1022 Emerging Infectious Diseases • Vol. 9, No. 8, August 2003

LETTERS

Carbapenem-Resistant

Pseudomonasaeruginosa–Carrying

VIM-2 Metallo-β-Lactamase

Determinants,Croatia

To the Editor: Carbapenem-hydrolyzing enzymes of the VIM-type (six different variants are known:VIM-1, VIM-2, VIM-3, VIM-4, VIM-5, and VIM-6) are new molecularclass B metallo-β-lactamases. Theseenzymes have recently been identifiedin carbapenem-resistant isolates ofPseudomonas aeruginosa and othergram-negative nonfermenters fromEuropean countries in theMediterranean basin (Italy, France,Greece, Spain, Portugal, and Turkey),as well as in Far East countries(Korea, Taiwan, and Singapore) andthe United States (1–3, Midilli et al.,GenBank accession no. AY144612,Koh et al., GenBank accession no.AY165025). Similar to blaIMP, blaVIMgenes are located on mobile gene cas-settes inserted in the variable regionsof integrons (1), a condition that pro-vides a wide potential for expressionand dissemination in gram-negativepathogens. VIM enzymes possess thebroadest range of substrate hydrolysisand can degrade virtually all β-lac-tams, except monobactams (4).

According to a recent report, theoverall resistance rate to imipenem inP. aeruginosa isolated from 17 repre-sentative laboratories in Croatia was11% (range 0%–20%) (5). However,molecular basis of carbapenem resist-ance was not investigated.

In October 2000, two P. aerugi-nosa isolates with an unusual resist-ance profile were isolated from twoCroatian patients (66 and 74 years ofage, respectively) who underwenthysterectomies at the Split UniversityHospital. Both isolates were cultured

from urine a week after surgery; a uri-nary catheter had been used for bothpatients who had become febrile andhad signs and symptoms of urinarytract infection. Analysis of themacrorestriction profiles of chromoso-mal DNA of the two isolates bypulsed-field gel electrophoresis, car-ried out as described previously (6),indicated that the two isolates wereclonally related (the two profiles wereapparently identical). In routine antibi-otic susceptibility testing, done bydisk diffusion, both isolates showed amultidrug-resistant phenotype, includ-ing ureidopenicillins, piperacillin,piperacillin-tazobactam, ceftazidime,cefoperazone, cefepime, aztreonam,ciprofloxacin, gentamicin, netilmicin,imipenem, and meropenem.

MICs to imipenem and meropen-em were high (>128 µg/mL). Thesefindings suggested production of anacquired carbapenemase. In fact,crude extracts of the two isolatesexhibited carbapenemase activity in aspectrophotometric assay (7) (imipen-em hydrolyzing–specific activity was,in either case, >170 nmol/min/mgprotein).

A colony blot hybridization, car-ried out as described with a blaIMP anda blaVIM probe (6), yielded a positiveresult with the latter probe.Polymerase chain reaction (PCR)amplification of the variable region ofclass 1 integrons, carried out asdescribed previously by using primersdesigned on the 5′- and 3′-conservedsegments of the integron (8), yielded a4-kb amplification product fromeither isolate. Direct sequencing ofthese amplification products showed,in both cases, the presence of a blaVIM-

2 allele located in a gene cassetteinserted in the attI site of a class 1integron.

The metallo-β-lactamase determi-nant was not transferred toEscherichia coli MKD135 or P.aeruginosa 10145/3 (9) in diparentalmating experiments conducted onsolid medium (the sensitivity of the

assay was >1x10-8 transconjugants perdonor). Plasmid extraction was per-formed with several techniques,including lysis with sodium dodecylsulfate (10) and alkaline lysis con-ducted with a conventional method(10) or with the Nucleobond BAC100system (Macherey-Nagel, Duren,Germany). Extraction of wholegenomic DNA was also performed, asdescribed (8). Plasmid DNA was notdetected in any of these preparations,either when analyzed by agarose gelelectrophoresis or after Southern blothybridization analysis with a blaVIM-2probe generated with PCR amplifica-tion of the entire blaVIM-2 gene. InSouthern blots, a hybridization signalwas only detectable in correspondenceof the band of chromosomal DNA.

To our knowledge, this isolation isthe first one of clinical strains produc-ing acquired metallo-β-lactamase inCroatia. A similar finding underscoresthe progressive emergence of thesedeterminants in different geographicareas and emphasizes the need for anearly recognition of these strains. Infact, monitoring dissemination of newantibiotic resistance determinants isessential to enforce adequate controlmeasures and adjust guidelines forantimicrobial chemotherapy in differ-ent hospital settings.

This work was supported by theEuropean research network on metallo-β-lactamases within the TMR program (con-tract no. FMRX-CT98-0232) and by grant“M.I.U.R” (no. 2001068755_003).

Sanda Sardelic,* Lucia Pallecchi,†Volga Punda-Polic,*

and Gian Maria Rossolini†*University Hospital and School ofMedicine Split, Split, Croatia; and†University of Siena, Siena, Italy

References

1. Nordmann P, Poirel L. Emerging carbapen-emases in gram-negative aerobes. ClinMicrobiol Infect 2002;8:321–31.

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2. Pournaras S, Tsakris A, Maniati M,Tzouvelekis LS, Maniatis AN. Novel vari-ant (bla(VIM-4)) of the metallo-β-lacta-mase gene bla(VIM-1) in a clinical strain ofPseudomonas aeruginosa. AntimicrobAgents Chemother 2002;46:4026–8.

3. Toleman MA, Rolston K, Jones RN, WalshTR. Molecular characterization of VIM-4, anovel metallo-β-lactamase isolated fromTexas: report from the CANCER surveil-lance program. In: 42nd ICAAC Abstracts,San Diego, CA, 2002 Sep 27–30;Washington: American Society forMicrobiology; 2002.

