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We characterized 20 Shigella isolates with decreased susceptibility to fluoroquinolones. Most patients (80%) from whom a travel history was obtained reported travel to South or Southeast Asia. Mutations within the quinolone resistance determining regions of gyrA and parC and plasmid-mediated resistance determinants (qnrB, qnrS, and aac(6′)-Ib-cr) were identified. The rise in antimicrobial resistance among Shigella isolates may necessitate the increased use of extended-spectrum cephalosporins or macrolides in some patients.
Shigellosis is a major source of gastroenteritis throughout the world, and severe infections may require antimicrobial treatment (16). Antimicrobial treatment can shorten the duration and severity of illness (12). However, the emergence of multidrug resistance has made the selection of effective antimicrobial therapy more difficult (1). Ampicillin and trimethoprim-sulfamethoxazole resistance is highly prevalent among Shigella strains in the United States and may necessitate the use of extended-spectrum cephalosporins or fluoroquinolones for the treatment of shigellosis (20). Recently, extended-spectrum cephalosporin resistance in Shigella has begun to emerge in the United States, threatening to leave fluoroquinolones as the mainstay of treatment. Importantly, fluoroquinolone resistance has been rising among Shigella strains, especially in Asia (4, 15). In this study, we characterize Shigella isolates collected in the United States with decreased susceptibility to ciprofloxacin.
State public health laboratories that participate in the National Antimicrobial Resistance Monitoring System (NARMS) submit every 20th Shigella isolate to the CDC for susceptibility testing. Broth microdilution (Sensititre; Trek Diagnostics, Westlake, OH) is used to determine the MIC for a panel of 15 antimicrobials. From 2006 to 2009, 2,026 routine surveillance Shigella isolates were submitted. Twenty isolates displayed decreased susceptibility (MIC ≥ 0.25 μg/ml) to the fluoroquinolone ciprofloxacin. Fifteen of these isolates (75%) were resistant (MIC ≥ 32 μg/ml) to the quinolone nalidixic acid (Table (Table1).1). Among the isolates, 10 were Shigella flexneri, six S. sonnei, three S. boydii, and one S. dysenteriae. The patients included 13 males, 5 females, and 2 for whom the sex was unknown; patients resided in 15 different states. The ages ranged from 1 to 76 years; the median age was 30.5 years. Additional information was available for 10 patients. Eight patients (80%) reported recent travel to India or Thailand. Nine patients reported diarrhea, and four reported fever and vomiting. None of the isolates were part of any recognized Shigella outbreaks.
Decreased susceptibility to ciprofloxacin among the Enterobacteriaceae is usually due to the acquisition of mutations in the quinolone resistance determining regions (QRDR) of the DNA gyrase (gyrA) and topoisomerase IV (parC) genes and/or plasmid-mediated quinolone resistance (PMQR) determinants, such as target protection genes (qnr), aminoglycoside acetyltransferase variants [aac(6′)-Ib-cr], or efflux genes (qepA). PCR was used to amplify the gyrA and parC genes from each isolate, and the QRDR region was sequenced (5). Fifteen isolates contained QRDR mutations (Table (Table1).1). All 15 had the common first-step gyrA mutation (Ser83Leu), 8 had a second gyrA mutation (Asp87Asn/Gly), and 13 had the common second-step parC mutation (Ser80Ile). The isolates containing QRDR mutations were nalidixic acid resistant, and nine were resistant to ciprofloxacin (≥4 μg/ml). PCR analysis was used to screen all the isolates for PMQRs [qnrA, qnrB, qnrD, qnrS, aac(6′)-Ib-cr, and qepA] (3, 14, 15, 19). Six isolates were PCR positive for qnr genes (four qnrS genes and two qnrB genes), and one qnrB-positive isolate was also positive for the aac(6′)-Ib gene (Table (Table2).2). Sequencing analysis determined that all four qnrS genes were qnrS1, while isolate AM29213 contained the qnrB19 gene. Isolate AM38988 contained the qnrB6 gene and the aac(6′)-Ib-cr variant (7, 18).
To determine the location of the PMQR genes, plasmids were purified from the six isolates and transferred by electroporation into Escherichia coli DH10B using 0.2 μg/ml of ciprofloxacin for selection (6). PCR analysis confirmed that all PMQRs transferred and the qnrB6 gene was located on the same plasmid as the aac(6′)-1b-cr gene. The transformants demonstrated higher MICs to nalidixic acid and ciprofloxacin than untransformed DH10B, and most of the plasmids conferred resistance to additional antimicrobial drugs, including ampicillin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline (Table (Table22).
PCR-based inc/rep typing (PBRT) and plasmid sizing determined that the qnrB19 plasmid was a 7-kb ColEPB plasmid, while the qnrB6 plus aac(6′)-1b-cr plasmid was a 60-kb IncN and ColE-type plasmid (2, 7, 13). Of the four remaining plasmids, two were 60 kb and two were 80 kb in size, and all were untypeable for the replicons currently included in the PBRT scheme. Interestingly, qnrB19-ColE plasmids, similar to those identified in isolate AM29213, were previously identified in Salmonella enterica Typhimurium isolated in the Netherlands and in commensal E. coli isolated from children living in different urban areas of Peru and Bolivia (9, 13). These plasmids apparently play a major role in the widespread dissemination of qnrB19 genes observed in both pathogenic and commensal enterobacteria.
Few studies have examined quinolone resistance among Shigella isolates in the United States and the resistance mechanisms involved (17, 20, 21). In this study, we have identified Shigella isolates with decreased susceptibility to ciprofloxacin. Isolates containing QRDR mutations (n = 15) displayed nalidixic resistance (MIC ≥ 32 μg/ml) and ciprofloxacin susceptibility of ≥0.25 μg/ml, while those containing PMQR determinants in the absence of QRDR mutations displayed nalidixic acid susceptibility (MIC ≤ 8 μg/ml) and ciprofloxacin MICs of 0.25 μg/ml. All ciprofloxacin-resistant isolates contained multiple QRDR mutations. This supports the idea that QRDR mutations are largely responsible for nalidixic acid and ciprofloxacin resistance among Shigella isolates. However, PMQR determinants are located on mobile genetic elements (i.e., plasmids), which may allow for dissemination among Shigella and possibly additional members of the Enterobacteriaceae. Also, it is believed that the presence of PMQR may facilitate the selection of QRDR mutations, resulting in higher levels of quinolone resistance (10).
Most of the patients (80%) from whom a travel history was obtained reported recent travel to Asia. Several reports have described the increasing prevalence of ciprofloxacin resistance among sporadic and epidemic Shigella isolates from China and India and in Asian travel-associated Shigella cases in the United States (4, 11, 15). This report supports the hypothesis that ciprofloxacin-resistant Shigella infections are imported into the United States from Asian countries. Physicians treating patients with diarrheal illness should always ask about recent travel because it may affect treatment decisions (8). The emergence of Shigella infections with decreased susceptibility to ciprofloxacin is a public health concern, especially since PMQR determinants may spread among other members of the Enterobacteriaceae. Continued surveillance for resistance to clinically important antimicrobials among Shigella isolates, as well as collection of exposure and travel history data and studies on the spread of resistance determinants, are necessary to guide public health interventions.
We thank the NARMS participating public health laboratories for submitting the isolates, Nancy Strockbine and Evangeline Sowers for confirming the Shigella species, Anne Whitney for DNA sequencing, and Rebecca Howie for assistance.
This work was supported by an interagency agreement between the CDC and the FDA Center for Veterinary Medicine.
Published ahead of print on 10 January 2011.