Journal of Antimicrobial Chemotherapy Advance Access published June 7, 2014
J Antimicrob Chemother doi:10.1093/jac/dku197
Presence of disinfectant resistance genes in Escherichia coli isolated from retail meats in the USA Likou Zou1,2, Jianghong Meng1, Patrick F. McDermott3, Fei Wang1, Qianru Yang4, Guojie Cao1, Maria Hoffmann1,3 and Shaohua Zhao3* 1
Department of Nutrition and Food Science, University of Maryland, College Park, MD, USA; 2The Laboratory of Microbiology, Sichuan Agricultural University, Dujiangyan, Sichuan, P. R. China; 3Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD, USA; 4Department of Food Science, Louisiana State University Agricultural Center, 111 Food Science Building, Baton Rouge, Louisiana, USA *Corresponding author. Tel: +1-301-210-4472; Fax: +1-301-210-4685; E-mail: [email protected]
Received 30 December 2013; returned 20 March 2014; revised 28 April 2014; accepted 11 May 2014 Objectives: To examine the distribution of all genes known to be responsible for resistance to quaternary ammonium compounds (QACs), and their association with resistance to QACs and other antimicrobials, in Escherichia coli recovered from retail meats. Methods: A total of 570 strains of E. coli isolated from US retail meats in 2006 were screened for the presence of 10 QAC resistance genes [qacE, qacED1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA and ydgE/ydgF]. The MICs of six common disinfectants were determined using an agar dilution method. Possible associations between the presence of the gene and bacterial resistance to QACs and antimicrobials were investigated. Results: emrE, sugE(c), mdfA and ydgE/ydgF were commonly present (77.2%–100%) in the E. coli isolates, but qac and sugE(p) were less prevalent (0.4%–22.3%). emrE-mdfA-sugE(c)-ydgE/F was the most common QAC resistance gene profile. A significant association was found between antimicrobial resistance and the presence of sugE(p) and qacED1 (P,0.05). Antimicrobial-resistant E. coli isolates tended to contain more diverse combinations of disinfectant resistance genes than susceptible ones. All isolates showed reduced susceptibility to five of six disinfectants compared with the control strains. Higher MICs were generally associated with the presence of qac and sugE(p) genes. Conclusions: The QAC resistance genes were commonly present among E. coli isolated from retail meats, and the qac and sugE(p) genes were highly associated with multidrug resistance phenotypes. Using QACs in the food industry may not be as effective as expected and could provide selection pressure for strains with acquired resistance to other antimicrobials. Keywords: quaternary ammonium compounds, qac genes, antimicrobial resistance, MICs
Introduction The quaternary ammonium compounds (QACs) are non-corrosive and non-irritating, with little toxicity and a high antimicrobial efficacy over a wide pH range, and are routinely used in both clinical and industrial settings.1 – 3 It has been suggested that the widespread use of QACs may contribute to the emergence of antimicrobialresistant bacteria.4,5 There are five QAC resistance genes, sugE(c), emrE, ydgE/ydgF and mdfA, that are known as chromosome-encoded genes conferring QAC resistance.6,7 Unlike mdfA, which belongs to the major facilitator superfamily (MFS), sugE(c), emrE and ydgE/ydgF are members of the small multidrug resistance (SMR) family. The sugE(c) gene mediates high levels of resistance to QACs.8,9 Likewise, emrE,
encoding a multidrug exporter, is associated with resistance to a wide range of toxic cationic hydrophobic compounds, including QACs.10 – 12 Unlike other resistance genes, ydgE and ydgF overlap on the Escherichia coli chromosome and confer resistance to QAC only when co-expressed.6,13 In addition, five QAC resistance genes [qacE, qacED1, qacF, qacG and sugE(p)] have been identified on mobile genetic elements in Gram-negative organisms.14 – 16 These genes belong to the SMR family15,17 and are generally plasmid and/or integron encoded, conferring efflux-mediated resistance to QACs. The qacE gene is located in the 3′ -CS of class 1 integrons in Gram-negative bacteria, and the qacED1 gene is a deletion mutation of qacE that has been associated with an increased MIC of benzalkonium chloride.14,18 The qacF gene displays a high degree of similarity (67.8% identity) to the qacE gene.16 The qacG gene has
Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2014. This work is written by US Government employees and is in the public domain in the US.
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been localized to class 1 integrons in Gram-negative bacteria,15 while sugE(p) is frequently present on an IncA/C multidrug resistance plasmid,19 which is common in Salmonella isolated from food animals and retail meats. Expression of the sugE gene confers high MICs of cetylpyridinium chloride (CTPC),8 a disinfectant used in food-animal processing for carcass washing, as well as a component of mouthwashes, toothpastes and lozenges. QACs are commonly used for disinfecting food-processing and production environments to ensure microbiologically safe food products.20 This raises questions about the possible role of QACs in promoting the development of antimicrobial resistance, in particular co- or cross-resistance to antimicrobials.21 Most E. coli strains are part of the normal intestinal flora but some strains, such as diarrhoeic E. coli, can cause enteric infections. Contamination of food with E. coli may occur during production, processing and distribution and at retail. Generally, meat products are one of the food categories that are most commonly contaminated with E. coli and also are major reservoirs of antimicrobial-resistant E. coli.22,23 E. coli isolates from clinical sources have been shown to exhibit a high MIC of QACs that was correlated with antibiotic resistance.24 While qacED1 is frequently presented in E. coli and other enteric bacteria, we undertook a more comprehensive study to examine the distribution of all known QAC resistance genes, and their association with resistance to QACs and other antimicrobial agents, in E. coli recovered from retail meat products.
Materials and methods Bacterial strains and antimicrobial susceptibility testing A total of 570 E. coli strains were included in this study. They were isolated in 2006 in the USA from retail pork chop (n ¼ 98), ground beef (n ¼ 114), ground turkey (n ¼ 176) and chicken breast (n ¼ 182) by the National Antimicrobial Resistance Monitoring System (NARMS) retail meat programme.25 MICs had previously been determined using the SensititreTM automated antimicrobial susceptibility system in accordance with the manufacturer’s instructions (Trek Diagnostic Systems, Cleveland, OH, USA) and the NARMS protocol. Fifteen antimicrobials were tested: amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, nalidixic acid, ciprofloxacin, gentamicin, kanamycin, amikacin, streptomycin, sulfisoxazole, trimethoprim/sulfamethoxazole and tetracycline. The results were interpreted in accordance with CLSI criteria26 with the exception of those for streptomycin (resistance breakpoint ≥64 mg/L). E. coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were used as quality control organisms.
PCR amplification The primers used to amplify qacE, qacED1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA and ydgE/ydgF were designed based on sequences from GenBank (Table S1, available as Supplementary data at JAC Online). The DNA template was prepared by suspending an overnight culture in 600 mL of reagent-grade water. The suspensions were heated at 1008C for 10 min and centrifuged at 13 000 rpm for 5 min. Each 25 mL of PCR mixture consisted of 2.5 mL of template, 5 mL of 5× PCR buffer, 1.5 mM MgCl2, 200 mM dNTP, 0.4 mM primers and 1.25 U of polymerase (Promega, Madison, WI, USA). Amplified PCR products were analysed on 2.0% (w/v) agarose gels. Appropriate positive and negative controls for amplification were selected from retail meat E. coli isolates. The positive controls that carried the QAC genes were confirmed using PCR followed
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by sequence analysis (GENEWIZ, Inc., Germantown, MD, USA). All results were confirmed by at least two independent experiments.
