LETTERS

7. Bonnin RA, Rotimi V, Al Hubail M, Gasiorowski E, Al Sweih N, Nordmann P, et al. Wide dissemination of GES-type carbapenemases in Acinetobacter bau- mannii in Kuwait. Antimicrob Agents Chemother. 2013;57:183–8. http://dx.doi. org/10.1128/AAC.01384-12 8. Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. The population structure of : expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS ONE. 2010;5:e10034. http://dx.doi. org/10.1371/journal.pone.0010034 9. Gogou V, Pournaras S, Giannouli M, Voulgari E, Piperaki ET, Zarrilli R, et al. Evolution of multidrug-resistant Aci- netobacter baumannii clonal lineages: a 10-year study in Greece (2000–09). J An- timicrob Chemother. 2011;66:2767–72. Figure. Results of Diversilab system (bioMérieux, La Balme-les-Grottes, France) analysis http://dx.doi.org/10.1093/jac/dkr390 of Acinetobacter baumannii isolates. Similarity line shows the cutoff that separates the 10. Poirel L, Bonnin RA, Boulanger A, Schren- different clones. zel J, Kaase M, Nordmann P. Tn125-re-

lated acquisition of blaNDM-like genes in identified mainly carbapenem-hydro- Acinetobacter baumannii. Antimicrob Rémy A. Bonnin, Gaëlle Cuzon, Agents Chemother. 2012;56:1087–9. lyzing carbapenemase OXA-58 (9). Laurent Poirel, http://dx.doi.org/10.1128/AAC.05620-11 Recently, we showed that the and Patrice Nordmann blaNDM-1 gene was carried by a com- Author affiliation: Hôpital de Bicêtre, Address for correspondence: Patrice Nordmann, posite transposon bracketed by 2 Kremlin-Bicêtre, France Service de Bactériologie-Virologie, Hôpital copies of ISAba125 in A. baumannii de Bicêtre, 78 Rue du Général Leclerc, 94275 DOI: http://dx.org/10.3201/eid1905.121618 (10). Cloning and sequencing of the Le Kremlin-Bicêtre Cedex, France; email: genetic context of the blaNDM-1 in the References [email protected] first isolate showed that transposon Tn125 was truncated at its 3′-end 1. Peleg AY, Seifert H, Paterson DL. Aci- netobacter baumannii: emergence of extremity by insertion sequence ISA- a successful . Clin Microbiol ba14, giving rise to a truncated Tn125 Rev. 2008;21:538–82. http://dx.doi. (ΔTn125). PCR mapping of all iso- org/10.1128/CMR.00058-07 Genomic Analysis lates showed that they possessed this 2. Poirel L, Bonnin RA, Nordmann P. Ge- netic basis of antibiotic resistance in patho- of truncated isoform of Tn125, which genic Acinetobacter . IUBMB Life. was therefore probably no longer 2011;63:1061–7. http://dx.doi.org/10.1002/ enterica Serovar functional. iub.532 Typhimurium The identification of several clini- 3. Nordmann P, Poirel L, Walsh TR, Liver- Definitive Phage cal A. baumannii isolates that possessed more DM. The emerging NDM car- bapenemases. Trends Microbiol. 2011; Type 104 the blaNDM-1 gene and originated from 19:588–95. http://dx.doi.org/10.1016/j. North Africa, with no obvious link to tim.2011.09.005 To the Editor: Salmonella en- the Indian subcontinent, strongly sug- 4. Bonnin RA, Poirel L, Naas T, Pirs M, Seme terica is among the leading causes gests that 1 NDM-producing A. bau- K, Schrenzel J, et al. Dissemination of New Delhi metallo-β-lactamase-1–producing Aci- of foodborne diseases worldwide. mannii clone is probably widespread in netobacter baumannii in Europe. Clin Micro- Multidrug-resistant S. enterica se- North Africa and that it might now act biol Infect. 2012;18:E362–5. http://dx.doi. rovar Typhimurium definitive phage as a reservoir for NDM-1. This finding org/10.1111/j.1469-0691.2012.03928.x type (DT) 104 emerged during the might indicate that control of spread 5. Boulanger A, Naas T, Fortineau N, Figueiredo S, Nordmann P. NDM- early 1990s in the United Kingdom of multidrug-resistant A. baumannii 1-producing Acinetobacter baumannii and spread worldwide thereafter (1). would have a primary role in control- from Algeria. Antimicrob Agents Che- This phage-type strain harbors a chro- ling spread of NDM-1. mother. 2012;56:2214–5. http://dx.doi. org/10.1128/AAC.05653-11 mosomally encoded genomic island, 6. Clinical Laboratory Standards Institute. Salmonella Genomic Island 1, which This work was funded by a grant Performance standards for antimicrobial is typically responsible for resis- from the Institut National de la Santé et de susceptibility testing. M100–S22. Wayne tance to , chloramphenicol, la Recherche Médicale. (PA): The Institute; 2012.

