jueves, 18 de agosto de 2016

Multidrug-Resistant Shigella Infections in Patients with Diarrhea, Cambodia, 2014–2015 - Volume 22, Number 9—September 2016 - Emerging Infectious Disease journal - CDC

Multidrug-Resistant Shigella Infections in Patients with Diarrhea, Cambodia, 2014–2015 - Volume 22, Number 9—September 2016 - Emerging Infectious Disease journal - CDC

Volume 22, Number 9—September 2016


Multidrug-Resistant Shigella Infections in Patients with Diarrhea, Cambodia, 2014–2015

Kamonporn Poramathikul, Ladaporn BodhidattaComments to Author , Sivhour Chiek, Wilawan Oransathid, Sirigade Ruekit, Panida Nobthai, Woradee Lurchachaiwong, Oralak Serichantalergs, Chanthap Lon, and Brett Swierczewski
Author affiliations: Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand (K. Poramathikul, L. Bodhidatta, W. Oransathid, S. Ruekit, P. Nobthai, W. Lurchachaiwong, O. Serichantalergs, B. Swierczewski);Battambang Provincial Referral Hospital, Battambang, Cambodia (S. Chiek)Armed Forces Research Institute of Medical Sciences, Phnom Penh, Cambodia (C. Lon)


We observed multidrug resistance in 10 (91%) of 11 Shigella isolates from a diarrheal surveillance study in Cambodia. One isolate was resistant to fluoroquinolones and cephalosporins and showed decreased susceptibility to azithromycin. We found mutations in gyrA, parC, β-lactamase, and mphA genes. Multidrug resistance increases concern about shigellosis treatment options.
Shigellosis is a major public health problem in developing countries. Antimicrobial therapy with fluoroquinolones is recommended to shorten the course of disease and fecal shedding. However, limitations on shigellosis treatment options have been a concern since 1993, when ciprofloxacin-resistant Shigella was documented (1), followed by reports of multidrug-resistant (MDR) Shigella and of Shigella that harbored extended-spectrum β-lactamase (ESBL) genes (2). We describe MDR Shigella isolated from patients with diarrhea in Cambodia during 2014–2015.