Determination of MICs of disinfectants The MICs of disinfectants for E. coli were determined using the agar dilution method recommended by the CLSI.26 E. coli suspensions were adjusted to a turbidity equivalent to that of a 0.5 McFarland standard and delivered to the surface of Mueller– Hinton agar plates using a multipoint inoculator (Oxoid, Lenexa, KS, USA). The final inoculum was 104 cfu per spot. Plates were incubated at 378C for 24 h. The disinfectants included were N-alkyl dimethyl benzyl ammonium chloride (ADBAC; Acros Organics, Morris Plains, NJ, USA), cetyltrimethylammonium bromide (CTAB; MP Biomedicals, Santa Ana, CA, USA), CTPC (Sigma, St Louis, MO, USA), hexadecyltrimethylammonium bromide (HAB; Sigma), chlorhexidine (CL; Sigma) and triclosan (TCL; Fisher Scientific, Pittsburgh, PA, USA). The range of concentrations used to determine the MICs of the disinfectants was 0.125 – 1024 mg/L. E. coli ATCC 10536 and S. aureus ATCC 25923 were used as quality control strains.
Data analysis The x2 or Fisher’s exact test and Student’s t-test were used to analyse the data using SAS 9.2 (SAS Institute, Cary, NC, USA). A P value ,0.05 was considered statistically significant for comparisons.
Nucleotide sequence accession numbers The nucleotide sequences of qacF, qacG and sugE(p) have been submitted to GenBank under accession numbers KC285363, KC285364 and KC285365, respectively.
Results Presence of disinfectant resistance genes The ydgE/ydgF genes were the most widespread QAC resistance genes, being found in 100% of isolates, followed by mdfA (96.5%; n¼550), sugE(c) (84.0%; n¼479), emrE (77.2%; n¼440), qacED1 (22.3%; n¼127), sugE(p) (6.8%; n¼39), qacF (1.8%; n¼10) and qacG (0.4%; n¼2). The qacE gene was not detected in any of the isolates. The top two QAC resistance genotypes were emrE-mdfA-sugE(c)-ydgE/F (54.6%) and mdfA-sugE(c)-ydgE/F (17.5%) (Table S2, available as Supplementary data at JAC Online).
Prevalence of QAC resistance genes in E. coli by meat type Regardless of the source, ydgE/ydgF (100%) and mdfA (99.5%) were commonly found in retail meat isolates. The prevalence of other QAC resistance genes varied by meat type. The qacED1 gene was more common (P,0.05) in E. coli isolated from poultry meats (chicken: 31.3%, n ¼ 57; ground turkey: 31.8%, n ¼ 56) than from ground beef (7.0%; n ¼ 8) and pork (6.1%; n ¼ 6). The sugE(p) gene was found in 10.2% (n¼18) of isolates from ground turkey, 7.7% (n¼14) from chicken and 3.5% (n¼4) from beef, but not in E. coli from pork. Surprisingly, the frequency of the sugE(c) gene was higher (P,0.05) in the beef (99.1%) and pork (98.0%) isolates than in the turkey (75.6%) and chicken (75.3%) isolates. The frequency of the qacF gene was slightly higher in E. coli from pork than beef and turkey, and no qacF gene was found in chicken
JAC
Disinfectant resistance genes in Escherichia coli
100.0%
Prevalence of QAC resistance genes
90.0% 80.0% Resistant (n = 399)
70.0%
Susceptible (n = 171)
60.0% 50.0% 40.0%
A
30.0% 20.0%
A
10.0% 0.0%
B ydgE/F
mdfA
emrE
sugE(c) qacEΔ1
B sugE(p)
qacF
qacG
Disinfectant resistance genes Figure 1. Percentage of QAC resistance genes in antibiotic-resistant and antibiotic-susceptible E. coli isolates. A/B, statistically significantly different (P , 0.01). The qacE gene was not detected in any of the isolates.
isolates (Table S2). The qacG gene was only found in two pork isolates, and the qacE gene was not detected in any of the isolates.
Association between disinfectant resistance genes and antimicrobial resistance The association between the presence of sugE(p) and qacED1 and antimicrobial resistance was significant (P, 0.05) (Figure 1). Interestingly, most sugE(p)-positive isolates (79.5%; n ¼ 31) were multidrug resistant (resistant to at least three or more classes of antimicrobial agents). Among the sugE(p)-positive isolates, 87.2% (n ¼ 34) were resistant to ampicillin, 74.4% (n ¼ 29) to amoxicillin/clavulanic acid, ceftriaxone, cefoxitin and ceftiofur, 66.7% (n ¼ 26) to tetracycline, 61.5% (n ¼ 24) to streptomycin and 46.2% (n ¼ 18) to sulfisoxazole. There were 22 different resistance patterns among the sugE(p)positive isolates. The most frequent resistance pattern was ampicillin-amoxicillin/clavulanic acid-ceftriaxone-cefoxitinceftiofur (20.5%; n ¼ 8), followed by ampicillin-amoxicillin/ clavulanic acid-ceftriaxone-cefoxitin-ceftiofur-streptomycin (7.7%; n ¼ 3) and ampicillin-amoxicillin/clavulanic acid-ceftriaxonecefoxitin-ceftiofur-streptomycin-tetracycline (7.7%; n¼3). Among the 127 qacED1-positive isolates, 93.7% (n¼ 119) were resistant to sulfisoxazole, 75.6% (n¼ 96) to gentamicin, 75.6% (n¼ 96) to tetracycline, 59.1% (n ¼ 75) to streptomycin and 30.7% (n ¼ 39) to ampicillin. Furthermore, many of the qacED1-positive isolates were resistant to multiple antimicrobials. For instance, 56.7% (n ¼ 72) were resistant to both sulfisoxazole and gentamicin, 43.3% (n ¼ 55) to sulfisoxazole, gentamicin and streptomycin, and 39.3% (n¼50) to sulfisoxazole, gentamicin and tetracycline. There were 57 different resistance patterns in the qacED1-positive isolates. The most frequent resistance pattern was sulfisoxazolegentamicin-streptomycin-tetracycline (15.0%, n ¼ 19), followed by ampicillin-sulfisoxazole-gentamicin-streptomycin-tetracycline (8.7%, n ¼ 11), sulfisoxazole-gentamicin-streptomycin (7.1%, n ¼ 9) and sulfisoxazole-gentamicin-tetracycline (7.1%, n ¼ 9). Only one resistance pattern, ampicillin-tetracycline, was found in
two qacG-positive isolates. Among 10 qacF-positive isolates, 90.0% (n ¼ 9) were resistant to sulfisoxazole and tetracycline, 60.0% (n¼6) to chloramphenicol and 40.0% (n¼4) to ampicillin. Two qacF-positive isolates were multidrug resistant, showing resistance to ampicillin-chloramphenicol-kanamycin-sulfisoxazole-streptomycin-tetracycline and amoxicillin/clavulanic acid-ampicillincefoxitin-chloramphenicol-kanamycin-sulfisoxazole-tetracycline, respectively. Seventeen different QAC resistance genotypes were identified in resistant and susceptible E. coli (Table S2). However, the genotypes in the resistant E. coli were more diverse than those found in the susceptible isolates. Among 399 antimicrobial-resistant isolates, there were 16 QAC resistance genotypes, whereas there were only seven genotypes among 171 susceptible isolates. mdfA-sugE(c)-ydgE-ydgF and emrE-mdfA-sugE(c)-ydgE-ydgF were the top two genotypes in both resistant and susceptible isolates, together accounting for 96.5% and 61.7% of each group, respectively.
