LETTERS

4. Chung KP, Tseng SP, Huang YT, Tsai TH, bla –positive were transported in Transystem (CO- Teng LJ, Hsueh PR. Arrival of Klebsi- NDM-1 PAN Italia S.p.A, Brescia, Italy) to ella pneumoniae carbapenemase (KPC)- Klebsiella 2 in Taiwan. J Antimicrob Chemother. preserve and DNA. The 20 2011;66:1182–4. Epub 2011 Feb 8. http:// pneumoniae from PCR swab specimens were squeezed dx.doi.org/10.1093/jac/dkr025 Environment, out into 0.5-mL volumes of sterile wa- 5. Centers for Disease Control and Preven- ter and centrifuged at 3,000 × g for 30 tion. Guidance for control of infections Vietnam with carbapenem-resistant or carbapen- seconds; 1 μL of the resulting suspen- emase-producing in To the Editor: The bla sion was then used as PCR template to acute care facilities. MMWR Morb Mortal NDM-1 detect bla as described (7). Two gene, which produces the New Delhi NDM-1 Wkly Rep. 2009;58:256–60. samples were positive for bla ; 6. Tacconelli E, Karchmer AW, Yokoe D, metallo-β-lactamase (NDM-1) en- NDM-1 D’Agata EMC. Preventing the infl ux zyme, confers resistance to the car- these 2 samples were collected from of vancomycin-resistant enterococci bapenem class of antimicrobial drugs the same river (Kim Nguu River) but into health care institutions, by a simple and can be transferred among different at sites 3 km apart. validated prediction rule. Clin Infect To isolate and identify the pheno- Dis. 2004;39:964–70. http://dx.doi. types of bacteria. NDM-1 was identi- type and genotype of bla –positive org/10.1086/423961 fi ed in 2008 in Sweden from a patient NDM-1 7. Balm MN, Ngan G, Jureen R, Lin RT, Teo from India who had been hospitalized bacteria, we repeatedly spread the 20 J. Molecular characterization of newly culture swab specimens onto Muller- emerged bla –producing Klebsiella in New Delhi (1). Since that report, KPC-2 Hinton agar (Nissui, Tokyo, Japan) pneumoniae in Singapore. J Clin Micro- blaNDM-1–positive bacteria have been biol. 2012;50:475–6. Epub 2011 Nov 23. identifi ed from patients in several containing 100 mg/L vancomycin http://dx.doi.org/10.1128/JCM.05914-11 countries; most of these patients had (Nakalai, Kyoto, Japan) plus 0.5 mg/L 8. Qi Y, Wei Z, Ji S, Du X, Shen P, Yu Y. a direct link with the Indian subconti- meropenem (LKT Laboratories, St. ST11, the dominant clone of KPC-pro- Paul, MN, USA) until single colonies ducing in China. J nent (2). The spread of blaNDM-1 among Antimicrob Chemother. 2011;66:307–12. bacterial is of concern not were obtained. Each colony was then http://dx.doi.org/10.1093/jac/dkq431 only because of resistance to carbapen- subcultured by plating onto MacCon- 9. Hsu LY, Tan TY, Tam VH, Kwa A, Fisher ems but also because such pathogens key agar (Nihon Seiyaku, Tokyo, DA, Koh TH, et al. Surveillance and cor- Japan) containing 0.5 mg/L merope- relation of antibiotic prescription and typically are resistant to multiple an- resistance of gram-negative bacteria in timicrobial drug classes, which leaves nem to ensure culture purity; colo- Singaporean hospitals. Antimicrob Agents few treatment choices available (3–5). nies were identifi ed by using API 20E Chemother. 