Detection of CTX-M-14 extended-spectrum β-lactamase in Shigella sonnei isolates from China

Journal of Infection (2007) 55, e125ee128

www.elsevierhealth.com/journals/jinf

CASE REPORT

Detection of CTX-M-14 extended-spectrum b-lactamase in Shigella sonnei isolates from China Zizhong Xiong a,b, Tao Li c, Yuanhong Xu c, Jun Li b,* a

Department of Infectious Diseases, The First Affiliated Hospital, Anhui Medical University, Hefei 230022, China School of Pharmacy, Anhui Medical University, Hefei 230032, China c Clinical Laboratory, The First Affiliated Hospital, Anhui Medical University, Hefei 230022, China b

Accepted 19 July 2007 Available online 4 September 2007

KEYWORDS Shigella sonnei; Resistance; Antimicrobial agents; Extended-spectrum b-lactamases

Summary Shigellosis is an important cause of acute diarrheal disease and multidrug-resistant phenotype has been reported in S. sonnei. In this study, we investigate the resistance and identify extended-spectrum b-lactamases (ESBLs) gene in 37 S. sonnei isolates by agar dilution procedure and the modified three-dimensional test, respectively. The bla genes of ESBL-producing isolates were detected by polymerase chain reaction (PCR) and sequencing. More than 50% of these strains were resistant to tetracycline, sulfamethoxazole-trimethoprim, ampicillin, ampicillin-sulbactam, or gentamicin. However, they were still susceptible to third generation cephalosporins, fluoroquinolones, and chloramphenicol. A total of 8.1% (3/37) of the isolates with intermediate susceptibility to ceftriaxone and cefotaxime were ESBL-producers, which produced CTX-M-14 ESBLs and TEM-1 b-lactamases. This is the first report of CTX-M-14 in S. sonnei isolates from China and it is important to closely monitor such strains. ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction Shigellosis is an important cause of acute diarrheal disease in both developing and industrialized countries.1 Shigella flexneri was the most predominant Shigella species in China, but Shigella sonnei has been more and more common, recently. Antimicrobial resistance of Shigella species

* Corresponding author. Tel./fax: þ86 551 516 1001. E-mail address: [email protected] (J. Li).

is increasing because of antibacterial agents used widely in clinical medicine.2e4 Multidrug-resistant (MDR) phenotype has been reported in S. sonnei, and a few of those strains were resistant to third generation cephalosporins, such as ceftriaxone, cefotaxime, in which antimicrobial therapy against shigellosis has become very limited.5 In the study described here, we have investigated the antimicrobial susceptibility of S. sonnei isolated from Outpatient Clinic of The First Affiliated Hospital, Anhui Medical University, Hefei, China from May to October, 2004. The extendedspectrum b-lactamase (ESBL), CTX-M-14, was determined in those strains.

0163-4453/$30 ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2007.07.017

e126 Table 1

Z. Xiong et al. Primers for PCR

Primer

Sequence

TEM universal primers

P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 P2 P1 P2 P1 P2

SHV universal primers CTX-M-1group universal primers CTX-M-2group universal primers CTX-M-9group universal primers

TEM entire gene primers CTX-M-9group entire gene primers

Case report Thirty-seven S. sonnei isolates were collected from patients in Outpatient Clinic of The First Affiliated Hospital, Anhui Medical University, Hefei, China from May to October, 2004. There were no replicate strains in this study. All patients came from the region of Hefei, China and have community acquired diarrheal disease. Antibiotic susceptibility of S. sonnei isolates were determined by standard agar dilution procedure6 on Mueller-Hinton agar (Oxoid, Basingstoke, UK), and Escherichia coli ATCC 25922 (susceptible strain), Klebsiella pneumoniae ATCC 700603 (ESBL-producing strain) were included for quality control. Results were interpreted according to NCCLS standards.6 Antimicrobial agents for susceptibility test included tetracycline, chloramphenicol, sulfamethoxazole-trimethoprim, ciprofloxacin, gentamicin, ampicillin, cefotaxime, ceftriaxone, ceftazidime (National Institute for Control of Pharmaceutical and Biological Products, Beijing, China), ampicillin-sulbactam (Pfizer, USA), levofloxacin (Daiichi Seiyaku, Tokyo, Japan), norfloxacin (The Second Shanghai Pharmaceutical Company, Shanghai,

Table 2

50 -TGCGGTATTATCCCGTGTTG-30 50 -TCGTCGTTTGGTATGGCTTC-30 50 -TCTCCCTGTTAGCCACCCTG-30 50 -CCACTGCAGCAGCTGC(A/C)GTT-30 50 -ACAGCGATAACGTGGCGATG-30 50 -TCGCCCAATGCTTTACCCAG-30 50 -TGGAAGCCCTGGAGAAAAGT-30 50 -CTTATCGCTCTCGCTCTGTT-30 50 -CTGCTTAATCAGCCTGTCGA-30 50 -TCAGTGCGATCCAGACGAAA-30 50 -TCTTACTTCGCCAACTTCT-30 50 -CCCTGGTAAATGCTTC-30 50 -GAGTAAACTTGGTCTG-30 50 -CGAAGCAGTCTAAATTCTTCGTGAAATAG-30 50 -GGGCCAGTTGGTGATTTGA-30

