LETTERS

2. Pournaras S, Tsakris A, Maniati M, Surveillance. Surveillance for antimicrobial different phylogeny. Antimicrob Agents Tzouvelekis LS, Maniatis AN. Novel vari- resistance in Croatia. Emerg Infect Dis Chemother 2000;44:1229–35. ant (bla(VIM-4)) of the metallo-β-lacta- 2002;8:14–8. 9. Riccio M, Pallecchi L, Fontana R, mase gene bla(VIM-1) in a clinical strain of 6. Cornaglia G, Mazzariol A, Lauretti L, Rossolini GM. In 70 of plasmid pAX22, a

Pseudomonas aeruginosa. Antimicrob Rossolini GM, Fontana R. Hospital out- blaVIM-1-containing integron carrying a new Agents Chemother 2002;46:4026–8. break of carbapenem-resistant aminoglycoside phosphotransferase gene 3. Toleman MA, Rolston K, Jones RN, Walsh producing VIM- cassette. Antimicrob Agents Chemother TR. Molecular characterization of VIM-4, a 1, a novel transferable metallo-β-lactamase. 2001;45:1249–53. novel metallo-β-lactamase isolated from Clin Infect Dis 2000;31:1119–25. 10. Sambrook J, Fritsch EF, Maniatis T. Texas: report from the CANCER surveil- 7. Lauretti L, Riccio L, Mazzariol G, Molecular cloning: a laboratory manual. lance program. In: 42nd ICAAC Abstracts, Cornaglia G, Amicosante G, Fontana R, et Cold Spring Harbor (NY): Cold Spring San Diego, CA, 2002 Sep 27–30; al. Cloning and characterization of blaVIM, a Harbor Laboratory Press; 1989. Washington: American Society for new integron-borne metallo-β-lactamase Microbiology; 2002. gene from Pseudomonas aeruginosa clini- Address for correspondence: Gian Maria 4. Docquier JD, Lamotte-Brasseur J, Galleni cal isolate. Antimicrob Agents Chemother M, Amicosante G, Frere JM, Rossolini GM. 1999;43:1584–90. Rossolini, Dipartimento di Biologia On functional and structural heterogeneity 8. Riccio ML, Franceschini N, Boschi L, Molecolare, Sez. Microbiologia, Università di β of VIM-type metallo- -lactamases. J Caravelli B, Cornaglia G, Fontana R, et al. Siena, Policlinico Le Scotte, 53100, Siena, Antimicrob Chemother 2003;51:257–66. Characterization of the metallo-β-lactamase Italy; fax: 39 0577 233325; email: 5. Tambic Andrasevic A, Tambic T, Kalenic S, determinant of Jankovic V, Working Group of the Croatian AC-54/97 reveals the existence of bla(IMP) [email protected] Committee for Antibiotic Resistance allelic variants carried by gene cassettes of

