-hydrolyzing β-lactamase gene. bla_VIM-12 is clusteredbetween bla_VIM-1 and bla_VIM-2; it differs from bla_VIM-1 by eightnucleotides that are all located at the 3' end and matches exactlythe nucleotides found at the corresponding positions in thebla_VIM-2 gene (16). The spread of VIM-12-producing K. pneumoniaein our hospital might be related to the frequent transfer ofpatients between wards. However, other factors, such as fecalor skin carriage by staff or patients, should not be ignored.Three of the patients had received imipenem and one had receivedmeropenem prior to the isolation of the VIM-12-producing K.pneumoniae strain. Treatment of two of the above patients withimipenem was also continued, given the subsequent laboratoryreports (Etest imipenem MIC of 3 µg/ml). The third patient(patient 5) died because of severe injuries soon after the recoveryof the imipenem-resistant K. pneumoniae isolate. In most cases,colistin was deemed one of the antimicrobial agents of choiceaccording to the results of the automated system (MICs of $≤$ 0.5µg/liter). It should be mentioned here that we observedsome discordant carbapenem susceptibility results between ourautomated system and the other susceptibility assays. It isof note that similar discordances have been described previouslyfor automated systems, which were also found to overestimateresistance rates (4). Strict control measures such as isolationof infected patients, hand hygiene, and restriction of carbapenemuse as monotherapy were taken, and the outbreak strain was notrecovered from other patients in the hospital after the initialoutbreak.

K. pneumoniae is recognized as an important pathogen mainlyinvolved in nosocomial infections and outbreaks. The spreadof MBL-encoding genes in K. pneumoniae and the emergence ofnew transferable MBL variants in our region may reflect a heavyusage of carbapenems for the treatment of multiresistant Acinetobacterbaumannii and Pseudomonas aeruginosa infections, which are commonin Greek hospitals. This evolution is worrisome and leaves limitedchoices for treatment regimens. The accurate and rapid detectionof these MBL-producing pathogens is necessary for therapeuticconsiderations and infection control purposes in order to preventfurther dissemination.

FOOTNOTES

* Corresponding author. Mailing address: Department of Clinical Microbiology, Hippokration General Hospital, Thessaloniki, Greece. Phone and fax: 30-2310-892050. E-mail: Sofianou{at}otenet.gr

Published ahead of print on 16 January 2008.

REFERENCES

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