4. Docquier JD, Lamotte-Brasseur J, GalleniM, Amicosante G, Frere JM, Rossolini GM.On functional and structural heterogeneityof VIM-type metallo-β-lactamases. JAntimicrob Chemother 2003;51:257–66.

5. Tambic Andrasevic A, Tambic T, Kalenic S,Jankovic V, Working Group of the CroatianCommittee for Antibiotic Resistance

Surveillance. Surveillance for antimicrobialresistance in Croatia. Emerg Infect Dis2002;8:14–8.

6. Cornaglia G, Mazzariol A, Lauretti L,Rossolini GM, Fontana R. Hospital out-break of carbapenem-resistantPseudomonas aeruginosa producing VIM-1, a novel transferable metallo-β-lactamase.Clin Infect Dis 2000;31:1119–25.

7. Lauretti L, Riccio L, Mazzariol G,Cornaglia G, Amicosante G, Fontana R, etal. Cloning and characterization of blaVIM, anew integron-borne metallo-β-lactamasegene from Pseudomonas aeruginosa clini-cal isolate. Antimicrob Agents Chemother1999;43:1584–90.

8. Riccio ML, Franceschini N, Boschi L,Caravelli B, Cornaglia G, Fontana R, et al.Characterization of the metallo-β-lactamasedeterminant of Acinetobacter baumanniiAC-54/97 reveals the existence of bla(IMP)allelic variants carried by gene cassettes of

different phylogeny. Antimicrob AgentsChemother 2000;44:1229–35.

9. Riccio M, Pallecchi L, Fontana R,Rossolini GM. In 70 of plasmid pAX22, ablaVIM-1-containing integron carrying a newaminoglycoside phosphotransferase genecassette. Antimicrob Agents Chemother2001;45:1249–53.

10. Sambrook J, Fritsch EF, Maniatis T.Molecular cloning: a laboratory manual.Cold Spring Harbor (NY): Cold SpringHarbor Laboratory Press; 1989.

Address for correspondence: Gian MariaRossolini, Dipartimento di BiologiaMolecolare, Sez. Microbiologia, Università diSiena, Policlinico Le Scotte, 53100, Siena,Italy; fax: 39 0577 233325; email:rossolini@unisi.it

Rickettsia felis inthe UnitedKingdom

To the Editor: Rickettsia felis is abacterium transmitted by the cat flea(Ctenocephalides felis), which alsoacts as a reservoir by means of transo-varial transmission (1–3). The distri-bution of R. felis is potentially as wideas that of its insect host, and to date,its presence has been confirmed in catflea populations in North and SouthAmerica and southern Europe (4,5).R. felis was first identified as a humanpathogen in 1994 (6), and cases of“flea-borne spotted fever,” whichhave signs and symptoms of febrileexanthema, have now been reportedin the United States, Mexico, Brazil,France, and Germany (7,8). To ourknowledge, reports on the presence ofR. felis, or indeed any other spottedfever group rickettsia, in the UnitedKingdom have not been published.

To determine whether R. felis ispresent in the United Kingdom, wesurveyed cat fleas collected from dogsand cats seen at veterinary practices insouthern England and NorthernIreland. A total of 31 dogs and 79 catsfrom veterinary practices in Bristol,Dorset, London, Devon, Gloucester-

shire, Hampshire, and Antrim wereincluded in our study. Fleas were col-lected by combing these animals for10 minutes. All fleas from each ani-mal were pooled in 70% ethanol. Atotal of 316 Ct. felis (Bouché, 1835),identified by using accepted morpho-logic criteria, were obtained, witheach animal yielding one to five fleas.DNA was extracted from each of the110 flea pools by using a standard sil-ica cartridge method (QiaAmp DNAmini kit, QIAGEN Ltd., Crawley,West Sussex, U.K.) using the manu-facturer’s instructions for tissue DNAextraction. The presence of rickettsialDNA was determined by using thepolymerase chain reaction (PCR) witholigonucleotide primers that targetrickettesial ompB (5) or gltA (2)genes. Positive control material wascultured R. felis. Rigorous controls tolimit contamination were carried out,including the use of separate, dedicat-ed rooms for DNA extraction, PCRsetup, and gel analysis. Amplificationproducts obtained from ompB andgltA PCRs were analyzed by usingDNA sequencing. Sequences obtainedwere edited by using BioEdit (avail-able from: URL: http://www.mbio.mncsu.edu/BioEdit/bioedit.html).Similarity to published sequences wasdetermined with the BLAST program

(available from: URL: http://www.ncbi.nlm.nih.gov) hosted by theNational Centre for BiotechnologyInformation.

Eighteen flea DNA pools werepositive for spotted fever group rick-ettsia. All 18 yielded PCR productswith both ompB and gltA-targetingPCRs. The ompB and gltA DNAsequences of all PCR products were100% identical to those published forR. felis, thereby providing evidencefor the presence of R. felis in fleas col-lected from >16% of the animals sur-veyed. PCR-positive fleas were col-lected from 4 dogs and 14 cats fromBristol, Hampshire, Dorset, andNorthern Ireland. Taking into accountthe number of fleas in each pool, weestimate that 6% to 12% of the fleascollected were infected with R. felis.

This study represents the firstdescription of a spotted fever grouprickettsia endemic to the UnitedKingdom. The species detected, R.felis, has clear public health implica-tions. The bacterium appears to bewidely distributed within the country,infecting a geographically dispersedpopulation of Ct. felis. Up to 12% ofCt. felis may be infected with R. felis,a flea that is by far the most commonspecies of ectoparasite encounteredon cats and dogs in the U.K. main-