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streptomycin, sulfonamide, and tetra- Table. Number of alleles and Simpson's index of diversity score in Salmonella enterica cycline (2). Multilocus variable-num- serovar Typhimurium definitive phage type 104 in humans* ber tandem-repeat analysis (MLVA) All, n = 266 DT104-group, n = 100 Locus No. alleles† D No. alleles† D is an established molecular epidemio- STTR9 5 0.62 1 0.00 logic tool; its high-resolution power STTR5 17 0.87 10 0.76 has been applied to the subtyping of STTR6 20 0.93 16 0.90 STTR10 25 0.90 20 0.93 a variety of bacterial species (3). An STTR3 12 0.73 2 0.20 MLVA system has been developed DT104o 24 0.60 23 0.92 for analyzing S. enterica serovar Ty- *D, Simpson’s index of diversity score. †Including the null allele. phimurium (4,5). The design of an MLVA system DT104o was specific for the DT104 isolates 1a and 1b from an outbreak dur- relies on the analyzed genome se- group, and all 100 DT104 group iso- ing 1996 and isolate 1c in 2007. Isolates quences. In this study, we found and lates displayed amplified products 1a and 1b were identical by MLVA6. evaluated a variable-number tandem- with 13–40 repeat copy numbers; the Isolate 1c was identical by MLVA5 repeat region, or locus, designated others showed the null allele. Focus- but not by MLVA6. In Setting 2, three DT104o. The locus is specific to S. ing only on the 100 DT104 group isolates obtained in different years also enterica ser. Typhimurium DT104, isolates, the discriminatory power of were identical by MLVA5, but differed according to the sequence of NCTC STTR9 and STTR3 were poor, where- from each other by MLVA6. This sug- 13348 (available from the Sanger as STTR5, STTR6, STTR10, and gests that MLVA6 could be useful in Institute, http://www.sanger.ac.uk/ DT104o displayed high discrimina- some epidemiologic settings such as in resources/downloads//salmo- tory powers (Table). In addition, using an outbreak investigation, though more nella.html). The repeat unit sequence the 5 common loci (MLVA5) in analy- extensive study would be required to was CTCAGAA/TTCTGAG, span- sis, we identified 66 types with a D confirm this suggestion. ning 1952121–1952274 on the refer- value of 0.974; use of MLVA5 plus the The DT104o locus is located at the ence genome or 22 repeats of 7 nt. DT104o locus (MLVA6) identified 83 proximal region of fragment 180 com- We used 266 apparently indepen- types with a D value of 0.984. These prised of a prophage structure, which dent isolates of S. enterica serovar results indicate that the DT104o locus was proven to be DT104-specific in a Typhimurium collected during 1981– is highly specific and therefore useful previous study (7). This finding is con- 2012; 103 were from human samples as an additional molecular epidemio- sistent with the results of our study. and 163 from non-human sources. logic marker for analyzing S. enterica In conclusion, development of an Bacteriophage typing was performed ser. Typhimurium DT104. MLVA system is dependent upon the according to Anderson’s method and Because DT104o was highly genome sequences available, and the scheme (6). Types of 100 isolates variable, 5 DT104 strains were tested system is usually used for molecular were in the DT104 group, comprising for the frequency of variants at each subtyping of a certain in a DT104, DT104B, and U302, the lat- locus after 5 serial passages by us- particular organism. However, a spe- ter being related to DT104 (2); MLVA ing liquid culture: cultures were di- cific group of strains could cause a was performed by using the 5 loci luted 1:1,000 at each passage. Sixteen