Rickettsia felis in shire, Hampshire, and Antrim were (available from: URL: http://www. included in our study. were col- ncbi.nlm.nih.gov) hosted by the the United lected by combing these animals for National Centre for Biotechnology Kingdom 10 minutes. All fleas from each ani- Information. To the Editor: felis is a mal were pooled in 70% ethanol. A Eighteen DNA pools were bacterium transmitted by the flea total of 316 Ct. felis (Bouché, 1835), positive for group rick- (Ctenocephalides felis), which also identified by using accepted morpho- ettsia. All 18 yielded PCR products acts as a reservoir by means of transo- logic criteria, were obtained, with with both ompB and gltA-targeting varial transmission (1–3). The distri- each animal yielding one to five fleas. PCRs. The ompB and gltA DNA bution of R. felis is potentially as wide DNA was extracted from each of the sequences of all PCR products were as that of its insect host, and to date, 110 flea pools by using a standard sil- 100% identical to those published for its presence has been confirmed in cat ica cartridge method (QiaAmp DNA R. felis, thereby providing evidence flea populations in North and South mini kit, QIAGEN Ltd., Crawley, for the presence of R. felis in fleas col- America and southern Europe (4,5). West Sussex, U.K.) using the manu- lected from >16% of the animals sur- R. felis was first identified as a human facturer’s instructions for tissue DNA veyed. PCR-positive fleas were col- in 1994 (6), and cases of extraction. The presence of rickettsial lected from 4 dogs and 14 from “flea-borne spotted fever,” which DNA was determined by using the Bristol, Hampshire, Dorset, and have signs and symptoms of febrile polymerase chain reaction (PCR) with Northern Ireland. Taking into account exanthema, have now been reported oligonucleotide primers that target the number of fleas in each pool, we in the United States, Mexico, Brazil, rickettesial ompB (5) or gltA (2) estimate that 6% to 12% of the fleas France, and Germany (7,8). To our genes. Positive control material was collected were infected with R. felis. knowledge, reports on the presence of cultured R. felis. Rigorous controls to This study represents the first R. felis, or indeed any other spotted limit contamination were carried out, description of a spotted fever group fever group rickettsia, in the United including the use of separate, dedicat- rickettsia endemic to the United Kingdom have not been published. ed rooms for DNA extraction, PCR Kingdom. The species detected, R. To determine whether R. felis is setup, and gel analysis. Amplification felis, has clear public health implica- present in the United Kingdom, we products obtained from ompB and tions. The bacterium appears to be surveyed cat fleas collected from dogs gltA PCRs were analyzed by using widely distributed within the country, and cats seen at veterinary practices in DNA sequencing. Sequences obtained infecting a geographically dispersed southern England and Northern were edited by using BioEdit (avail- population of Ct. felis. Up to 12% of Ireland. A total of 31 dogs and 79 cats able from: URL: http://www.mbio. Ct. felis may be infected with R. felis, from veterinary practices in Bristol, mncsu.edu/BioEdit/bioedit.html). a flea that is by far the most common Dorset, London, Devon, Gloucester- Similarity to published sequences was species of ectoparasite encountered determined with the BLAST program on cats and dogs in the U.K. main-