China). ESBLs producing strains were confirmed by the modified three-dimensional test described previously,7 and Ceftriaxone (30 mg), cefotaxime (30 mg), and cefoxitin (30 mg) disc (Oxoid, Basingstoke, UK) were used for the test. Partial bla genes of ESBL-producing isolates were detected by PCR using universal primers for TEM, SHV, CTX-M-1group, CTXM-2group, CTX-M-9group, respectively.8,9 If the partial genes were positive, entire genes were also tested by PCR.9,10 All primers for PCR were shown in Table 1. The PCR was performed using a commercially available PCR kit (Takara, Dalian, China) and the Biometra PCR Thermal Cycler (Germany). Standard strains encoding different b-lactamase (TEM-1, SHV-18, CTX-M-3, Toho-1, CTX-M-24) employed as positive controls and E. coli ATCC25922 as the negative control. PCR product (5 ml) was subjected to electrophoresis on 1.2% agarose gel (Invitrogen, California, USA) to identify the amplified DNA fragment. The purified PCR products of entire genes from the isolates were sequenced with an ABI 3100 genetic analyzer (Shanghai Biotechnology Company, Shanghai, China) and continued by primers walking on both DNA strands. For sequence comparison, the NCBI BLAST program

Susceptibility of Shigella sonnei isolates to antimicrobial agents

Antimicrobial agent

Shigella sonnei (n Z 37) R

Tetracycline Sulfamethoxazole-trimethoprim Chloramphenicol Levofloxacin Ciprofloxacin Norfloxacin Gentamicin Ampicillin Ampicillin-sulbactam Ceftriaxone Cefotaxime Ceftazidime R, resistant; I, intermediate; and S, susceptible.

97.3% 94.6% 2.7% 0% 0% 0% 62.2% 64.9% 51.4% 0% 0% 0%

I (36/37) (35/37) (1/37) (0/37) (0/37) (0/37) (23/37) (24/37) (19/37) (0/37) (0/37) (0/37)

0% 0% 0% 2.7% 2.7% 2.7% 0% 0% 10.8% 8.1% 8.1% 0%

S (0/37) (0/37) (0/37) (1/37) (1/37) (1/37) (0/37) (0/37) (4/37) (3/37) (3/37) (0/37)

2.7% 5.4% 97.3% 97.3% 97.3% 97.3% 37.8% 35.1% 37.8% 91.9% 91.9% 100%

(1/37) (2/37) (36/37) (36/37) (36/37) (36/37) (14/37) (13/37) (14/37) (34/37) (34/37) (37/37)

ESBL in Shigella sonnei Table 3

e127

Susceptibility of 3 ESBL-producing isolates of Shigella sonnei to antimicrobial agents

Antimicrobial agent

Ampicillin Ampicillin-sulbactam Ceftriaxone Cefotaxime Ceftazidime Ciprofloxacin Levofloxacin Norfloxacin Gentamicin Chloramphenicol Tetracycline Sulfamethoxazole-trimethoprim

MIC (mg/L) Shigella sonnei 04-1446

Shigella sonnei 04-1650

Shigella sonnei 04-1768

>128 128 32 16 0.5 0.125 0.5 0.5 >128 4 >128 >1216/64

>128 64 32 16 0.5 0.125 0.25 0.5 64 4 64 >1216/64

>128 64 32 32 0.5 0.25 0.25 0.5 >128 8 >128 >1216/64

and facilities of the TIGR Comprehensive Microbial Resource (http://www.tigr.org) were utilized. About 97.3%, 94.6%, 64.9%, 51.4%, and 62.2% of the 37 S. sonnei isolates were resistant to tetracycline, sulfamethoxazole-trimethoprim, ampicillin, ampicillin-sulbactam, and gentamicin respectively. Most strains were susceptible to third generation cephalosporins, fluoroquinolones, chloramphenicol, but 3 of 37 (8.1%) isolates were intermediate to third generation cephalosporins (Tables 2 and 3). According to the result of the modified three-dimensional test, the 3 isolates were ESBL-producing strains. Results of PCR and sequencing revealed that the 3 isolates carried TEM-1, CTX-M-14 genes.

Discussion The overuse and misuse of antimicrobial agents in treatment of diarrhea have lead to an increase of antimicrobial resistance, which is becoming a critical area of concern in clinic, especially in developing countries.5 In this study, results of susceptibility testing showed that S. sonnei isolates were multidrug resistant, especially to tetracycline, gentamicin, sulfamethoxazole-trimethoprim, ampicilin, and ampicilin-sulbactam (more than 50% were resistant). Only third generation cephalosporins, fluoroquinolones, and chloramphenicol showed high activity and could be used for the treatment of the infection caused by those strains. In this locality, third generation cephalosporins are generally used in the treatment of severe shigellosis cases, but resistance to third generation cephalosporins in S. sonnei was recently reported abroad5 and we also detected 3 S. sonnei isolates with intermediate susceptibility to ceftriaxone and cefotaxime in our study, which strongly suggested the production of ESBLs in those strains. ESBLs are the major mechanism for resistance to third generation cephalosporins, such as cefotaxime, and aztreonam, and have been found in Enterobacteriaceae worldwide, especially in E. coli and K. pneumonia.11 Only a few cases of ESBL production have been reported in Shigella species,5 but results susceptibility testing and the modified three-dimensional test in present study showed that 3 S. sonnei isolates produced ESBLs, which were resistant to most b-lactams. PCR and sequencing confirmed that these isolates produced CTX-M-14 ESBL and