The Study

During July 2014–April 2015, we examined stool specimens collected from patients 3 months–5 years of age and 18–60 years of age who were seen for or admitted with acute diarrhea at 3 healthcare settings in Battambang, Cambodia, as part of ongoing hospital-based surveillance of diarrhea etiology. Stool specimens were processed for identification of enteric pathogens by standard microbiology, ELISA, and PCR. Shigella species were identified by standard biochemical tests and the API 20E system (bioMérieux, Marcy l’Étoile, France) and serotyped by commercial antisera (Denka Seiken Co, Ltd., Tokyo, Japan). Antimicrobial drug susceptibility testing was performed with the standard Kirby-Bauer disk diffusion method by using commercially available antimicrobial disks (Becton Dickinson, Franklin Lakes, NJ, USA). Antimicrobial drugs tested for susceptibility were ampicillin, azithromycin (AZM), cefotaxime (CTX), ceftazidime (CAZ), ceftriaxone (CRO), ciprofloxacin (CIP), nalidixic acid (NAL), tetracycline, and trimethoprim/sulfamethoxazole. Susceptibility results were interpreted according to Clinical and Laboratory Standards Institute guidelines (3). We used zone diameter interpretive standards for Enterobacteriaceae for all antimicrobial drugs tested, except AZM, for which we applied the standard for Staphylococcus spp.
Shigella spp. were isolated from 11 (5%) of 212 diarrhea stool samples. Antimicrobial drug susceptibility testing showed that 10 (91%) of the 11 Shigellaisolates were resistant to ampicillin, tetracycline, trimethoprim/sulfamethoxazole, and NAL. We selected the 10 MDR isolates for further characterization and determined MICs of AZM and CIP by Etest (bioMérieux). ESBL production was tested by using Neg Combo Panel Type 50 on the MicroScan WalkAway plus System (Siemens Healthcare Diagnostics, Newark, DE, USA). PCR and sequencing were used to characterize resistance genes (gyrA and parC) in the quinolone-resistance determining region (QRDR), the AZM resistance gene (mphA), and β-lactamase genes (47).
Of the 10 MDR isolates, 2 were S. flexneri 2a; 1 was an S. flexneri 2 variant; 6 were S. flexneri 3a; and 1 was S. sonnei (Table 1). CIP resistance was detected in 5 (50%) of the 10 isolates. Sequence analysis showed mutations of gyrA and parC genes with the amino acid substitutions in the QRDR (Table 2). All NAL-resistant isolates susceptible to CIP had a single mutation in gyrA. Isolates resistant to both NAL and CIP contained multiple mutations in gyrA andparC.
The most common mechanism of quinolone resistance in the Shigella spp. was mutation of gyrA, typically at codon 83 or 87, and of parC at codon 80 (7). All isolates in our study had the common mutation in gyrA at position 83 (Ser83→Leu); 1 isolate had another common mutation at position 87 (Asp87→Gly). A mutation in parC at position 80 (Ser80→Ile), detected in the S. sonnei isolate, was previously reported in an Sdysenteriae serotype 1 isolate in India (7) and in Asia travel-associated S. sonnei and S. flexneri isolates in the United States (8). A mutation at position 57 (Ser57→Arg) was detected in all 4 CIP-resistant S. flexneri 3a isolates, but this mutation’s role in CIP resistance is unclear because position 57 is outside the QRDR region. Characterization of plasmid-mediated quinolone resistance (PMQR) genes should be further investigated because coexistence of mutations in the QRDR and PMQR genes has been reported in Shigella isolates with decreased susceptibility to fluoroquinolones (8). PMQR may facilitate the selection of QRDR mutations, resulting in higher levels of quinolone resistance.
No clinical breakpoints for AZM have been clearly defined for Shigella spp., but CDC’s National Antimicrobial Resistance Monitoring System for Enteric Bacteria (http://www.cdc.gov/narms/index.html) recommends using the term “decreased susceptibility” for reporting. We detected decreased susceptibility to AZM in S. flexneri 3a (isolate no. 9) with a MIC of 32 µg/mL. This isolate was found to carry the mphA gene encoding a macrolide 2′-phosphotransferase that inactivates macrolide antimicrobial drugs and has been reported to reduce AZM susceptibility in Shigella isolates (5). Emergence of decreased susceptibility to AZM may affect treatment options for shigellosis, especially for pediatric cases because ceftriaxone is administered parenterally by injection and fluoroquinolones are not encouraged for use in children.
We detected >1 β-lactamase gene in all 10 Shigella isolates; 2 isolates that were resistant to cephalosporins revealed ESBL production (Table 2). The 8 isolates that carried β-lactamase–producing genes TEM-1 or TEM-1 and OXA-1 were cephalosporin susceptible, suggesting that TEM-1 and OXA-1 may not play a role in increased resistance to third-generation cephalosporins. Of the remaining 2 isolates, 1 S. flexneri (isolate no. 9), which harbored CTX-M-27 and TEM-1, showed resistance to CRO and CTX but not to CAZ, and 1 S. sonnei (isolate no. 10), which carried CTX-M-55, was resistant to all cephalosporins tested.
A key element that increased ceftazidimase activity was a single amino acid substitution from Asp to Gly at position 240; this substitution was identified in CTX-M-15, CTX-M-16, CTX-M-27, and CTX-M-32. CTX-M-27, first reported from France in 2003, differed from its parental enzyme, CTX-M-14, by substitution of Asp240Gly (9). Reports suggest that this Gly-240–harboring CTX-M-27 confers higher levels of resistance to CAZ in Escherichia coliinfections, but we did not detect this characteristic in the Shigella isolates we examined. CTX-M-55 was first reported in ESBL-producing E. coli andKlebsiella pneumoniae isolates in Thailand in 2007; it was associated with high resistance to CRO, CTX, and CAZ (10) and was subsequently reported in other Asia countries, including Cambodia. Among fecal samples collected from children in Cambodia, 88% carried E. coli harboring ESBL genes containingblaCTX-M variants, including CTX-M-15, CTX-M-55, and CTX-M-14 (11). A case of ESBL-producing S. sonnei harboring CTX-M-55 was also reported in a woman traveling from Korea to China (12).
The CDC Health Alert Network has distributed a health advisory on CIP- and AZM-nonsusceptible Shigella infection in the United States (13). Three separate outbreaks of MDR shigellosis among men who have sex with men, international travelers, and children in daycare centers have been reported (13). We found 2 ESBL-producing, fluoroquinolone-resistant Shigella isolates. Moreover, S. flexneri 3a (isolate no. 9), which had decreased susceptibility to AZM, was also resistant to nearly all oral and parenteral drugs considered for shigellosis treatment. This isolate can ferment sorbitol, a feature found in 7% of Shigella spp. and possibly causing misidentification of Shigella spp. as other species (14). S. sonnei (isolate no. 10) belongs to biotype g (i.e., with biochemical reactions ONPG+ [o-nitrophenyl-β-D-galactopyranose], rhamnose–, and xylose–), which has been shown to carry integrons with multiple gene cassettes, leading to multidrug resistance (15).