2010;54:1173–8. http:// In 2011, spread of bla –positive strips (bioMérieux, Basingstoke, UK). dx.doi.org/10.1128/AAC.01076-09 NDM-1 MICs of these isolates for 13 antimi- 10. Urban C, Bradford PA, Tuckman M, bacteria in an environmental setting in Segal-Maurer S, Wehbeh W, Grenner L, New Delhi was reported (6). crobial drugs were calculated by using et al. Carbapenem-resistant Escherichia The possible appearance of bac- Etest (bioMérieux), and susceptibility coli harboring Klebsiella pneumoniae teria harboring bla in Vietnam is data were interpreted by using Clini- carbapenemase β-lactamases associated NDM-1 cal and Laboratory Standards Institute with long-term care facilities. Clin In- of concern because cultural and eco- fect Dis. 2008;46:e127–30. http://dx.doi. nomic links between Vietnam and In- guidelines (www.clsi.org). org/10.1086/588048 dia are strongly established, including We harvested several species of extensive person-to-person exchanges bacteria from the 2 seepage samples Address for correspondence: Indumathi positive for bla : Acinetobacter that could enable easy exchange of NDM-1 Venkatachalam, National University Hospital– pathogens. In addition, Vietnam faces baumannii, Klebsiella pneumoniae, Infectious Diseases, 5 Lower Kent Ridge Rd, a serious problem of antimicrobial , P. fl uore- Singapore City 119074, Singapore; email: drug resistance because drugs are free- scens/putida, and P. luteola. These [email protected] ly available and used in an indiscrimi- isolates were placed onto media con- taining 0.5 mg/L meropenem, and nate fashion. Thus, once blaNDM-1– positive bacteria colonize persons in bacterial DNA was extracted and Vietnam, they would be able to spread used for the template for PCR analy- sis to detect bla as described (7). easily and pose a serious public health NDM-1 bla was detected in 3 K. pneu- threat. NDM-1 During September 2011, we col- moniae isolates from each of the 2 lected paired swab samples (1 for positive samples (6 isolates total); this PCR, 1 for culture) of seepage water result was confi rmed by sequencing. from 20 sites (rivers, lakes, and water All 6 isolates were highly resistant pools in streets) within a 10-km radius to all β-lactam antimicrobial drugs, of central Hanoi, Vietnam. Samples including carbapenems (Table). To

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References Table. Resistance to 13 antimicrobial drugs of blaNDM-1–positive Klebsiella pneumoniae isolates from the Kim Nguu River, Hanoi, Vietnam* MIC, mg/L 1. Yong D, Toleman MA, Giske CG, Cho Antimicrobial drug Site X Site Y HS, Sundman K, Lee K, et al. Character- Piperacillin/tazobactam 64–>256 64–>256 ization of a new metallo-beta-lactamase Cefotaxime 48–>256 96–128 gene, bla (NDM-1), and a novel erythro- Ceftazidime >256 >256 mycin esterase gene carried on a unique Ceftriaxone 96–>256 128–>256 genetic structure in Klebsiella pneumoniae Meropenem 8–>32 12–>32 sequence type 14 from India. Antimicrob Doripenem 4–>32 8–>32 Agents Chemother. 2009;53:5046–54. Imipenem 6–>32 >32 http://dx.doi.org/10.1128/AAC.00774-09 Fosfomycin 3–8 8 2. Nordmann P, Nass T, Poirel L. Global Gentamicin >1,024 >1,024 spread of carbapenem-producing En- Tobramycin 384–>1,024 256–384 terobacteriaceae. Emerg Infect Dis. Ciprofloxacin 0.064–1.5 0.064 2011;17:1791–8. Colistin 0.19–2 0.125–0.38 3. Kumarasamy KK, Toleman MA, Walsh Tgecycline 1.5–3 0.5–1.5 TR, Bagaria J, Butt F, Balakrishnan R, *MICs were interpreted by using Clinical and Laboratory Standards Institute guidelines et al. Emergence of a new antibiotic re- (www.clsi.org). sistance mechanism in India, Pakistan, and the UK: a molecular, biological, detect another β-lactamase, multiplex Our results show that blaNDM-1– and epidemiological study. Lancet In- PCRs were carried out as described positive K. pneumoniae sequence fect Dis. 2010;10:597–602. http://dx.doi. org/10.1016/S1473-3099(10)70143-2 (8); genetic variants blaTEM, blaSHV, type 283 is present in the Kim Nguu bla , bla , bla , bla , and River, which fl ows through the cen- 4. Walsh TR. Emerging carbapenemases: OXA CTX-M IMP VIM a global perspective. Int J Antimicrob blaKPC were not detected in any of the tral part of Hanoi at 2 sites. The iso- Agents. 