MICs of disinfectants for selected E. coli isolates In general, we observed high MICs of ADBAC, CTAB, CTPC and HAB, and low MICs of CL and TCL (Table S3, available as Supplementary data at JAC Online). Compared with the reference strains, the isolates showed reduced susceptibility to all QACs except for TCL. TCL, however, is a polychlorophenoxy phenol and does not belong to the QAC disinfectant family. High MICs of QAC were observed for the isolates that carried qacG (the R1 genotype). The MICs of CTAB, CTPC and HAB were 512–1024 mg/L, 1024 mg/L and 1024 mg/L, respectively. Isolates containing emrE, mdfA, ydgE/F, sugE(c) and sugE(p) (the R2 genotype) also showed a higher MIC (512 mg/L) of CTPC and HAB. Those with genotypes R3 and R4, carrying qacF, displayed higher MICs (by 2- to 4-fold) of all the disinfectants except TCL, compared with the control strains. Interestingly, the MICs of QACs were not significantly different between qacED1-positive and qacED1-negative isolates among the antimicrobial-resistant isolates. However, pan-susceptible isolates that carried qacED1
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(types S1 and S2) had a higher MIC of CTAB (1024 mg/L) compared with pan-susceptible isolates without qacED1. In general, the higher MICs of QAC were associated with qac and sugE(p).
Discussion Despite the widespread use of chemical interventions, E. coli is commonly present on retail meats,25 and many E. coli exhibit resistance to common antimicrobials. We hypothesized that the use of QACs in the food-processing environment might select for strains with acquired QAC resistance that also carry genes encoding resistance to medically important antimicrobial agents.27 If so, the use of QAC disinfectants might influence the types of antimicrobial-resistant organisms that reach consumers via foods. Reduced susceptibilities to disinfectants have been reported among bacteria isolated from food or food production environments.27 – 29 However, the prevalence of QAC resistance genes in E. coli, the mechanisms of disinfectant resistance and the association between QAC resistance and the antimicrobial susceptibility of E. coli isolated from food were not clear. The present study demonstrated that a variety of QAC resistance genes were present in E. coli. Among the QAC resistance genes, five genes, qacE, qacED1,14 qacF,16,30 qacG 15 and sugE(p),19,31 are located in mobile genetic elements and linked (co-exist) with different antibiotic resistance genes. The other five genes, emrE,12,32 sugE(c),33 mdfA,7 ydgE and ydgF, are chromosome-encoded genes and have shown cross-resistance to different antimicrobials.6,34 Based on previous descriptions of the location of QAC resistance genes, we found that the chromosome-encoded efflux pump genes6,7,9,10,17 were commonly present in E. coli isolated from retail meat. However, mobile-element-encoded QAC genes were relatively low in frequency. Similar findings were reported in Salmonella, with 27.0% positive for qacED1 and 0% positive for qacE.11 Kucken et al.14 reported that the qacED1 gene was detected in 10.0% of isolates and that the qacE gene was found in only 1.0% of isolates. In addition, qacF and qacG, which had also previously been found in Enterobacter aerogenes and Pseudomonas spp.,15,16,30 were present only in 1.8% (n ¼ 10) and 0.4% (n ¼ 2), respectively, of isolates in our study. These genes have been reported to be located in class 1 integrons.15,21,35 Several studies have shown that genes encoding efflux pumps promote bacterial resistance to a narrow range of QACs without conferring cross-resistance to antimicrobials.6,9,36 We examined the association between antimicrobial resistance and the presence of different QAC resistance genes. Most notably, qacED1 and sugE(p) were highly associated (P, 0.05) with antimicrobial resistance. Studies have shown that the qacED1 gene is common in enteric bacteria and is located in the 3′ -conserved segment of class 1 integrons that carry sul1 (sulphonamide resistance determinant).14,37 The qacED1 and sul1 genes usually co-exist in integrons, which could explain the fact that 93.7% (n ¼ 119) of qacED1-positive isolates showed co-resistance to sulphonamides. In addition, 75.6% (n¼ 96) of qacED1-positive isolates were resistant to gentamicin and 56.7% (n ¼ 72) to both sulfisoxazole and gentamicin. Whether or not these resistance genes were present with the qacED1 gene in class 1 integrons was not determined in this study. It is known that multiple gene cassettes can be arranged in tandem within these elements and more than 60 distinct cassettes have been identified to date, including
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aminoglycoside resistance genes.38,39 Cassette-associated genes also have been shown to confer resistance to b-lactams, phenicols, trimethoprim and streptothricin, as well as QACs. Therefore, it was not surprising that the presence of the qacED1 gene in our isolates was frequently associated with resistance to sulfisoxazole, gentamicin, tetracycline, streptomycin and ampicillin. Likewise, many sugE(p)-positive isolates were resistant to ampicillin (87.2%), amoxicillin/clavulanic acid, ceftriaxone, cefoxitin, ceftiofur (74.4%), tetracycline (66.7%), streptomycin (61.5%) and sulfisoxazole (46.2%). The sugE(p) gene in IncA/C (E. coli) and pSN254 (Salmonella) plasmids has been linked with other resistance genes, such as blaCMY-2, sul1, sul2, floR, aadA, aacC and tet.19,31,40 Since most sugE(p)-positive isolates were multidrug resistant, it is likely that the sugE(p) gene is linked with the multidrug resistance IncA/C plasmid and other types of multidrug resistance plasmids. Our findings indicated that using QACs in the food-processing environment could select for strains with acquired QAC resistance that also carry resistance genes to medically important antimicrobial agents such as ceftriaxone, an important drug to treat salmonellosis. Compared with the reference strains, E. coli isolates with different QAC genotypes showed reduced susceptibility to QACs (Table S3). With the exception of mdfA, which belongs to the MFS family, the other genes are members of the SMR family. These resistance genes encode efflux pumps conferring resistance to QACs via an electrochemical proton gradient.17 Several studies have shown that different chromosome-encoded QAC resistance genes have contributed to resistance to QACs.6,7,9,10,36 E. coli isolates harbouring mdfA have been shown to be resistant to benzalkonium.7 The sugE and emrE genes have been demonstrated to provide resistance to CTPC, HAB, cetylpyridinium ammonium bromide, cetyldimethylethyl ammonium bromide and benzalkonium.8,9,11 In addition, the expression of E. coli ydgE/ydgF genes confers host resistance to benzalkonium.11 Our study showed that these genes were not only commonly present in E. coli, but also associated with a decrease in susceptibility to all QACs. Since each bacterial strain has more than one QAC resistance gene, we were not able to assess how much or what level of QAC resistance was contributed by each qac gene or by different qac gene combinations. However, among the six disinfectants tested, regardless of the source of the E. coli isolates, the resistance status and the genotype combinations of QAC resistance genes, TCL remained the most effective disinfectant against E. coli. In conclusion, this study demonstrated that QAC resistance genes were commonly present among E. coli isolated from retail meats. Therefore, using QACs for the decontamination of the environment may not be as effective as expected. Several genes, including qacF, qacG and sugE(p), were newly described in E. coli. The qac and sugE(p) genes were highly associated with multidrug resistance phenotypes. Therefore, using QACs in the food-processing environment could provide selection pressure for strains with acquired resistance to other antimicrobials.
Acknowledgements We are grateful to Maureen Davidson for helpful comments and manuscript review prior to submission.
Disinfectant resistance genes in Escherichia coli
Funding The study was conducted as part of our routine work.
Transparency declarations None to declare.