pandemic and become a target of pub- (STTR3, STTR5, STTR6, STTR9, colonies of each strain were tested by lic health concern, as was S. enterica and STTR10) with slight modifica- using MLVA6 (online Technical Ap- ser. Typhimurium DT104. The MLVA tions (4,5). The DT104o locus was pendix Table 1, wwwnc.cdc.gov/EID/ system could be improved by adding tested by using primers o-for (5′-GT- article/19/5/12-1395-Techapp1.pdf). loci based on the genome sequence of CAACATGAACTGCCCCTCA-3′), No variants were observed in STTR3, such pandemic strains. In this study, labeled with NED, and o-rev (5′-TTT- STTR9, or STTR10. STTR5, STTR6, we showed that the newly identified GCTCTTCGCTCTTAGCAATC-3′); and DT104o each showed 1 variant of DT104o locus could be useful in iden- this spanned 1952367–1952043 on the 80 colonies. The results suggest that tification and subtyping of S. enterica reference sequence, resulting in a 325- DT104o would not be less stable than ser. Typhimurium DT104. bp product with 171-bp offset. other loci. For all 266 isolates tested, the We also found that DT104o could Acknowledgment number of alleles and the Simpson’s provide more discriminatory power to We thank the staff at all the municipal index of diversity score (D) identified MLVA5 in some settings (online Tech- and prefectural public health institutes of in each locus are summarized in the nical Appendix Table 2). We compared Japan for providing the isolates. We also Table. The 5 common and DT104o loci 2 settings using isolates from non- thank Nobuko Takai and Jiyoung Lee for displayed high discriminatory power: human samples. Setting 1 comprised their technical assistance.

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This study was partly supported 5. Larsson JT, Torpdahl M, Petersen RF, populations in Camargue, southern by grants-in-aid from the Ministry of Sorensen G, Lindstedt BA, Nielsen EM. France, indicated an A. phagocytophi- Development of a new nomenclature for Health, Labour and Welfare of Japan Salmonella typhimurium multilocus vari- lum seroprevalence of ≈10% (3). To (H22-Shokuhin-Ippan-012, H23-Shinko- able number of tandem repeats analysis investigate the prevalence and diver- Shitei-020, H24-Shokuhin-Ippan-007, and (MLVA). Euro Surveill. 2009;14:19174 sity of A. phagocytophilum bacteria in H24-Shokuhin-Ippan-008). http://www.eurosurveillance.org/View- ticks in Camargue, we collected quest- Article.aspx?ArticleId=19174 6. Anderson ES, Ward LR, Saxe MJ, de ing ticks from horse pastures and feed- Hidemasa Izumiya, Sa JD JD.. Bacteriophage-typing designa-designa- ing ticks from horses. Jun Terajima, Shouji Yamamoto, tions of Salmonella typhimurium. J Hyg Ticks feeding on horses were col- (Lond). 1977;78:297–300. http://dx.doi. lected in randomly selected stables Makoto Ohnishi, org/10.1017/S0022172400056187 Haruo Watanabe, Akemi Kai, 7. Hermans AP, Abee T, Zwietering MH, during 2007 (84 stables), 2008 (72 sta- Takayuki Kurazono, Aarts HJ. Identification of novelSalmonel - ble), and 2010 (19 stables). The stables Masumi Taguchi, Tetsuo Asai, la enterica serovar Typhimurium DT104- were chosen among those where evi- specific prophage and nonprophage dence of A. phagocytophilum serocon- Masato Akiba, Yuko Matsumoto, chromosomal sequences among serovar and Yutaka Tamura Typhimurium isolates by genomic sub- version in horses had been previously Author affiliations: National Institute of- In tractive hybridization. Appl Environ Mi- found (3). In 2008 and 2010, questing crobiol. 