2010;36:S8–14. http://dx.doi. isolates other than K. pneumoniae. All lates we obtained were also positive org/10.1016/S0924-8579(10)70004-2 5. Moellering RC Jr. NDM-1—a cause 6 K. pneumoniae isolates were posi- for 2 other β-lactamases, blaTEM-1 and tive for bla and bla variants by bla , were highly resistant to for worldwide concern. N Engl J TEM CTX-M CTX-M-3 Med. 2010;363:2377–9. http://dx.doi. PCR; these variants were confi rmed as aminoglycosides related to rmtB, and org/10.1056/NEJMp1011715 blaTEM-1 and blaCTX-M-3 by sequencing. showed mild elevation of MIC against 6. Walsh TR, Weeks J, Livermore DM, Aminoglycosides are often used ciprofl oxacin up to 1.5 mg/L. Wide- Toleman MA. Dissemination of NDM-1 in the management of severe infec- scale surveillance of environmental positive bacteria in the New Delhi envi- ronment and its implications for human tious diseases caused by gram-negative and clinical samples in Vietnam and health: an environmental point prevalence pathogens. 16S rRNA methylases were establishment of a strategy to prevent study. Lancet Infect Dis. 2011;11:355– 62. http://dx.doi.org/10.1016/S1473- found to confer high levels of resistance further spread of blaNDM-1 are urgently to aminoglycosides such as amikacin, needed. 3099(11)70059-7 7. Pfeifer Y, Wilharm G, Zander E, Wichel- tobramycin, and gentamicin. The 6 K. haus TA, Gottig S, Hunfeld KP, et al. pneumoniae isolates we found were Rie Isozumi, Molecular characterization of blaNDM-1 in highly resistant to gentamicin (MIC Kumiko Yoshimatsu, an strain iso- >1,024 mg/L) and tobramycin (MIC Tetsu Yamashiro, lated in Germany in 2007. J Antimicrob Futoshi Hasebe, Chemother. 2011;66:1998–2001. http:// 256–>1,024 mg/L) (Table). Therefore, dx.doi.org/10.1093/jac/dkr256 we screened genetic elements of 16S Binh Minh Nguyen, 8. Dallenne C, Da Costa A, Decre D, Fa- rRNA methylases (rmtB, rmtC, and Tuan Cuong Ngo, vier C, Arlet G. Development of a set of armA) by PCR and detected rmtB in Shumpei P. Yasuda, multiplex PCR assays for the detection of Takaaki Koma, Kenta Shimizu, genes encoding important beta-lactamases all 6 isolates (9). Multilocus sequence in Enterobacteriaceae. J Antimicrob Che- typing was applied for these 6 isolates; and Jiro Arikawa mother. 2010;65:490–5. http://dx.doi. all were identifi ed as K. pneumoniae Author affi liations: Hokkaido University, org/10.1093/jac/dkp498 sequence type 283 (10), which had not Sapporo, Japan (R. Isozumi, K. Yoshimat- 9. Doi Y, Arakawa Y. 16S ribosomal RNA su, S.P. Yasuda, T. Koma, K. Shimizu, J. methylation: emerging resistance mecha- been reported as harboring blaNDM-1. nism against aminoglycosides. Clin In- The azide-resistant Arikawa); Nagasaki University, Nagasaki, fect Dis. 2007;45:88–94. http://dx.doi. strain J53 has been used as recipient Japan (T. Yamashiro, F. Hasebe); and Na- org/10.1086/518605 for conjugation assay, which had been tional Institute of Hygiene and Epidemi- 10. Diancourt L, Passet V, Verhoef J, Grimont ology, Hanoi, Vietnam (B.M.. Nguyen, T. PA, Brisse S. Multilocus sequence typing reported previously (6), but we found of Klebsiella pneumoniae nosocomial iso- C. Ngo) no transconjugant strain with blaNDM-1 lates. J Clin Microbiol. 2005;43:4178–82. on MacConkey agar containing 100 DOI: http://dx.doi.org/10.3201/eid1808.111816 http://dx.doi.org/10.1128/JCM.43.8.4178- mg/L sodium azide and 0.5 mg/L me- 4182.2005 ropenem.