TEM-1 b-lactamase. The CTX-M group, a new family of plasmid-mediated ESBLs, increasingly reported from many countries, preferentially hydrolyzes cefotaxime.11,12 So far, CTX-M-3 and CTX-M-14 were the most prevalent type of ESBLs in China.8e10 In this study, we also detected CTXM-14 in S. sonnei isolates from patients in Outpatient Clinic of our hospital. To our knowledge, this is the first report in China (the nucleotide sequence reported here has been registered with the EMBL database under accession no. DQ350884). Although CTX-M-14 possess the 70SXXK73, 130 SDN132, E-166, 234KTG236 sequences that are typical of class A enzymes, it contains no Lys-240 or Arg-240 acid residue which cause ceftazidime resistance.10,12,13 Various surveys have reported ESBL-producing strains in nosocomial infections and these organisms are also isolated with increasing frequency from patients with community-onset disease14,15 like our study here. Although ESBL-producing strains comprise less than 10% of all S. sonnei isolates in the present study, they can pose a significant threat to patients and presents a challenge for disease management, especially with S. sonnei as the increasing cause of shigellosis.

Acknowledgments We thank Professor Demei Zhu for her assistance in antimicrobial susceptibility testing. This research was supported by grants from Natural Science Fund of Anhui Province (050430804) and the Department of Education of Anhui Province (KJ2007B145), China, China Postdoctoral Science Fund (20060400202), and Postdoctoral Science Fund of Anhui Province, China (2006e8).

Reference 1. Turner SA, Luck SN, Sakellaris H, Rajakumar K, Adler B. Role of attP in integrase-mediated integration of the Shigella resistance locus pathogenicity island of Shigella flexneri. Antimicrob Agents Chemother 2004;48:1028e31. 2. Nguyen TV, Le PV, Le CH, Weintraub A. Antibiotic resistance in diarrheagenic Escherichia coli and Shigella strains isolated from children in Hanoi, Vietnam. Antimicrob Agents Chemother 2005;49:816e9.

e128 3. Salam MA, Bennish ML. Antimicrobial therapy for shigellosis. Rev Infect Dis 1991;13:S332e41. 4. Vasilev V, Japheth R, Yishai R, Andorn N. Antimicrobial resistance of Shigella flexneri serotypes in Israel during a period of three years: 2000e2002. Epi Infect 2004;132:1049e54. 5. Kim S, Kim J, Kang Y, Park Y, Lee B. Occurrence of extendedspectrum b-lactamases in members of the genus Shigella in the Republic of Korea. J Clin Microbiol 2004;42:5264e9. 6. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing: ninth information supplement (M100eS14). National Committee for Clinical Laboratory Standards, Wayne. Pa 2004. 7. Shahid M, Malik A, Agrawal M, Singhal S. Phenotypic detection of extended-spectrum and AmpC b-lactamases by a new spotinoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test. J Antimicrob Chemother 2004;54:684e7. 8. Xiong Z, Zhu D, Wang F, Zhang Y, Okamoto R, Inoue M. Investigation of extended-spectrum b-lactamase in Klebsiellae pneumoniae and Escherichia coli from China. Diagn Microbiol Infect Dis 2002;44:195e200. 9. Xiong Z, Zhu D, Wang F, Zhang Y, Okamoto R, Inoue M. A Klebsiellae pneumoniae producing three kinds of class A b-lactamases

Z. Xiong et al.

10.

11.

12.

13.

14.

15.

encoded by one single plasmid isolated from a patient in Huashan Hospital, Shanghai, China. Int J Antimicrob Agents 2004;23:262e7. Xiong Z, Zhu D, Wang F, Zhang Y. CTX-M-14, CTX-M-24 and resistance in Escherichia coli and Klebsiellae pneumoniae clinical isolates. Chin Med J 2006;119:160e4. Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14: 933e51. Du Bois SK, Marriot MS, Amyes SG. TEM- and SHV-derived extendedspectrum b-lactamases: relationship between selection, structure and function. J Antimicrob Chemother 1995;35:7e22. Bonnet R. Growing group of extended-spectrum b-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004;48: 1e14. Pai HE, Choi E, Lee H, Hong JY, Jacoby GA. Identification of CTX-M-14 extended spectrum b-lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J Clin Microbiol 2001;39:3747e9. Pitout JDD, Gregson DB, Church DL, Elsayed S, Laupland KB. Community-wide outbreaks of clonally related CTX-M-14 b-lactamase-producing Escherichia coli strains in the Calgary health region. J Clin Microbiol 2005;43:2844e9.