2005;71:4979–85. http://dx.doi. fectious Diseases, Tokyo, Japan (H. Izumi- ticks were collected by the dragging org/10.1128/AEM.71.9.4979-4985.2005 ya, J. Terajima, S. Yamamoto, M. Ohnishi, method in 19 pastures, around bushes, and in areas where horses spent the H. Watanabe); Tokyo Metropolitan Institute Address for correspondence: Hidemasa most time. Surveys were conducted in of Public Health, Tokyo (A. Kai); Saitama Izumiya, 1-23-1 Toyama, Shinjuku-ku, Tokyo, the spring, which represents the peak Institute of Public Health, Saitama, Japan 162-8640, Japan; email: [email protected] (T. Kurazono); Osaka Prefectural Institute activity time of Ixodes ticks. of Public Health, Osaka, Japan (M. Tagu- A total of 406 adult ticks were chi); National Veterinary Assay Laboratory, collected, representing 6 species: Tokyo (T. Asai); National Institute of Animal Rhipicephalus bursa, R. sanguineus, Health, Ibaraki, Japan (M. Akiba); Yokoha- Single Genotype R. turanicus, R. pusillus, Dermacen- ma City Institute of Health, Kanagawa, Ja- tor marginatus, and Hyalomma mar- pan (Y. Matsumoto); and Rakuno Gakuen of Anaplasma ginatum. Tick species were identified University, Hokkaido, Japan (Y. Tamura) phagocytophilum by morphologic criteria and molecu- lar analyses based on mitochondrial DOI: http://dx.doi.org/10.3201/eid1905.121395 identified from 12S rDNA sequences (4). Total DNA Ticks, Camargue, was extracted from the ticks by us- References France ing the NucleoSpin Tissue Kit (Ma- cherey-Nagel, Düren, Germany) (5). 1. Threlfall EJ. Epidemic Salmonella To the Editor: Granulocytic typhimurium DT 104-a truly interna- A. phagocytophilum was detected by tional multiresistant clone. J Antimicrob is a tickborne zoonosis nested PCR targeting the 16S rDNA Chemother. 2000;46:7–10. http://dx.doi. caused by Anaplasma phagocytophi- (online Technical Appendix 1, ww- org/10.1093/jac/46.1.7 lum bacteria, which are emerging in wnc.cdc.gov/EID/article/19/5/12- 2. Briggs CE, Fratamico PM. Molecular Europe. Besides infecting humans, A. characterization of an antibiotic resistance 1003-Techapp1.pdf). gene cluster of Salmonella typhimurium phagocytophilum infect a wide range Of the 406 ticks, 40 were infected DT104. Antimicrob Agents Chemother. of wild and domestic mammals (1). with A. phagocytophilum. The infected 1999;43:846–9. In Europe, the Ixodes ricinus tick is group included ticks from all 6 collect- 3. van Belkum A. Tracing isolates of bacte- the main vector for the bacteria, but rial species by multilocus variable number ed species except R. pusillus. Infection of tandem repeat analysis (MLVA). FEMS A. phagocytophilum has also been rates among the species ranged from 0 Immunol Med Microbiol. 2007;49:22–7. detected in association with Rhipi- to 22% (online Technical Appendix 2, http://dx.doi.org/10.1111/j.1574- cephalus and Dermacentor spp. ticks wwwnc.cdc.gov/EID/article/19/5/12- 695X.2006.00173.x (2). The climate and biotopes of the 4. Lindstedt BA, Vardund T, Aas L, 1003-Techapp2.pdf). The prevalence Kapperud G. Multiple-locus variable- Mediterranean region are particularly of A. phagocytophilum infection did number tandem-repeats analysis of favorable for several species of ticks not differ significantly between -spe Salmonella enterica subsp. enterica serovar and, therefore, for tickborne diseases. cies (logistic regression model, p Typhimurium using PCR multiplexing and Although I. ricinus ticks are rare multicolor capillary electrophoresis. J Mi- = 0.76) but was higher among quest- crobiol Methods. 2004;59:163–72. http:// or absent in the Mediterranean Ba- ing ticks than feeding ticks (p<0.001; dx.doi.org/10.1016/j.mimet.2004.06.014 sin, serosurveys performed on equine odds ratio 1.15).

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