Supplementary data Tables S1, S2 and S3 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 Bore E, Hebraud M, Chafsey I et al. Adapted tolerance to benzalkonium chloride in Escherichia coli K-12 studied by transcriptome and proteome analyses. Microbiology 2007; 153: 935–46. 2 Buffet-Bataillon S, Tattevin P, Bonnaure-Mallet M et al. Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds-a critical review. Int J Antimicrob Agents 2012; 39: 381–9. 3 Langsrud S, Sundheim G. Factors contributing to the survival of poultry associated Pseudomonas spp. exposed to a quaternary ammonium compound. J Appl Microbiol 1997; 82: 705–12. 4 Langsrud S, Sundheim G, Borgmann-Strahsen R. Intrinsic and acquired resistance to quaternary ammonium compounds in food-related Pseudomonas spp. J Appl Microbiol 2003; 95: 874–82. 5 Buffet-Bataillon S, Le Jeune A, Le Gall-David S et al. Molecular mechanisms of higher MICs of antibiotics and quaternary ammonium compounds for Escherichia coli isolated from bacteraemia. J Antimicrob Chemother 2012; 67: 2837– 42. 6 Bay DC, Turner RJ. Diversity and evolution of the small multidrug resistance protein family. BMC Evol Biol 2009; 9: 140. 7 Edgar R, Bibi E. MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol 1997; 179: 2274– 80. 8 Chung YJ, Saier MH Jr. Overexpression of the Escherichia coli sugE gene confers resistance to a narrow range of quaternary ammonium compounds. J Bacteriol 2002; 184: 2543– 5. 9 Sikora CW, Turner RJ. SMR proteins SugE and EmrE bind ligand with similar affinity and stoichiometry. Biochem Biophys Res Commun 2005; 335: 105–11. 10 Ma C, Chang G. Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli. Proc Natl Acad Sci USA 2007; 104: 3668. 11 Nishino K, Yamaguchi A. Analysis of a complete library of putative drug transporter genes in Escherichia coli. J Bacteriol 2001; 183: 5803– 12. 12 Yerushalmi H, Lebendiker M, Schuldiner S. EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and H+ and is soluble in organic solvents. J Biol Chem 1995; 270: 6856– 63.
JAC 16 Ploy MC, Courvalin P, Lambert T. Characterization of In40 of Enterobacter aerogenes BM2688, a class 1 integron with two new gene cassettes, cmlA2 and qacF. Antimicrob Agents Chemother 1998; 42: 2557– 63. 17 Bay DC, Rommens KL, Turner RJ. Small multidrug resistance proteins: a multidrug transporter family that continues to grow. Bioch Biophy Acta 2008; 1778: 1814– 38. 18 Chuanchuen R, Khemtong S, Padungtod P. Occurrence of qacE/qacED1 genes and their correlation with class 1 integrons in Salmonella enterica isolates from poultry and swine. SE Asian J Trop Med Public Health 2007; 38: 855– 62. 19 Welch TJ, Fricke WF, McDermott PF et al. Multiple antimicrobial resistance in plague: an emerging public health risk. PLoS One 2007; 2: e309. 20 Chaidez C, Lopez J, Castro-del Campo N. Quaternary ammonium compounds: an alternative disinfection method for fresh produce wash water. J Water Health 2007; 5: 329–33. 21 Chapman JS. Disinfectant resistance mechanisms, cross-resistance, and co-resistance. Int Biodeter Biodegr 2003; 51: 271–6. 22 Azap OK, Arslan H, Serefhanoglu K et al. Risk factors for extendedspectrum b-lactamase positivity in uropathogenic Escherichia coli isolated from community-acquired urinary tract infections. Clin Microbiol Infect 2010; 16: 147–51. 23 Xia X, Meng J, McDermott PF et al. Escherichia coli from retail meats carry genes associated with uropathogenic Escherichia coli, but are weakly invasive in human bladder cell culture. J Appl Microbiol 2011; 110: 1166– 76. 24 Buffet-Bataillon S, Branger B, Cormier M et al. Effect of higher minimum inhibitory concentrations of quaternary ammonium compounds in clinical E. coli isolates on antibiotic susceptibilities and clinical outcomes. J Hosp Infect 2011; 79: 141– 6. 25 Zhao S, Blickenstaff K, Bodeis-Jones S et al. Comparison of the prevalences and antimicrobial resistances of Escherichia coli isolates from different retail meats in the United States, 2002 to 2008. Appl Environ Microbiol 2012; 78: 1701– 7. 26 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15. CLSI, Wayne, PA, USA, 2010. 27 Sidhu MS, Sorum H, Holck A. Resistance to quaternary ammonium compounds in food-related bacteria. Microb Drug Resist 2002; 8: 393– 9. 28 Sundheim G, Langsrud S, Heir E et al. Bacterial resistance to disinfectants containing quaternary ammonium compounds. Int Biodeter Biodegr 1998; 41: 235–9. 29 Heir E, Sundheim G, Holck AL. Identification and characterization of quaternary ammonium compound resistant staphylococci from the food industry. Int J Food Microbiol 1999; 48: 211– 9. 30 Kung VL, Ozer EA, Hauser AR. The accessory genome of Pseudomonas aeruginosa. Microbiol Mol Biolo Rev 2010; 74: 621– 41. 31 Call DR, Singer RS, Meng D et al. blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids. Antimicrob Agents Chemother 2010; 54: 590–6.
13 Rapp M, Granseth E, Seppala S et al. Identification and evolution of dual-topology membrane proteins. Nat Struct Mol Biol 2006; 13: 112–6.
32 Rotem D, Schuldiner S. EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry. J Biol Chem 2004; 279: 48787– 93.
14 Kucken D, Feucht H, Kaulfers P. Association of qacE and qacED1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. FEMS Microbiol Lett 2000; 183: 95– 8.
33 He GX, Zhang C, Crow RR et al. SugE, a new member of the SMR family of transporters, contributes to antimicrobial resistance in Enterobacter cloacae. Antimicrob Agents Chemother 2011; 55: 3954 –7.
15 Li D, Yu T, Zhang Y et al. Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river. Appl Environ Microbiol 2010; 76: 3444– 51.
34 Poole K. Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 2005; 56: 20–51. 35 Chen L, Chavda KD, Fraimow HS et al. Complete nucleotide sequence of blaKPC-4 and blaKPC-5 harboring IncN and IncX plasmids from Klebsiella
5 of 6
Zou et al.
pneumoniae strains isolated in New Jersey. Antimicrob Agents Chemother 2012; 57: 269–76.
Escherichia coli isolated from catfish. Foodborne Pathog Dis 2009; 6: 553– 9.
36 Piddock LJ. Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Microbiol 2006; 4: 629–36.
39 Reyes A, Bello H, Dominguez M et al. Prevalence and types of class 1 integrons in aminoglycoside-resistant Enterobacteriaceae from several Chilean hospitals. J Antimicrob Chemother 2003; 51: 317–21.
37 Kazama H, Hamashima H, Sasatsu M et al. Distribution of the antiseptic-resistance gene qacED1 in Gram-positive bacteria. FEMS Microbiol Lett 1998; 165: 295– 9. 38 Nawaz M, Khan AA, Khan S et al. Molecular characterization of tetracycline-resistant genes and integrons from avirulent strains of
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40 Zhao S, Blickenstaff K, Glenn A et al. b-Lactam resistance in Salmonella strains isolated from retail meats in the United States by the National Antimicrobial Resistance Monitoring System between 2002 and 2006. Appl Environ Microbiol 2009; 75: 7624– 30.
