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 A NTIMICROBIAL  A GENTS AND CHEMOTHERAPY, Sept. 2010, p. 3991–3992 Vol. 54, No. 90066-4804/10/$12.00 doi:10.1128/AAC.00225-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Plasmid-Mediated Resistance to Cephalosporins inSalmonella enterica Serovar Typhi

Masatomo Morita,1 Nobuko Takai,1 Jun Terajima,1 Haruo Watanabe,1 Manabu Kurokawa,2

Hiroko Sagara,3 Kenji Ohnishi,4 and Hidemasa Izumiya1*

 Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan1 ; Department of Microbiology, Kobe Institute of  Health, Kobe, Japan2 ; Yokohama Municipal Citizen’s Hospital, Yokohama, Japan3 ; and Department of 

 Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, Tokyo, Japan4

Received 16 February 2010/Returned for modification 17 May 2010/Accepted 20 June 2010

This study characterized a cephalosporin-resistant   Salmonella enterica  serovar Typhi isolate. The or-ganism possessed a plasmid encoding the CTX-M-15 extended-spectrum--lactamase. This plasmid is thedeterminant for the phenotype of cephalosporin resistance and is transferrable among   Enterobacteriaceae.

Typhoid fever is a systemic infection caused by  Salmonella enterica serovar Typhi and has become a predominantly travel-

associated disease in developed countries (3, 6). Fluoroquino-lones have been the most effective drugs for treatment sincethe spread of multidrug-resistant (resistant to ampicillin, chlor-amphenicol, and trimethoprim-sulfamethoxazole)   S.   Typhistrains. Furthermore, in the era of emergence of fluoroquin-olone-resistant  S.  Typhi, it appears that alternative antimicro-bial agents are necessary to ensure therapy for typhoid fever (7,8). Actually, expanded-spectrum cephalosporins and macro-lide antibiotics are considered to be viable alternatives (12).However, a few cephalosporin-resistant  S.  Typhi strains, whichproduce extended-spectrum  -lactamases (ESBLs), have beenreported (1, 9, 11).

Forty-nine clinical isolates of  S.  Typhi were collected from

48 patients with typhoid fever in 2008. All isolates wereidentified by regional public health centers and sent to theDepartment of Bacteriology I, National Institute of Infec-tious Diseases. Isolates were routinely phage typed by thestandard technique with the phage set kindly provided bythe Health Protection Agency, London, United Kingdom,and MICs of antimicrobials were determined by the brothmicrodilution method. The results were interpreted accord-ing to the criteria of the Clinical and Laboratory Standards

Institute (CLSI) (2, 5). One isolate with the phage type E1(080049Ty) displayed a high level of resistance to ceftazi-

dime, cefotaxime, and ceftriaxone. This isolate was alsoresistant to nalidixic acid, showed reduced susceptibility tofluoroquinolones, and was susceptible to imipenem, aztreo-nam, kanamycin, gentamicin, chloramphenicol, tetracycline,and trimethoprim-sulfamethoxazole. The MICs of ceftazi-dime and cefotaxime decreased significantly in the presenceof clavulanic acid at a fixed concentration of 4 mg liter1

(Table 1). A plasmid profile of the resistant  S. Typhi isolateshowed one specific band, which was absent in the suscep-tible strain (080048Ty) with the same phage type, E1 (Fig.1A). The plasmid DNA prepared by alkaline lysis with SDS was dissolved in Milli -Q water at a suitable concentrationand was used for electroporation of  Escherichia coli  W3110.

Transformants were selected on LB agar containing ampi-cillin. The resultant transformant produced a  -lactam re-sistance pattern similar to that produced by  S. Typhi isolate080049Ty (Table 1). These results suggest that a certaingene(s) located on the plasmid conferred the phenotype of resistance against expanded-spectrum cephalosporins. Thepresence of the gene conferring the ESBL phenotype was verified by PCR from the plasmid DNA with the primer pairfor detection of the CTX-M-type  -lactamase gene (5-CA 

* Corresponding author. Mailing address: Department of Bacteriol-ogy I, National Institute of Infectious Diseases, Toyama 1-23-1, Shin-

 juku-ku, Tokyo, 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1163. E-mail: izumiya@nih.go.jp.

