Emergence of Plasmid-Mediated Quinolone Resistance among Non-Typhi Salmonella enterica Isolates from Humans in the United States

Abstract

Plasmid-mediated quinolone resistance determinants are emerging among gram-negative pathogens. Here we report results of a retrospective study investigating the prevalence of aac(6′)-Ib-cr, qepA, and qnr genes among 19,010 human isolates of non-Typhi Salmonella enterica collected in the United States from 1996 to 2006.

Approximately 1.4 million people in the United States are infected with non-Typhi Salmonella enterica (NTS) annually, resulting in 15,000 hospitalizations and >400 deaths (14). Severe invasive infections are commonly treated with the fluoroquinolone ciprofloxacin (CIP). Although endogenous topoisomerase mutations are an important source of fluoroquinolone resistance in Enterobacteriaceae (6), three recently described plasmid-mediated mechanisms confer decreased susceptibility to ciprofloxacin: QepA efflux, QNR proteins, and AAC(6′)-Ib-cr (12). The last is a mutant aminoglycoside acetyltransferase [AAC(6′)-Ib] which modifies CIP and norfloxacin (13). Here we report the prevalence of plasmid-mediated quinolone resistance mechanisms among human isolates of NTS submitted to the National Antimicrobial Resistance Monitoring System (NARMS) from 1996 to 2006.

(Portions of the data were presented at the ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens, 15 to 18 June 2008, Copenhagen, Denmark.)

State and local public health laboratories participating in NARMS submitted every 10th (1996 to 2002) or every 20th (2003 to 2006) NTS isolate that they received to the Centers for Disease Control and Prevention for susceptibility testing. MICs were determined for 14 to 17 antimicrobial agents by broth microdilution (Sensititre; Trek Diagnostics, Westlake, OH). Isolates that exhibited decreased susceptibility to CIP (≥0.25 mg/liter) were included in the study. Data for 2006 are preliminary and may be subject to change.

The qepA and qnr genes were detected as previously described (5, 10). A SYBR green-based real-time PCR assay using previously described primers was developed to screen isolates for the aac(6′)-Ib gene (9). PCR mixtures contained 1× SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA), 0.4 μM (each) primer, 2 μl template DNA, and sterile water to a final volume of 25 μl. Thermal cycling conditions were as follows: 10 min at 95°C, followed by 35 cycles of 95°C for 20 s, 55°C for 30 s, and 72°C for 30 s. Finally, a melt-curve analysis between 70 and 90°C was performed. Amplification, data acquisition, and data analysis were carried out in a RotorGene 6000 (Corbett Research, Mortlake, NSW, Australia). The presence of the aac(6′)-Ib-cr variant was confirmed by direct sequencing of PCR products. Chromosomal topoisomerase mutations were detected as previously described (5).

Among the 19,010 NTS isolates submitted to NARMS between 1996 and 2006, 283 (1.5%) displayed decreased susceptibility to CIP. Of these, 273 were available for PCR screening. Eight (2.9%) isolates, including isolates of serotypes Typhimurium (n = 1), Newport (n = 1), Senftenberg (n = 4), Typhimurium var. O:5− (n = 1), and Cubana (n = 1), harbored the aac(6′)-Ib gene. Seven isolates contained wild-type aac(6′)-Ib; one contained the two point mutations (Trp102Arg and Asp179Tyr) characteristic of the aac(6′)-Ib-cr variant (Table 1). This isolate displayed a CIP MIC of 1.0 mg/liter and contained in addition to aac(6′)-Ib-cr a mutation in the gyrA gene (Asp87Tyr). The isolate was serotype Typhimurium var. O:5− and was isolated from a 4-year-old child in Massachusetts in 2005 (Table 1). The patient's family reported a history of travel to China. The patient did not receive antibiotic treatment and recovered without complications.

TABLE 1.

Characteristics of Salmonella isolates harboring qnr or the aac(6′)-Ib-cr gene

Isolate no.	Yr of collection	Salmonella enterica serotype	Submitting statea	Resistance phenotypeb	CIP MIC (mg/liter)	aac(6′)-Ib/qnr variant
AM20376	2004	Corvallis	MI	STR, SUL, TET	0.5	qnrS1
AM19383	2004	Montevideo	NY	SXT	0.5	qnrS1
AM21420	2004	Telelkebir	RI	NDc	0.5	qnrB5
AM25008	2005	Typhimurium var. O:5−	MA	AMP, CHL, KAN, NAL, SUL, SXT, TET	1	aac(6′)-Ib-cr
AM25736	2006	Corvallis	IL	STR, SUL, TET	0.25	qnrS1
AM25927	2006	Corvallis	WI	ND	0.25	qnrS1
AM25798	2006	Enteritidis	CT	AMP, STR	0.5	qnrS1
AM30594	2006	Saintpaul	PA	TET	0.5	qnrS1
AM26790	2006	Saintpaul	OR	SXT, TET	0.5	qnrS1
AM27315	2006	Typhimurium	MI	STR, SUL, TET	0.5	qnrS1
AM27549	2006	Typhimurium	PA	STR, SUL, TET	0.5	qnrS1
AM29365	2006	Typhimurium	NY	ND	0.5	qnrS1
AM29869	2006	Typhimurium	WA	ND	0.5	qnrS1
AM27031	2006	Kiambu	CA	NAL	0.5	qnrB5
AM26209	2006	Aqua	TN	ND	0.25	qnrB5
AM27125	2006	Typhimurium	NY	AMP, CHL, STR, SUL, TET	0.5	qnrB5
AM28764	2006	Cubana	NY	GEN, KAN, NAL, STR, SUL, SXT	0.5	qnrB2
AM28704	2006	Montevideo	IL	KAN, SUL, SXT, TET	0.25	qnrA1