J Antimicrob Chemother doi:10.1093/jac/dku197
Presence of disinfectant resistance genes in Escherichia coli isolated from retail meats in the USA Likou Zou1,2, Jianghong Meng1, Patrick F. McDermott3, Fei Wang1, Qianru Yang4, Guojie Cao1, Maria Hoffmann1,3 and Shaohua Zhao3* 1
Department of Nutrition and Food Science, University of Maryland, College Park, MD, USA; 2The Laboratory of Microbiology, Sichuan Agricultural University, Dujiangyan, Sichuan, P. R. China; 3Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD, USA; 4Department of Food Science, Louisiana State University Agricultural Center, 111 Food Science Building, Baton Rouge, Louisiana, USA *Corresponding author. Tel: +1-301-210-4472; Fax: +1-301-210-4685; E-mail: [email protected]
Received 30 December 2013; returned 20 March 2014; revised 28 April 2014; accepted 11 May 2014 Objectives: To examine the distribution of all genes known to be responsible for resistance to quaternary ammonium compounds (QACs), and their association with resistance to QACs and other antimicrobials, in Escherichia coli recovered from retail meats. Methods: A total of 570 strains of E. coli isolated from US retail meats in 2006 were screened for the presence of 10 QAC resistance genes [qacE, qacED1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA and ydgE/ydgF]. The MICs of six common disinfectants were determined using an agar dilution method. Possible associations between the presence of the gene and bacterial resistance to QACs and antimicrobials were investigated. Results: emrE, sugE(c), mdfA and ydgE/ydgF were commonly present (77.2%–100%) in the E. coli isolates, but qac and sugE(p) were less prevalent (0.4%–22.3%). emrE-mdfA-sugE(c)-ydgE/F was the most common QAC resistance gene profile. A significant association was found between antimicrobial resistance and the presence of sugE(p) and qacED1 (P,0.05). Antimicrobial-resistant E. coli isolates tended to contain more diverse combinations of disinfectant resistance genes than susceptible ones. All isolates showed reduced susceptibility to five of six disinfectants compared with the control strains. Higher MICs were generally associated with the presence of qac and sugE(p) genes. Conclusions: The QAC resistance genes were commonly present among E. coli isolated from retail meats, and the qac and sugE(p) genes were highly associated with multidrug resistance phenotypes. Using QACs in the food industry may not be as effective as expected and could provide selection pressure for strains with acquired resistance to other antimicrobials. Keywords: quaternary ammonium compounds, qac genes, antimicrobial resistance, MICs
Introduction The quaternary ammonium compounds (QACs) are non-corrosive and non-irritating, with little toxicity and a high antimicrobial efficacy over a wide pH range, and are routinely used in both clinical and industrial settings.1 – 3 It has been suggested that the widespread use of QACs may contribute to the emergence of antimicrobialresistant bacteria.4,5 There are five QAC resistance genes, sugE(c), emrE, ydgE/ydgF and mdfA, that are known as chromosome-encoded genes conferring QAC resistance.6,7 Unlike mdfA, which belongs to the major facilitator superfamily (MFS), sugE(c), emrE and ydgE/ydgF are members of the small multidrug resistance (SMR) family. The sugE(c) gene mediates high levels of resistance to QACs.8,9 Likewise, emrE,
encoding a multidrug exporter, is associated with resistance to a wide range of toxic cationic hydrophobic compounds, including QACs.10 – 12 Unlike other resistance genes, ydgE and ydgF overlap on the Escherichia coli chromosome and confer resistance to QAC only when co-expressed.6,13 In addition, five QAC resistance genes [qacE, qacED1, qacF, qacG and sugE(p)] have been identified on mobile genetic elements in Gram-negative organisms.14 – 16 These genes belong to the SMR family15,17 and are generally plasmid and/or integron encoded, conferring efflux-mediated resistance to QACs. The qacE gene is located in the 3′ -CS of class 1 integrons in Gram-negative bacteria, and the qacED1 gene is a deletion mutation of qacE that has been associated with an increased MIC of benzalkonium chloride.14,18 The qacF gene displays a high degree of similarity (67.8% identity) to the qacE gene.16 The qacG gene has
Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2014. This work is written by US Government employees and is in the public domain in the US.
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been localized to class 1 integrons in Gram-negative bacteria,15 while sugE(p) is frequently present on an IncA/C multidrug resistance plasmid,19 which is common in Salmonella isolated from food animals and retail meats. Expression of the sugE gene confers high MICs of cetylpyridinium chloride (CTPC),8 a disinfectant used in food-animal processing for carcass washing, as well as a component of mouthwashes, toothpastes and lozenges. QACs are commonly used for disinfecting food-processing and production environments to ensure microbiologically safe food products.20 This raises questions about the possible role of QACs in promoting the development of antimicrobial resistance, in particular co- or cross-resistance to antimicrobials.21 Most E. coli strains are part of the normal intestinal flora but some strains, such as diarrhoeic E. coli, can cause enteric infections. Contamination of food with E. coli may occur during production, processing and distribution and at retail. Generally, meat products are one of the food categories that are most commonly contaminated with E. coli and also are major reservoirs of antimicrobial-resistant E. coli.22,23 E. coli isolates from clinical sources have been shown to exhibit a high MIC of QACs that was correlated with antibiotic resistance.24 While qacED1 is frequently presented in E. coli and other enteric bacteria, we undertook a more comprehensive study to examine the distribution of all known QAC resistance genes, and their association with resistance to QACs and other antimicrobial agents, in E. coli recovered from retail meat products.
Materials and methods Bacterial strains and antimicrobial susceptibility testing A total of 570 E. coli strains were included in this study. They were isolated in 2006 in the USA from retail pork chop (n ¼ 98), ground beef (n ¼ 114), ground turkey (n ¼ 176) and chicken breast (n ¼ 182) by the National Antimicrobial Resistance Monitoring System (NARMS) retail meat programme.25 MICs had previously been determined using the SensititreTM automated antimicrobial susceptibility system in accordance with the manufacturer’s instructions (Trek Diagnostic Systems, Cleveland, OH, USA) and the NARMS protocol. Fifteen antimicrobials were tested: amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, nalidixic acid, ciprofloxacin, gentamicin, kanamycin, amikacin, streptomycin, sulfisoxazole, trimethoprim/sulfamethoxazole and tetracycline. The results were interpreted in accordance with CLSI criteria26 with the exception of those for streptomycin (resistance breakpoint ≥64 mg/L). E. coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were used as quality control organisms.
PCR amplification The primers used to amplify qacE, qacED1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA and ydgE/ydgF were designed based on sequences from GenBank (Table S1, available as Supplementary data at JAC Online). The DNA template was prepared by suspending an overnight culture in 600 mL of reagent-grade water. The suspensions were heated at 1008C for 10 min and centrifuged at 13 000 rpm for 5 min. Each 25 mL of PCR mixture consisted of 2.5 mL of template, 5 mL of 5× PCR buffer, 1.5 mM MgCl2, 200 mM dNTP, 0.4 mM primers and 1.25 U of polymerase (Promega, Madison, WI, USA). Amplified PCR products were analysed on 2.0% (w/v) agarose gels. Appropriate positive and negative controls for amplification were selected from retail meat E. coli isolates. The positive controls that carried the QAC genes were confirmed using PCR followed
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by sequence analysis (GENEWIZ, Inc., Germantown, MD, USA). All results were confirmed by at least two independent experiments.