MDR Shigella is an emerging problem that raises concern about shigellosis treatment worldwide, including in Cambodia. Health authorities should implement systematic surveillance of antimicrobial drug resistance and controlled antimicrobial drug use to increase understanding of the problem and minimize unnecessary antimicrobial drug use, which contributes to increased resistance.
Ms. Poramathikul is a medical research technologist in the bacteriology section, Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. Her interests include the surveillance of enteric pathogens and antimicrobial drug resistance.


We thank David Saunders, Carl J. Mason, and Sovann Ly for supervision of this surveillance study. We also thank Samon Nou for subject enrollment and Chhoeun Sophal and Poch Vathna for laboratory technical assistance.
This work is supported by the Global Emerging Infectious Disease Surveillance, a division of the Armed Forces Health Surveillance Center.


  1. Horiuchi SInagaki YYamamoto NOkamura NImagawa YNakaya RReduced susceptibilities of Shigella sonnei strains isolated from patients with dysentery to fluoroquinolones. Antimicrob Agents Chemother1993;37:24869DOIPubMed
  2. Zaidi MBEstrada-García TShigella: a highly virulent and elusive pathogen. Curr Trop Med Rep2014;1:817.PubMed
  3. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement (M100–S25). Wayne (PA): The Institute; 2015.
  4. Dallenne CDa Costa ADecré DFavier CArlet GDevelopment of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother2010;65:4905DOIPubMed
  5. Howie RLFolster JPBowen ABarzilay EJWhichard JMReduced azithromycin susceptibility in Shigella sonnei, United States. Microb Drug Resist.2010;16:2458DOIPubMed
  6. Giraud EBrisabois AMartel JLChaslus-Dancla EComparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field. Antimicrob Agents Chemother1999;43:21317.PubMed
  7. Dutta SKawamura YEzaki TNair GBIida KYoshida SAlteration in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant Shigella dysenteriae serotype 1 clinical isolates from Kolkata, India. Antimicrob Agents Chemother2005;49:16601DOIPubMed
  8. Folster JPPecic GBowen ARickert RCarattoli AWhichard JMDecreased susceptibility to ciprofloxacin among Shigella isolates in the United States, 2006 to 2009. Antimicrob Agents Chemother2011;55:175860DOIPubMed
  9. Bonnet RRecule CBaraduc RChanal CSirot DDe Champs CEffect of D240G substitution in a novel ESBL CTX-M-27. J Antimicrob Chemother.2003;52:2935DOIPubMed
  10. Kiratisin PApisarnthanarak ASaifon PLaesripa CKitphati RMundy LMThe emergence of a novel ceftazidime-resistant CTX-M extended-spectrum beta-lactamase, CTX-M-55, in both community-onset and hospital-acquired infections in Thailand. Diagn Microbiol Infect Dis.2007;58:34955DOIPubMed
  11. Stoesser NSheppard AEMoore CEGolubchik TParry CMNget PModernizing Medical Microbiology Informatics GroupExtensive within-host diversity in fecally carried extended-spectrum-beta-lactamase-producing Escherichia coli isolates: implications for transmission analyses. J Clin Microbiol2015;53:212231DOIPubMed
  12. Lee WChung HSLee HYum JHYong DJeong SHCTX-M-55-type extended-spectrum β-lactamase-producing Shigella sonnei isolated from a Korean patient who had travelled to China. Ann Lab Med2013;33:1414DOIPubMed
  13. Centers for Disease Control and Prevention. Ciprofloxacin- and azithromycin-nonsusceptible shigellosis in the United States. 2015 Jun 4 [cited 2015 Oct 6]. http://emergency.cdc.gov/han/han00379.asp
  14. BioMérieux SA. Apiweb technical brochure: biochemical information for identification software. API/ID 32 database update. APIWEB Ref. 40 011 Version 1.3.0 Update. 2015 [cited 2016 Jun 30].https://techlib.biomerieux.com/wcm/techlib/techlib/documents/docLink/User_Manual_Supplements/40096001-40097000/User_Manual_Supplements_-_161150-295_-_A_-_en_-_APIWEB_-_Technical_Brochure.pdf
  15. Ud-Din AIWahid SULatif HAShahnaij MAkter MAzmi IJChanging trends in the prevalence of Shigella species: emergence of multi-drug resistant Shigella sonnei biotype g in Bangladesh. PLoS One2013;8:e82601DOIPubMed


DOI: 10.3201/eid2209.152058

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