Emerging Infectious Diseases • Vol. 9, No. 8, August 2003 1023 LETTERS land. Furthermore, Ct. felis often Acknowledgments Rickettsia felis in Ctenocephalides spp. feeds on humans. We thank Alex Davies and Anne fleas, Brazil. Emerg Infect Dis 2002;8:317–9. Clinicians encountering patients Seabright for assistance with collecting 5. Marquez FJ, Munain MA, Perez JM, with fever or rash (or both) and a his- and processing the fleas and D. Raoult for Panchon J. Presence of Rickettsia felis in tory of cat contact or flea bites should providing Rickettsia felis. the cat flea from southwestern Europe. consider a diagnosis of R. felis. Emerg Infect Dis 2002;8:89–91. Novartis UK provided financial 6. Schriefer ME, Sacci JB Jr, Dumler JS, Laboratory confirmation of infection assistance with this project. Bullen MG, Azad AF. Identification of a is not easy, but in vitro culture of R. novel rickettsial infection in a patient diag- felis, and hence material for a sero- nosed with murine . J Clin Microbiol logic assay for the diagnosis of Martin J. Kenny,* 1994;32:949–54. Richard J. Birtles,† 7. Richter J, Fournier PE, Petridou J, human R. felis infections, has recent- Haussinger D, Raoult D. Rickettsia felis ly been described, and serology Michael J. Day,* infection acquired in Europe and docu- appears to be an accurate indicator of and Susan E. Shaw* mented by polymerase chain reaction. exposure (9). As with other spotted *University of Bristol, Langford, Somerset, Emerg Infect Dis 2002;8:207–8. 8. Zavala-Velazquez JE, Ruiz-Sosa JA, fever group rickettsial infections, United Kingdom; and †University of Liverpool, Leahurst, Neston, Cheshire, Sanchez-Elias RA, Becerra-Carmona G, molecular diagnostics may provide a United Kingdom Walker DH. Rickettsia felis in useful alternative approach to detect- Yucatan. Lancet 2000;356:1079–80. ing and identifying R. felis in infected 9. La Scola B, Meconi S, Fenollar F, Rolain References JM, Roux V, Raoult D. Emended descrip- tissues. In culture, R. felis has been tion of Rickettsia felis (Bouyer et al. 2001), 1. Azad AF, Sacci JB Jr, Nelson WM, Dasch shown to be resistant to erythromycin a temperature-dependent cultured bacteri- GA, Schmidtman ET, Carl M. Genetic um. Int J Syst Evol Microbiol (unlike other rickettsia), gentamicin, characterization and transovarial transmis- 2002;52:2035–41. amoxicillin, and trimethoprim-sul- sion of a novel typhus-like rickettsia found 10. Rolain JM, Stuhl L, Maurin M, Raoult D. in cat fleas. Proc Natl Acad Sci U S A famethoxazole. Thus, infection with Evaluation of antibiotic susceptibilities of 1992;89:43–62. this bacterium should be considered three rickettsial species including Rickettsia 2. Higgins JA, Radulovic S, Schriefer ME, felis by a quantitative PCR DNA assay. in cases of antibiotic-insensitive fever Azad AF. Rickettsia felis: a new species of Antimicrob Agents Chemother 2002; with a rash, especially in young, old, pathogenic rickettsia isolated from cat and immunosuppressed persons. The fleas. J Clin Microbiol 1996;34:671–4. 46:2747–51. organism is sensitive to doxycycline, 3. Bouyer DH, Stenos J, Crocquet-Valdes P, Moron CG, Popov VL, Zavala-Velazquez Address for correspondence: Susan E. Shaw, rifampicin, thiamphenicol, and fluo- JE, et al. Rickettsia felis: molecular charac- Department of Clinical Veterinary Sciences, roquinolones (10) terization of a new member of the spotted University of Bristol, Langford, Somerset, fever group. Int J Syst Evol Microbiol 2001;51:339–47. BS40 5DU, United Kingdom; fax: +44 (0)117 4. Oliveira RP, Galvao MA, Mafra CL, 9289505; email: [email protected] Chamone CB, Calic SB, Silva SU, et al.

Community studies detected ESBL in the evaluat- antibiotics was determined by the ed population (1,2). We performed disk-diffusion test, following recom- Transmission of three survey studies to determine the mendations of the National Extended-Spectrum incidence of Committee for Clinical Laboratory β-Lactamase strains producing ESBLs in the stools Standards (4,5). The interpretative of outpatients attending our hospital. reading of the antibiogram was per- To the Editor: The spread of mul- The first study was performed during formed according to standard guide- tiresistant gram-negative in a 4-month period (February–May lines (4–6). The MICs of cefotaxime the general population is a problem of 2001), the second during a 3 month- and ceftazidime, with and without paramount importance, but the period (April–June 2002), and the clavulanic acid, were later determined responsible mechanisms are poorly third during 1 month (October 2002). by Etest (AB Biodisk, Solna, understood. Several studies have Stool samples were spread onto Sweden). Strains producing ESBL focused on β-lactam resistance in plates of MacConkey agar containing were defined as strains showing syn- Enterobacteriaceae isolated from 2 mg/L of cefotaxime. A colony of ergism between amoxicillin-clavulan- stools in healthy people, but they did each distinct morphotype was ana- ic acid and cefotaxime, ceftazidime, not specifically investigate the lyzed further. Species were identified cefepime, or aztreonam (4,5). extended-spectrum β-lactamases according to conventional methods All strains suspected of carrying a (ESBL). Furthermore, none of these (3). The susceptibility to β-lactam resistance pattern compatible with

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