Determination of MICs of disinfectants The MICs of disinfectants for E. coli were determined using the agar dilution method recommended by the CLSI.26 E. coli suspensions were adjusted to a turbidity equivalent to that of a 0.5 McFarland standard and delivered to the surface of Mueller– Hinton agar plates using a multipoint inoculator (Oxoid, Lenexa, KS, USA). The final inoculum was 104 cfu per spot. Plates were incubated at 378C for 24 h. The disinfectants included were N-alkyl dimethyl benzyl ammonium chloride (ADBAC; Acros Organics, Morris Plains, NJ, USA), cetyltrimethylammonium bromide (CTAB; MP Biomedicals, Santa Ana, CA, USA), CTPC (Sigma, St Louis, MO, USA), hexadecyltrimethylammonium bromide (HAB; Sigma), chlorhexidine (CL; Sigma) and triclosan (TCL; Fisher Scientific, Pittsburgh, PA, USA). The range of concentrations used to determine the MICs of the disinfectants was 0.125 – 1024 mg/L. E. coli ATCC 10536 and S. aureus ATCC 25923 were used as quality control strains.
Data analysis The x2 or Fisher’s exact test and Student’s t-test were used to analyse the data using SAS 9.2 (SAS Institute, Cary, NC, USA). A P value ,0.05 was considered statistically significant for comparisons.
Nucleotide sequence accession numbers The nucleotide sequences of qacF, qacG and sugE(p) have been submitted to GenBank under accession numbers KC285363, KC285364 and KC285365, respectively.
Results Presence of disinfectant resistance genes The ydgE/ydgF genes were the most widespread QAC resistance genes, being found in 100% of isolates, followed by mdfA (96.5%; n¼550), sugE(c) (84.0%; n¼479), emrE (77.2%; n¼440), qacED1 (22.3%; n¼127), sugE(p) (6.8%; n¼39), qacF (1.8%; n¼10) and qacG (0.4%; n¼2). The qacE gene was not detected in any of the isolates. The top two QAC resistance genotypes were emrE-mdfA-sugE(c)-ydgE/F (54.6%) and mdfA-sugE(c)-ydgE/F (17.5%) (Table S2, available as Supplementary data at JAC Online).
Prevalence of QAC resistance genes in E. coli by meat type Regardless of the source, ydgE/ydgF (100%) and mdfA (99.5%) were commonly found in retail meat isolates. The prevalence of other QAC resistance genes varied by meat type. The qacED1 gene was more common (P,0.05) in E. coli isolated from poultry meats (chicken: 31.3%, n ¼ 57; ground turkey: 31.8%, n ¼ 56) than from ground beef (7.0%; n ¼ 8) and pork (6.1%; n ¼ 6). The sugE(p) gene was found in 10.2% (n¼18) of isolates from ground turkey, 7.7% (n¼14) from chicken and 3.5% (n¼4) from beef, but not in E. coli from pork. Surprisingly, the frequency of the sugE(c) gene was higher (P,0.05) in the beef (99.1%) and pork (98.0%) isolates than in the turkey (75.6%) and chicken (75.3%) isolates. The frequency of the qacF gene was slightly higher in E. coli from pork than beef and turkey, and no qacF gene was found in chicken
JAC
Disinfectant resistance genes in Escherichia coli
100.0%
Prevalence of QAC resistance genes
90.0% 80.0% Resistant (n = 399)
70.0%
Susceptible (n = 171)
60.0% 50.0% 40.0%
A
30.0% 20.0%
A
10.0% 0.0%
B ydgE/F
mdfA
emrE
sugE(c) qacEΔ1
B sugE(p)
qacF
qacG
Disinfectant resistance genes Figure 1. Percentage of QAC resistance genes in antibiotic-resistant and antibiotic-susceptible E. coli isolates. A/B, statistically significantly different (P , 0.01). The qacE gene was not detected in any of the isolates.
isolates (Table S2). The qacG gene was only found in two pork isolates, and the qacE gene was not detected in any of the isolates.
Association between disinfectant resistance genes and antimicrobial resistance The association between the presence of sugE(p) and qacED1 and antimicrobial resistance was significant (P, 0.05) (Figure 1). Interestingly, most sugE(p)-positive isolates (79.5%; n ¼ 31) were multidrug resistant (resistant to at least three or more classes of antimicrobial agents). Among the sugE(p)-positive isolates, 87.2% (n ¼ 34) were resistant to ampicillin, 74.4% (n ¼ 29) to amoxicillin/clavulanic acid, ceftriaxone, cefoxitin and ceftiofur, 66.7% (n ¼ 26) to tetracycline, 61.5% (n ¼ 24) to streptomycin and 46.2% (n ¼ 18) to sulfisoxazole. There were 22 different resistance patterns among the sugE(p)positive isolates. The most frequent resistance pattern was ampicillin-amoxicillin/clavulanic acid-ceftriaxone-cefoxitinceftiofur (20.5%; n ¼ 8), followed by ampicillin-amoxicillin/ clavulanic acid-ceftriaxone-cefoxitin-ceftiofur-streptomycin (7.7%; n ¼ 3) and ampicillin-amoxicillin/clavulanic acid-ceftriaxonecefoxitin-ceftiofur-streptomycin-tetracycline (7.7%; n¼3). Among the 127 qacED1-positive isolates, 93.7% (n¼ 119) were resistant to sulfisoxazole, 75.6% (n¼ 96) to gentamicin, 75.6% (n¼ 96) to tetracycline, 59.1% (n ¼ 75) to streptomycin and 30.7% (n ¼ 39) to ampicillin. Furthermore, many of the qacED1-positive isolates were resistant to multiple antimicrobials. For instance, 56.7% (n ¼ 72) were resistant to both sulfisoxazole and gentamicin, 43.3% (n ¼ 55) to sulfisoxazole, gentamicin and streptomycin, and 39.3% (n¼50) to sulfisoxazole, gentamicin and tetracycline. There were 57 different resistance patterns in the qacED1-positive isolates. The most frequent resistance pattern was sulfisoxazolegentamicin-streptomycin-tetracycline (15.0%, n ¼ 19), followed by ampicillin-sulfisoxazole-gentamicin-streptomycin-tetracycline (8.7%, n ¼ 11), sulfisoxazole-gentamicin-streptomycin (7.1%, n ¼ 9) and sulfisoxazole-gentamicin-tetracycline (7.1%, n ¼ 9). Only one resistance pattern, ampicillin-tetracycline, was found in
two qacG-positive isolates. Among 10 qacF-positive isolates, 90.0% (n ¼ 9) were resistant to sulfisoxazole and tetracycline, 60.0% (n¼6) to chloramphenicol and 40.0% (n¼4) to ampicillin. Two qacF-positive isolates were multidrug resistant, showing resistance to ampicillin-chloramphenicol-kanamycin-sulfisoxazole-streptomycin-tetracycline and amoxicillin/clavulanic acid-ampicillincefoxitin-chloramphenicol-kanamycin-sulfisoxazole-tetracycline, respectively. Seventeen different QAC resistance genotypes were identified in resistant and susceptible E. coli (Table S2). However, the genotypes in the resistant E. coli were more diverse than those found in the susceptible isolates. Among 399 antimicrobial-resistant isolates, there were 16 QAC resistance genotypes, whereas there were only seven genotypes among 171 susceptible isolates. mdfA-sugE(c)-ydgE-ydgF and emrE-mdfA-sugE(c)-ydgE-ydgF were the top two genotypes in both resistant and susceptible isolates, together accounting for 96.5% and 61.7% of each group, respectively.