Published ahead of print on 28 June 2010.

TABLE 1. MICs for the strains used in this study

StrainMIC (mg liter1) of  a:

 AMP (32) CAZ (32) CAZ-CLA CTX (32) CTX-CLA CRO (32) NAL (32)

S.  Typhi 080049Ty (phage type E1)   64 64 0.25   128   0.125   128   64S.  Typhi 080048Ty (phage type E1)   2 1 0.25 0.125   0.125 0.125   64Transformant ( E. coli  W3110 derivative)   64 64 0.25   128   0.125   128 32Transconjugant (S. Typhi 080048Ty derivative)   64 64 0.25   128   0.125   128   64

 a AMP, ampicillin; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; NAL, nalidixic acid; CLA, clavulanic acid. Numbers in parentheses are CLSI breakpointsfor resistance.

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 AGCGCAGGTGGGCGACAGCAC-3   and 5-CTTTTGCCGTCTAAGGCGATAAAC-3; data not shown) and sub-sequent Southern hybridization of undigested plasmid DNA  with a digoxigenin-labeled probe generated with DIG-HighPrime (Roche Diagnostics) according to the manufacturer’sinstructions (Fig. 1B). In addition, sequence analysis re- veal ed that this  blaCTX-M was 100% identical to  blaCTX-M-15(data not shown). Most of the  blaCTX-M-15  genes have been

identified in   Enterobacteriaceae, mainly in  E. coli  and  Kleb- siell a   spp., and CTX-M-15-producing   E. coli   strains wereisolated from healthy people as well (4, 10). Therefore, thecephalosporin-resistant   S.   Typhi isolate in this study mayhave acquired the transferable plasmid containing blaCTX-M-15 from another enteric bacterium in the patient’sintestine.   E. coli   W3110 harboring the above transformedplasmid with  blaCTX-M-15 was used as the donor of the plas-mid in the transconjugation analysis to confirm the trans-ferability. The expanded-spectrum cephalosporin-suscepti-ble   S.   Typhi isolate 080048Ty was used as the recipientstrain; this strain was also resistant to nalidixic acid. A mixture of each bacterial culture was incubated overnight at

37°C. Transconjugants were isolated on LB agar plates sup-plemented with ampicillin and nalidixic acid, and the pres-ence of a plasmid with the same size as that of the donor was verified (Fig. 1). The doubly resistant colonies appeared with frequencies of 104 to 103 (defined as the number of ampicillin- and nalidixic acid-resistant  S.  Typhi colonies di- vided by the number of nalidixic acid-resistant   S. Typhicolonies). These results indicate the transfer of a plasmidcontaining  blaCTX-M-15   from  E. coli  to  S.  Typhi at a signifi-cant frequency.

This report showed the plasmid-mediated transferabilityof the ESBL phenotype in   S.   Typhi, thus suggesting the

transfer of the plasmid between enteric bacteria. Anothertransferable plasmid containing   blaCTX-M-15   was also ob-served in  S.  Typhi from an Iraqi patient (9). Obviously, thedissemination of the plasmids as an ESBL phenotype deter-minant, in addition to the emergence of fluoroquinolone-resistant S.  Typhi, would make typhoid fever treatment in-creasingly difficult. Therefore, continued and carefulobservations and investigations regarding the characteriza-tion of   S.   Typhi isolates are necessary, especially in theidentification of drug resistance profiles, and the develop-ment of novel therapeutic options should also be reconsid-ered.

We thank all the municipal and prefectural public health institutesin Japan for providing us with the above-described isolates.

This work was supported in part by a grant-in-aid from the Ministryof Health, Labor, and Welfare (H20-Shinko-Ippan-013, H20-Shinko-Ippan-015).

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FIG. 1. Plasmid profiles of cephalosporin-resistant   S.   Typhi

(080049Ty; lane 1), cephalosporin-susceptible   S.   Typhi (080048Ty;lane 2), an  E. coli   transformant (lane 3), and an  S.  Typhi transconju-gant (lane 4). (A) Staining by ethidium bromide. (B) Southern blotanalysis with a digoxigenin-labeled probe specific for CTX-M-15.

3992 MORITA ET AL. A  NTIMICROB. A GENTS CHEMOTHER.

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