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Address for correspondence: Rie Isozumi, (3,4). R. felis can also infect at least 10 collected in human dwellings were Hokkaido University, Kita15, Nishi7, Kita-ku, other species of , including P. irritans, and 3 specimens were C. Sapporo, Hokkaido, 060-8638, Japan; email: P. irritans fl eas, trombiculid and felis. A screening by amplifi cation [email protected] mesostygmata , hard and soft using primers and probes specifi c , and booklice (5,6). for the 16S–23S internal transcribed In Africa, the presence of R. felis in spacer of spp. (7) produced fl eas has been documented in Algeria, no positive results. Tunisia, Egypt, Ethiopia, Gabon, We screened rickettsial DNA by Côte d’Ivoire, and the Democratic using qPCR with a -specifi c, Republic of Congo (5). Recent studies gltA gene-based RKND03 system conducted in Senegal (3) and Kenya (8) and a bioB-based qPCR system Rickettsia felis in (4) have shown that as much as 4.4% specifi c for R. felis. We found that the , Southern and 3.7%, respectively, of acute 3 specimens of C. felis fl eas contained Ethiopia, 2010 febrile diseases in these regions may the DNA of R. felis; however, 23 be caused by R. felis infections. We (43%) of 53 P. irritans specimens To the Editor: Fleas (order conducted a study to determine the also contained DNA of R. felis. We Siphonaptera) are obligate distribution and prevalence of R. felis amplifi ed and sequenced nearly the hematophagous insects. They are in fl eas in Ethiopia. entire rickettsial gltA gene from 3 C. laterally fl attened, holometabolous, In our study, 55 fl eas were felis and 10 P. irritans specimens and and wingless ectoparasites. More collected in 2010 in 2 villages in found that the sequence was identical than 2,500 species of fl ea, belonging Ethiopia; 25 fl eas were collected to that of R. felis (GenBank accession to 16 families and 238 genera, have from Tikemit Eshet (6°51′837″N and no. NC_007111). been described. A minority of these 35°51′348″E; altitude2,121 m), and During the fi eld collection of the genera live in close association with 30 fl eas were collected from Mizan fl eas, the conservation of specimens humans (synanthropic), including Teferi (6°59′640″N and 35°35′507″E; may be diffi cult. Degradation of fl eas of these species: Pulex altitude 1,700 m). The specimens specimens may pose a problem for the irritans, Ctenocephalides felis, were collected by using a plate fi lled ensuing morphologic identifi cation. Ctenocephalides canis, Xenopsylla with soapy water with a candle in the For fl eas, a specifi c preparation is cheopis, Nosopsyllus fasciatus, middle of the plate. Because fl eas are required that destroys internal organs , and thermotropic, they jumped toward the and produces a chitin complex of the Tunga penetrans (1). Many fl eas are candle and fell onto the plate, where insect. This type of preparation makes capable of transmitting the following they rapidly drowned in the soapy it diffi cult, and sometimes impossible, pathogens to their hosts: bacteria water. The fl eas were identifi ed by to use the insect later for molecular (e.g., , R. felis, morphologic features and stored in studies. The development of qPCR , and many Bartonella 90% ethanol until DNA extraction. specifi c for P. irritans and C. felis spp.); viruses (e.g., myxoma virus); To confi rm the phenotypic fl eas facilitated the identifi cation of protozoa (e.g., Trypanosoma spp.); identifi cation, we designed primers and damaged samples and also precluded or helminths (e.g., Hymenolepis spp.) probes for quantitative real-time PCR the laborious and time-consuming (2). Ctenocephalides spp. fl eas are of (qPCR) that were specifi c for 2 species procedure of identifi cation by special interest as main reservoirs and of fl ea (P. irritans and C. felis) based morphologic features. vectors of R. felis, because this agent on the sequences of mitochondrial We conclude that the reservoirs causes an emerging disease, fl eaborne cytochrome oxidase gene published of R. felis in Ethiopia include both C. . The distribution and in GenBank (Table). All of the felis and P. irritans fl eas. In Ethiopia, prevalence of this disease have not identifi cations made by morphologic P. irritans fl eas have been reported been well studied. Symptoms of this appearance were confi rmed by to be prevalent (9). P. irritans fl eas disease range from mild to moderate qPCR because some specimens were have been shown to be infected by and include fever, cutaneous rash, damaged and diffi cult to identify. We R. felis in several locations, notably and sometimes an inoculation eschar found that most (52/55) of the fl eas in the Democratic Republic of the

Table. Sequences of primers and probes used to identify fleas by quantitative real-time PCR, southern Ethiopia, 2010 Species Forward primer, 3co5c Reverse primer, 3co5c Fluorescent probe P. irritans CGAATACTTTTAGAAAGCCAAAACA CATTGATGACCAATAGATTTTAGAGTG TTGCTTTACCGTCTTTACGTTT C. felis TCGTTATTTACTTGAAAGACAAAATG TCATTGATGACCAATTGCTTT TGCTTTACCTTCTCTTCGACTTTT *P., Pulex; C., Ctenocephalides.

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