MICs of disinfectants for selected E. coli isolates In general, we observed high MICs of ADBAC, CTAB, CTPC and HAB, and low MICs of CL and TCL (Table S3, available as Supplementary data at JAC Online). Compared with the reference strains, the isolates showed reduced susceptibility to all QACs except for TCL. TCL, however, is a polychlorophenoxy phenol and does not belong to the QAC disinfectant family. High MICs of QAC were observed for the isolates that carried qacG (the R1 genotype). The MICs of CTAB, CTPC and HAB were 512–1024 mg/L, 1024 mg/L and 1024 mg/L, respectively. Isolates containing emrE, mdfA, ydgE/F, sugE(c) and sugE(p) (the R2 genotype) also showed a higher MIC (512 mg/L) of CTPC and HAB. Those with genotypes R3 and R4, carrying qacF, displayed higher MICs (by 2- to 4-fold) of all the disinfectants except TCL, compared with the control strains. Interestingly, the MICs of QACs were not significantly different between qacED1-positive and qacED1-negative isolates among the antimicrobial-resistant isolates. However, pan-susceptible isolates that carried qacED1
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(types S1 and S2) had a higher MIC of CTAB (1024 mg/L) compared with pan-susceptible isolates without qacED1. In general, the higher MICs of QAC were associated with qac and sugE(p).
Discussion Despite the widespread use of chemical interventions, E. coli is commonly present on retail meats,25 and many E. coli exhibit resistance to common antimicrobials. We hypothesized that the use of QACs in the food-processing environment might select for strains with acquired QAC resistance that also carry genes encoding resistance to medically important antimicrobial agents.27 If so, the use of QAC disinfectants might influence the types of antimicrobial-resistant organisms that reach consumers via foods. Reduced susceptibilities to disinfectants have been reported among bacteria isolated from food or food production environments.27 – 29 However, the prevalence of QAC resistance genes in E. coli, the mechanisms of disinfectant resistance and the association between QAC resistance and the antimicrobial susceptibility of E. coli isolated from food were not clear. The present study demonstrated that a variety of QAC resistance genes were present in E. coli. Among the QAC resistance genes, five genes, qacE, qacED1,14 qacF,16,30 qacG 15 and sugE(p),19,31 are located in mobile genetic elements and linked (co-exist) with different antibiotic resistance genes. The other five genes, emrE,12,32 sugE(c),33 mdfA,7 ydgE and ydgF, are chromosome-encoded genes and have shown cross-resistance to different antimicrobials.6,34 Based on previous descriptions of the location of QAC resistance genes, we found that the chromosome-encoded efflux pump genes6,7,9,10,17 were commonly present in E. coli isolated from retail meat. However, mobile-element-encoded QAC genes were relatively low in frequency. Similar findings were reported in Salmonella, with 27.0% positive for qacED1 and 0% positive for qacE.11 Kucken et al.14 reported that the qacED1 gene was detected in 10.0% of isolates and that the qacE gene was found in only 1.0% of isolates. In addition, qacF and qacG, which had also previously been found in Enterobacter aerogenes and Pseudomonas spp.,15,16,30 were present only in 1.8% (n ¼ 10) and 0.4% (n ¼ 2), respectively, of isolates in our study. These genes have been reported to be located in class 1 integrons.15,21,35 Several studies have shown that genes encoding efflux pumps promote bacterial resistance to a narrow range of QACs without conferring cross-resistance to antimicrobials.6,9,36 We examined the association between antimicrobial resistance and the presence of different QAC resistance genes. Most notably, qacED1 and sugE(p) were highly associated (P, 0.05) with antimicrobial resistance. Studies have shown that the qacED1 gene is common in enteric bacteria and is located in the 3′ -conserved segment of class 1 integrons that carry sul1 (sulphonamide resistance determinant).14,37 The qacED1 and sul1 genes usually co-exist in integrons, which could explain the fact that 93.7% (n ¼ 119) of qacED1-positive isolates showed co-resistance to sulphonamides. In addition, 75.6% (n¼ 96) of qacED1-positive isolates were resistant to gentamicin and 56.7% (n ¼ 72) to both sulfisoxazole and gentamicin. Whether or not these resistance genes were present with the qacED1 gene in class 1 integrons was not determined in this study. It is known that multiple gene cassettes can be arranged in tandem within these elements and more than 60 distinct cassettes have been identified to date, including
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aminoglycoside resistance genes.38,39 Cassette-associated genes also have been shown to confer resistance to b-lactams, phenicols, trimethoprim and streptothricin, as well as QACs. Therefore, it was not surprising that the presence of the qacED1 gene in our isolates was frequently associated with resistance to sulfisoxazole, gentamicin, tetracycline, streptomycin and ampicillin. Likewise, many sugE(p)-positive isolates were resistant to ampicillin (87.2%), amoxicillin/clavulanic acid, ceftriaxone, cefoxitin, ceftiofur (74.4%), tetracycline (66.7%), streptomycin (61.5%) and sulfisoxazole (46.2%). The sugE(p) gene in IncA/C (E. coli) and pSN254 (Salmonella) plasmids has been linked with other resistance genes, such as blaCMY-2, sul1, sul2, floR, aadA, aacC and tet.19,31,40 Since most sugE(p)-positive isolates were multidrug resistant, it is likely that the sugE(p) gene is linked with the multidrug resistance IncA/C plasmid and other types of multidrug resistance plasmids. Our findings indicated that using QACs in the food-processing environment could select for strains with acquired QAC resistance that also carry resistance genes to medically important antimicrobial agents such as ceftriaxone, an important drug to treat salmonellosis. Compared with the reference strains, E. coli isolates with different QAC genotypes showed reduced susceptibility to QACs (Table S3). With the exception of mdfA, which belongs to the MFS family, the other genes are members of the SMR family. These resistance genes encode efflux pumps conferring resistance to QACs via an electrochemical proton gradient.17 Several studies have shown that different chromosome-encoded QAC resistance genes have contributed to resistance to QACs.6,7,9,10,36 E. coli isolates harbouring mdfA have been shown to be resistant to benzalkonium.7 The sugE and emrE genes have been demonstrated to provide resistance to CTPC, HAB, cetylpyridinium ammonium bromide, cetyldimethylethyl ammonium bromide and benzalkonium.8,9,11 In addition, the expression of E. coli ydgE/ydgF genes confers host resistance to benzalkonium.11 Our study showed that these genes were not only commonly present in E. coli, but also associated with a decrease in susceptibility to all QACs. Since each bacterial strain has more than one QAC resistance gene, we were not able to assess how much or what level of QAC resistance was contributed by each qac gene or by different qac gene combinations. However, among the six disinfectants tested, regardless of the source of the E. coli isolates, the resistance status and the genotype combinations of QAC resistance genes, TCL remained the most effective disinfectant against E. coli. In conclusion, this study demonstrated that QAC resistance genes were commonly present among E. coli isolated from retail meats. Therefore, using QACs for the decontamination of the environment may not be as effective as expected. Several genes, including qacF, qacG and sugE(p), were newly described in E. coli. The qac and sugE(p) genes were highly associated with multidrug resistance phenotypes. Therefore, using QACs in the food-processing environment could provide selection pressure for strains with acquired resistance to other antimicrobials.
Acknowledgements We are grateful to Maureen Davidson for helpful comments and manuscript review prior to submission.
Disinfectant resistance genes in Escherichia coli
Funding The study was conducted as part of our routine work.
Transparency declarations None to declare.
