Plasmid-Mediated Quinolone Resistance in Different Diarrheagenic Escherichia coli Pathotypes Responsible for Complicated, Noncomplicated, and Traveler's Diarrhea Cases

LETTER

Diarrheagenic Escherichia coli (DEC) organisms are important agents of endemic and epidemic diarrhea worldwide, as well as significant contributors of traveler's diarrhea (TD) in industrialized countries (1, 2). Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), further divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) are considered the most important DEC pathotypes (2). STEC are food-borne pathogens responsible for important outbreaks of hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS) in industrialized countries (2). EAEC, ETEC, EPEC, and EIEC are generally considered major causes of TD in adults from developed countries and the leading causes of infant diarrhea in developing ones (2).

The first-choice agents for treating DEC infections are quinolones (3), although their use concretely in complicated STEC infections remains controversial (4). However, plasmid-mediated quinolone resistance (qnr) genes encoding small pentapeptide repeat proteins that protect type II DNA topoisomerases from quinolones, including five qnr families (qnrA1-7, qnrB1-74, qnrC, qnrD1-2, and qnrS1-9), have been described. qnr genes by themselves are able to confer only a low level of quinolone resistance, but they have been proposed to promote the emergence of chromosomal mutations leading to levels of resistance of clinical significance (5). Although their occurrence has been widely documented in extraintestinal E. coli isolates (6), studies concerning qnr occurrence in DEC isolates are scarce.

A routine screening for susceptibility to 13 different antimicrobials was carried out with 54 STEC, 16 aEPEC, 9 EAEC, 6 ETEC, and 2 EIEC strains (87 strains in total) isolated from complicated (HC and HUS) and noncomplicated endemic diarrhea and TD cases in the Spanish National Reference Laboratory (SNRL) during 2012 and 2013. The susceptibility testing was performed by the disk diffusion method as previously described (7), and results were interpreted according to CLSI guidelines. For strains showing a decrease in the diameter of the inhibition halo of ciprofloxacin (≤27 mm), the MICs of ciprofloxacin and nalidixic acid were determined by Etests. Additionally, to evaluate the possible association between qnr genes and the production of extended-spectrum β-lactamases (ESBLs), the ESBL phenotype was detected by the double-synergy test. Resistance genes were identified by PCR and DNA sequencing, plasmid analysis was carried out by S1 nuclease–pulsed-field gel electrophoresis (S1-PFGE) and PCR-based replicon typing, and conjugation assays were performed to link resistance genes to plasmids, as previously described (7).

Overall, four DEC strains out of 87 (4.6%) exhibited a decreased ciprofloxacin susceptibility (MIC, 0.38 to 1.5 μg/ml), with three of them being still susceptible to nalidixic acid (MIC, 6 to 16 μg/ml) (Table 1). Among them, qnrB19 was identified in an EAEC strain isolated from an adult with diarrhea traveling from Mexico and also in an STEC O157:H7 strain isolated from a 7-year-old boy suffering from HUS after diarrhea (Table 1). Likewise, qnrS1 was detected in an aEPEC strain isolated from a 1-year-old boy with noncomplicated diarrhea and also in an EIEC strain isolated from an adult with diarrhea traveling from Southeast Asia (Table 1). This EIEC strain showed a resistance phenotype indicating ESBL production and harbored bla_CTX-M-15 (Table 1). Conjugation experiments were positive for the EAEC, aEPEC, and EIEC strains. Plasmid analysis showed that qnrB19 was transferred on a ColE_Tp plasmid of ∼3 kb in the EAEC strain (Table 1). In the aEPEC strain, qnrS1 was transferred on a nontypeable plasmid of ∼48 kb, and cotransfer of the bla_TEM1 gene was observed (Table 1). In the ESBL-producing EIEC strain, qnrS1 was transferred with bla_CTX-M-15 and bla_TEM1 on an IncK plasmid of ∼97 kb (Table 1). Finally, in the STEC O157:H7 strain, qnrB19 was harbored on a nonconjugative ColE_Tp plasmid of ∼3.5 kb (Table 1).

TABLE 1.

Features of the four qnr-positive diarrheagenic Escherichia coli strains^a

Strain	Pathotype	Origin	Serotype	qnr gene	Resistance phenotype or genotype	MIC (μg/ml) of NAL, CIP	Plasmid size (kb), incompatibility group
2384/12	EAEC	TD	O65/O71:H1b	qnrB19	AMP CHL TET AMC	12, 0.38	3, ColETp
4425/12	STEC	CD	O157:H7	qnrB19	AMP SSS STR TET SXT	16, 0.38	3.5, ColETp
4472/12	aEPEC	NCD	O49:H−	qnrS1	AMP SSS NAL TET blaTEM1	>256, 1.5	48, NT
2113/13	EIEC	TD	O96:H19	qnrS1	AMP SSS STR CEF CTX SXT AMC blaTEM1 blaCTX-M-15	6, 0.38	97, IncK

^aNAL, nalidixic acid; CIP, ciprofloxacin; EAEC, enteroaggregative E. coli; STEC, Shiga toxin-producing E. coli; aEPEC, atypical enteropathogenic E. coli; EIEC, enteroinvasive E. coli; TD, traveler's diarrhea; CD, complicated endemic diarrhea; NCD, noncomplicated endemic diarrhea; H−, nonmotile; AMP, ampicillin; CHL, chloramphenicol; TET, tetracycline; AMC, amoxicillin-clavulanic acid; SSS, sulfonamides; STR, streptomycin; SXT, trimethoprim-sulfamethoxazole; CEF, cephalothin; CTX, cefotaxime; NT, nontypeable.

^bThis strain cross-reacted with the respective O antisera.

To our knowledge, this is the first report of the occurrence of qnr genes in STEC, aEPEC, and EIEC clinical strains. Our study also confirms the occurrence of qnr genes in EAEC strains reported by Riveros et al. (8) and Kim et al. (9), which might have contributed to the increasing trend of fluoroquinolone resistance recently observed in this E. coli pathotype worldwide (8, 10). As for the plasmids, although qnrB19 has previously been found in ColE-like plasmids (8, 11), qnrS1 has rarely been identified in IncK plasmids. The presence of bla_CTX-M-15 in IncK plasmids from E. coli has been recently reported (12), despite these plasmids being involved mainly in the spreading of bla_CTX-M-14 (13), but to our knowledge, no IncK plasmid simultaneously harboring qnrS1 and bla_CTX-M-15 has been reported yet. Although the clinical implications of our findings are still unknown, it may be speculated that qnr genes might play a significant role in therapeutic failures in DEC infections. In addition, epidemiologic surveillance and correct use of antimicrobial agents are needed to limit the spread of plasmid-mediated quinolone resistances.

Funding Statement

This work was supported by grants MPY-1042/14 and PI14CIII/00051 from the Fondo de Investigaciones Sanitarias from the Spanish Ministry of Economy and Competitiveness. Sergio Sánchez acknowledges the Miguel Servet Programme of the Fondo de Investigaciones Sanitarias, Spanish Ministry of Economy and Competitiveness, for his research contract (CP13/00237).