Extended-spectrum ß-lactamase-mediated third-generation cephalosporin resistance in Shigella isolates in Bangladesh

Mahbubur Rahman*, Shereen Shoma, Harunur Rashid, A. K. Siddique, G. B. Nair and D. A. Sack

ICDDR,B: Centre for Health and Population Research, GPO Box 128, Dhaka 1000, Bangladesh

Keywords: ESBLs , multidrug resistance , ceftriaxone , shigellosis

Sir,

Emergence of multidrug-resistant (MDR) strains of Shigella is a growing concern across the globe.1 Third-generation cephalosporins are used for treating infections caused by MDR Shigella.2 However, resistance to third-generation cephalosporins due to extended-spectrum ß-lactamases (ESBLs), which confer resistance to all ß-lactams except cephamycins and carbapenems, has emerged as a new problem.3 Although >150 different ESBLs have been described worldwide in bacteria, there are only six case reports on ESBL-producing Shigella species from six countries and no report in Bangladesh.35 In this study, we report for the first time in Bangladesh the presence of ESBL- and AmpC-like ß-lactamase-mediated ceftriaxone resistance in clinical isolates of MDR Shigella.

The strains were isolated from patients with shigellosis by standard methods, in Dhaka, Bangladesh during 2001–2002. Antimicrobial susceptibility was carried out by the disc diffusion method according to NCCLS guidelines. MICs of antibiotics were determined by Etest (AB Biodisk, Solna, Sweden). The ESBL was detected by the double disc diffusion synergy test (DDST).3 Conjugal transfer of resistance plasmids to nalidixic acid-resistant Escherichia coli K-12 was carried out and transconjugants were selected on MacConkey agar containing ceftriaxone (6 mg/L). In a second approach, conjugal transfer of resistance plasmids to a ceftriaxone-susceptible and trimethoprim/sulfamethoxazole-resistant wild-type Shigella sonnei isolate was carried out. Like wild-type strains, all transconjugants were tested for antimicrobial susceptibility, plasmid profiles and ESBL production.

Of 160 Shigella isolates (82 S. flexneri, 31 S. boydii, 21 S. dysenteriae and 26 S. sonnei) tested, 90% were resistant to one or more drugs. The resistance rates were: ampicillin (60%), trimethoprim/sulfamethoxazole (75%), tetracycline (80%), azithromycin (62%), nalidixic acid (58%), mecillinam (3%) and ceftriaxone/cefixime (2.5%). Four of 96 ampicillin-resistant isolates were resistant to ceftriaxone and cefixime; one isolate was resistant and 42 isolates (44% of 96) were intermediate to co-amoxiclav. Of the four ceftriaxone-resistant isolates, two S. sonnei were highly resistant (MIC >256 mg/L) whereas the other two isolates (S. boydii and S. flexneri) exhibited distinctly lower MICs of ceftriaxone (24 mg/L). All four isolates were MDR (Table 1) and exhibited decreased ciprofloxacin susceptibility (MIC = 0.125–0.5 mg/L). Three ceftriaxone-resistant isolates (two S. sonnei and one S. boydii) were positive by DDST and were resistant to ß-lactams, but susceptible to ß-lactam/ß-lactamase-inhibitor combination, cefoxitin, aztreonam and imipenem indicating the presence of a class A ESBL (Bush group 2). In contrast, the S. flexneri isolate was resistant to co-amoxiclav, aztreonam and cefoxitin, but susceptible to imipenem and negative in DDST, thus suggesting the presence of an AmpC-like ß-lactamase (Bush group 1).3 ß-Lactamase production was transferred to E. coli and S. sonnei from all four ceftriaxone-resistant isolates. The first-generation E. coli and second-generation S. sonnei transconjugants harboured a 50 MDa R plasmid, as did three parent ESBL-producing strains. However, the gene coding for the AmpC-like ß-lactamase was located on a large 94 MDa plasmid in S. flexneri and its transconjugants. The ß-lactamase phenotype was transferred at a frequency of 10–4, the transconjugants became resistant to ß-lactams and remained susceptible to other drugs (Table 1).


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Table 1. Characteristics of ceftriaxone-resistant Shigella isolates

 
The first SHV-11 ESBL-producing S. dysenteriae strain was recorded in India in 1999.4 We report here an ESBL-producing S. boydii, and an AmpC-like ß-lactamase-producing S. flexneri detected for the first time in Bangladesh. The two other ESBL-producing S. sonnei isolates were also detected for the first time in Bangladesh. Though we have not characterized these ESBLs at the molecular level, the resistance phenotypes of S. sonnei and S. boydii seem to suggest the ESBLs as CTX-M types and the ß-lactamase of S. flexneri as AmpC type.

ESBL-producing MDR Shigella spp. pose an important threat in the treatment of dysentery especially in children. Infections caused by MDR which carry plasmid-borne genes for ESBLs or AmpC-like ß-lactamases should be considered as a warning message to limit irrational use of antibiotics. Recently, fluoroquinolone (ciprofloxacin)-resistant S. dysenteriae type 1 strains were reported from India and Bangladesh further complicating the treatment options.6 Notably, ceftriaxone-resistant strains in our study had increased MICs of ciprofloxacin, which is the first step to complete resistance. The patient suffering from an infection caused by the ceftriaxone-resistant S. flexneri isolate recovered completely during ciprofloxacin therapy, and pivmecillinam (oral form of mecillinam) was effective for the other three cases. An outbreak of shigellosis by such strains or their widespread dissemination in the community will represent a therapeutic challenge in the near future for treating shigellosis.

Acknowledgements

The work was supported by the United States Agency for International Development, Washington, DC, USA.

Footnotes

* Corresponding author. Tel: +880-2-8811751; Fax: +880-2-8812529; Email: mahbubur{at}icddrb.org

References

1 . Sack, R. B., Rahman, M., Yunus, M. et al. (1997). Antimicrobial resistance in organisms causing diarrheal diseases. Clinical Infectious Diseases 24, Suppl. 1, S102–5.[ISI][Medline]

2 . Varsano, I., Elditz-Marcus, T., Nussinotich, M. et al. (1991). Comparative efficacy of ceftriaxone and ampicillin for treatment of severe shigellosis in children. Journal of Pediatrics 118, 627–32.[ISI][Medline]

3 . Bradford, P. A. (2001). Extended-spectrum ß-lactamases in the 21st century: characterisation, epidemiology and detection of this important resistant threat. Clinical Microbiology Reviews 14, 933–51.[Abstract/Free Full Text]

4 . Ahmed, J. & Kundu, M. (1999). Molecular characterization of the SHV-11 ß-lactamase of Shigella dysenteriae. Antimicrobial Agents and Chemotherapy 43, 2081–3.[Abstract/Free Full Text]

5 . Ziya, C. A., Zeynep, G., Meral, B. et al. (2003). CTX-M-3 extended-spectrum ß-lactamase in a Shigella sonnei clinical isolate: first report from Turkey. Scandinavian Journal of Infectious Diseases 35, 503–5.[CrossRef][ISI][Medline]

6 . Bhattacharya, S. K., Sarkar, K., Nair, G. B. et al. (2003). Multidrug-resistant Shigella dysenteriae type 1 in south Asia. Lancet Infectious Diseases 3, 755.[CrossRef][ISI][Medline]





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