Supplementary data Tables S1, S2 and S3 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 Bore E, Hebraud M, Chafsey I et al. Adapted tolerance to benzalkonium chloride in Escherichia coli K-12 studied by transcriptome and proteome analyses. Microbiology 2007; 153: 935–46. 2 Buffet-Bataillon S, Tattevin P, Bonnaure-Mallet M et al. Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds-a critical review. Int J Antimicrob Agents 2012; 39: 381–9. 3 Langsrud S, Sundheim G. Factors contributing to the survival of poultry associated Pseudomonas spp. exposed to a quaternary ammonium compound. J Appl Microbiol 1997; 82: 705–12. 4 Langsrud S, Sundheim G, Borgmann-Strahsen R. Intrinsic and acquired resistance to quaternary ammonium compounds in food-related Pseudomonas spp. J Appl Microbiol 2003; 95: 874–82. 5 Buffet-Bataillon S, Le Jeune A, Le Gall-David S et al. Molecular mechanisms of higher MICs of antibiotics and quaternary ammonium compounds for Escherichia coli isolated from bacteraemia. J Antimicrob Chemother 2012; 67: 2837– 42. 6 Bay DC, Turner RJ. Diversity and evolution of the small multidrug resistance protein family. BMC Evol Biol 2009; 9: 140. 7 Edgar R, Bibi E. MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol 1997; 179: 2274– 80. 8 Chung YJ, Saier MH Jr. Overexpression of the Escherichia coli sugE gene confers resistance to a narrow range of quaternary ammonium compounds. J Bacteriol 2002; 184: 2543– 5. 9 Sikora CW, Turner RJ. SMR proteins SugE and EmrE bind ligand with similar affinity and stoichiometry. Biochem Biophys Res Commun 2005; 335: 105–11. 10 Ma C, Chang G. Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli. Proc Natl Acad Sci USA 2007; 104: 3668. 11 Nishino K, Yamaguchi A. Analysis of a complete library of putative drug transporter genes in Escherichia coli. J Bacteriol 2001; 183: 5803– 12. 12 Yerushalmi H, Lebendiker M, Schuldiner S. EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and H+ and is soluble in organic solvents. J Biol Chem 1995; 270: 6856– 63.
JAC 16 Ploy MC, Courvalin P, Lambert T. Characterization of In40 of Enterobacter aerogenes BM2688, a class 1 integron with two new gene cassettes, cmlA2 and qacF. Antimicrob Agents Chemother 1998; 42: 2557– 63. 17 Bay DC, Rommens KL, Turner RJ. Small multidrug resistance proteins: a multidrug transporter family that continues to grow. Bioch Biophy Acta 2008; 1778: 1814– 38. 18 Chuanchuen R, Khemtong S, Padungtod P. Occurrence of qacE/qacED1 genes and their correlation with class 1 integrons in Salmonella enterica isolates from poultry and swine. SE Asian J Trop Med Public Health 2007; 38: 855– 62. 19 Welch TJ, Fricke WF, McDermott PF et al. Multiple antimicrobial resistance in plague: an emerging public health risk. PLoS One 2007; 2: e309. 20 Chaidez C, Lopez J, Castro-del Campo N. Quaternary ammonium compounds: an alternative disinfection method for fresh produce wash water. J Water Health 2007; 5: 329–33. 21 Chapman JS. Disinfectant resistance mechanisms, cross-resistance, and co-resistance. Int Biodeter Biodegr 2003; 51: 271–6. 22 Azap OK, Arslan H, Serefhanoglu K et al. Risk factors for extendedspectrum b-lactamase positivity in uropathogenic Escherichia coli isolated from community-acquired urinary tract infections. Clin Microbiol Infect 2010; 16: 147–51. 23 Xia X, Meng J, McDermott PF et al. Escherichia coli from retail meats carry genes associated with uropathogenic Escherichia coli, but are weakly invasive in human bladder cell culture. J Appl Microbiol 2011; 110: 1166– 76. 24 Buffet-Bataillon S, Branger B, Cormier M et al. Effect of higher minimum inhibitory concentrations of quaternary ammonium compounds in clinical E. coli isolates on antibiotic susceptibilities and clinical outcomes. J Hosp Infect 2011; 79: 141– 6. 25 Zhao S, Blickenstaff K, Bodeis-Jones S et al. Comparison of the prevalences and antimicrobial resistances of Escherichia coli isolates from different retail meats in the United States, 2002 to 2008. Appl Environ Microbiol 2012; 78: 1701– 7. 26 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15. CLSI, Wayne, PA, USA, 2010. 27 Sidhu MS, Sorum H, Holck A. Resistance to quaternary ammonium compounds in food-related bacteria. Microb Drug Resist 2002; 8: 393– 9. 28 Sundheim G, Langsrud S, Heir E et al. Bacterial resistance to disinfectants containing quaternary ammonium compounds. Int Biodeter Biodegr 1998; 41: 235–9. 29 Heir E, Sundheim G, Holck AL. Identification and characterization of quaternary ammonium compound resistant staphylococci from the food industry. Int J Food Microbiol 1999; 48: 211– 9. 30 Kung VL, Ozer EA, Hauser AR. The accessory genome of Pseudomonas aeruginosa. Microbiol Mol Biolo Rev 2010; 74: 621– 41. 31 Call DR, Singer RS, Meng D et al. blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids. Antimicrob Agents Chemother 2010; 54: 590–6.
13 Rapp M, Granseth E, Seppala S et al. Identification and evolution of dual-topology membrane proteins. Nat Struct Mol Biol 2006; 13: 112–6.
32 Rotem D, Schuldiner S. EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry. J Biol Chem 2004; 279: 48787– 93.
14 Kucken D, Feucht H, Kaulfers P. Association of qacE and qacED1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. FEMS Microbiol Lett 2000; 183: 95– 8.
33 He GX, Zhang C, Crow RR et al. SugE, a new member of the SMR family of transporters, contributes to antimicrobial resistance in Enterobacter cloacae. Antimicrob Agents Chemother 2011; 55: 3954 –7.
15 Li D, Yu T, Zhang Y et al. Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river. Appl Environ Microbiol 2010; 76: 3444– 51.
34 Poole K. Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 2005; 56: 20–51. 35 Chen L, Chavda KD, Fraimow HS et al. Complete nucleotide sequence of blaKPC-4 and blaKPC-5 harboring IncN and IncX plasmids from Klebsiella
5 of 6
Zou et al.
pneumoniae strains isolated in New Jersey. Antimicrob Agents Chemother 2012; 57: 269–76.
Escherichia coli isolated from catfish. Foodborne Pathog Dis 2009; 6: 553– 9.
36 Piddock LJ. Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Microbiol 2006; 4: 629–36.
39 Reyes A, Bello H, Dominguez M et al. Prevalence and types of class 1 integrons in aminoglycoside-resistant Enterobacteriaceae from several Chilean hospitals. J Antimicrob Chemother 2003; 51: 317–21.
37 Kazama H, Hamashima H, Sasatsu M et al. Distribution of the antiseptic-resistance gene qacED1 in Gram-positive bacteria. FEMS Microbiol Lett 1998; 165: 295– 9. 38 Nawaz M, Khan AA, Khan S et al. Molecular characterization of tetracycline-resistant genes and integrons from avirulent strains of
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40 Zhao S, Blickenstaff K, Glenn A et al. b-Lactam resistance in Salmonella strains isolated from retail meats in the United States by the National Antimicrobial Resistance Monitoring System between 2002 and 2006. Appl Environ Microbiol 2009; 75: 7624– 30.