^aCA, California; CT, Connecticut; FL, Florida; IL, Illinois; MA, Massachusetts; MI, Michigan; MN, Minnesota; NY, New York; OR, Oregon; PA, Pennsylvania; RI, Rhode Island; TN, Tennessee; WA, Washington; WI, Wisconsin.

^bAMP, ampicillin; CHL, chloramphenicol; GEN, gentamicin; NAL, nalidixic acid; KAN, kanamycin; STR, streptomycin; SUL, sulfamethoxazole or sulfisoxazole; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline.

^cND, none detected.

The prevalence of qnr genes among NTS isolates submitted to NARMS between 1996 and 2003 has been reported elsewhere (5). Among NTS isolates exhibiting decreased susceptibility to CIP collected in 2004 to 2006, 17 (9.8%) carried qnr genes: 11 isolates harbored qnrS1, four carried qnrB5, one carried qnrB2, and one isolate carried qnrA1 (Table 1). Typhimurium (n = 5), Corvallis (n = 3), Montevideo (n = 2), and Saintpaul (n = 2) were the most common serotypes harboring qnr genes. None of the isolates in the present study carried the qepA gene.

The aac(6′)-Ib-cr mechanism was originally reported in 2003 in a clinical isolate of Escherichia coli collected in Shanghai, China (16). Since then the aac(6′)-Ib-cr variant has appeared in several countries in Asia, North America, and Europe (1, 2, 4, 9, 11, 15). Interestingly, the aac(6′)-Ib-cr-positive NTS isolate described here was isolated after travel to Shanghai, China. In China, the gene has been detected in both human and veterinary Enterobacteriaceae isolates, with high frequency (>50%) among clinical E. coli isolates (7, 8, 13).

The low prevalence (0.4%) of aac(6′)-Ib-cr among human isolates of NTS contrasts with a previous U.S. report in which 44 of 313 (14%) isolates of Klebsiella pneumoniae, E. coli, and Enterobacter spp. carried the gene (9). The difference in aac(6′)-Ib-cr prevalence among Enterobacteriaceae might be due to several factors. First, the gene might have been introduced recently into Salmonella populations; the fact that the first aac(6′)-Ib-cr-positive NARMS isolate of Salmonella appeared in 2005 supports the recent emergence of the mechanism as opposed to late discovery of an established mechanism. Second, the different prevalences might be due to instability of the genetic element hosting the gene or a higher fitness cost associated with gene carriage in Salmonella compared with other Enterobacteriaceae. Finally, selective pressures facilitating selection and maintenance of aac(6′)-Ib-cr (e.g., use of aminoglycosides and fluoroquinolones) may differ among various Enterobacteriaceae reservoirs. Further studies of Salmonella from humans and animals are necessary to determine sources of aac(6′)-Ib-cr and its first appearance in Salmonella populations.

A previous study reported 10 (0.08%) qnr-positive NTS NARMS isolates from 1996 to 2003 (5). Here we report 17 (0.3%) qnr-positive isolates among 6,057 isolates submitted in 2004 to 2006. The fact that 14 of these were collected in 2006 and originated from 10 different states is of concern, as it indicates that qnr genes may be increasing among NTS isolates in the United States. The fact that qnr genes were found in four serotypes (Berta, Mbandaka, Bovismorbificans, and Anatum) in 1996 to 2003 and in nine additional serotypes (Typhimurium, Corvallis, Saintpaul, Montevideo, Telelkebir, Kiambu, Enteritidis, Aqua, and Cubana) in 2004 to 2006 further supports the dissemination of the genes.

This is the first description of qnrA among NARMS human isolates of NTS. This gene has previously been reported among isolates of serotype Enteritidis in China (3). In the United States, qnrA has primarily been found among isolates of Klebsiella pneumoniae and Enterobacter spp. (12).

In conclusion, we show that the aac(6′)-Ib-cr gene is present among NTS isolates in the United States collected from humans, although the mechanism appears to be rare. Furthermore, we report a notable increase in qnr genes among NTS isolates submitted to NARMS in 2006. Vigilant surveillance and prospective studies are necessary to determine changes in prevalence and the basis for continued appearance. Judicious use of antimicrobial agents in human and veterinary medicine will be crucial for limiting further spread of plasmid-mediated quinolone resistance among Enterobacteriaceae.