Antibiotic therapy can affect not only the pathogenic bacteria, but also commensal microorganisms in the gut flora of humans, which might serve as a reservoir of antimicrobial resistance genes.Citation1 Escherichia coli can be found widely in nature, and can also be implicated in human infectious diseases. Therefore, fecal E. coli is often considered as a good indicator for selection pressure imposed by antimicrobial use.Citation2 Recently, many researchers began showing interest in the surveillance of antimicrobial resistance in normal flora of humans.Citation3–Citation5 In China, however, very little is known about the prevalence of antibiotic resistant genes in intestinal tracts of healthy people. The aim of our study is to investigate the prevalence of antimicrobial resistance genes and diversity of integrons in fecal E. coli strains recovered from healthy persons in China.
In 2009, we performed a research on the fecal E. coli strains isolated from asymptomatic healthy unrelated human subjects in Fuzhou, China.Citation6 And in this study, 150 fecal E. coli strains were randomly selected from those that were collected in 2009. E. coli ATCC 25922 was used as a positive control.
Susceptibility to antibiotics was assessed by the disc diffusion method according to the CLSI.Citation7 The following 18 antimicrobial agents were tested in this study: ampicillin (AMP, 10 μg), ampicillin plus sulbactam (SAM, 10/10 μg), cefazolin (CZO, 30 μg), cefuroxime (CXM, 30 μg), ceftazidime (CAZ, 30 μg), cefotaxime (CTX, 30 μg), cefepime (FEP, 30 μg), cefoxitin (FOX), aztreonam (ATM, 30 μg), imipenem (IPM, 10 μg), amikacin (AMK, 30 μg), kanamycin (KAN, 30 μg), streptomycin (STR, 10 μg), nalidixic acid (NA), ciprofloxacin (CIP, 5 μg), trimethoprim-sulfamethoxazole (SXT, 1·25/23·75 μg), chloramphenicol (CHL, 30 μg), and tetracycline (TET, 30 μg). The discs were obtained from Oxoid Ltd (Basingstoke, Hampshire, England).
The presence of genes associated with ampicillin (blaTEM, blaSHV, blaOXA-1), tetracycline (tetA–tetE), streptomycin (aadA), sulphonamide (sul1, sul2 and sul3), kanamycin (aphA1 and aphA2), chloramphenicol resistance (cmlA and floR), and plasmid-borne quinolone resistance genes (qnrA, qnrB, and qnrS) was also analyzed by PCR.Citation8,Citation9 The presence of class 1, 2, and 3 integrons was characterized by PCR in all E. coli strains.Citation8 The characterization of the variable region of class 1 and 2 integrons was performed by PCR and subsequent DNA sequencing.
As shown in , the resistances to STR (79·3%), NA (53·3%), TET (50%), AMP (44%), and SXT (32%) were higher than those of the other antimicrobials (0–19·3%). Of these, streptomycin and tetracycline have been widely used as growth promoters or prophylactic agents in animal husbandry.Citation10 The high resistance of gut flora may be due to the extensive and long-term use of these antibiotics in humans and in livestock. Our study also found that the resistance rate of human fecal E. coli strains to third-generation cephalosporins such as CTX (15·3%) and CAZ (4·0%) was higher than that in other countries.Citation11,Citation12 In China, beta-lactams, aminoglycosides, and quinolones are often used to cure Enterobacteriaceae infections. Therefore, it is not surprising that the high resistance of commensal flora may be due to the extensive use of these antimicrobials.
Table 1. Association between antibiotic profile and integrons in 150 fecal E.coli strainsa
Of the 150 E. coli strains tested, 40 (26·7%) carried integrons, among which 39 (97·5%) carried intI1 and only 1 strain carried intI2. Class 3 integrons were not detected in the study. The prevalence of the integrase gene (26·7%) in this study was similar to that in Spain (29%),Citation11 however, it is lower than that found in clinical Enterobacteriaceae isolates in China (59·9%).Citation13 All integron-positive strains showed resistance to at least three antimicrobial agents, as found in previous studies.Citation11,Citation13,Citation14
Six different gene cassette arrangements were demonstrated in 24 of the 39 intI1-positive isolates and were as follows (number of isolates): dfrA17-aadA5 (8), aadA1(6), aadA2(4), aadA5(2), dfrA5(2), and dfrA7(2). Only one E. coli isolate in this study containing class 2 integrons presented the gene cassette array in its variable region: dfrA1- sat2- aadA1, also being frequent in other study.Citation8 And the most frequently detected resistance genes (aadA family and dfr family) in this study were aminoglycoside adenylyltransferase genes and dihydrofolate reductase genes that confer resistance to streptomycin, spectinomycin, and trimethoprim. The high frequencies of aadA and dfr family detection in integrons are similar to other studies.Citation11,Citation13,Citation14
Integrons were significantly associated with AMP, SAM, SXT, KAN, NA, CHL, and TET (), and all 40 integron-positive isolates showed resistance to at least three antimicrobial agents. Higher percentages of resistance to some antimicrobial agents (STR, AMP, TET, SXT, CHL, and NA) were observed among integron-positive isolates with respect to integron-negative isolates. shows the antimicrobial resistance genes detected in our isolates. The blaTEM gene was detected in 27 of the 74 ampicillin-resistant isolates and the blaOXA-1 gene in one additional isolate. Regarding tetracycline resistance, the tetA gene was the predominant one in those strains, and it has been previously suggested that tetA gene and class 1 integrons are often located on the same conjugative plasmid.Citation15
Table 2. Genes of resistance detected among commensal E. coli strains with and without integrons
Higher percentage of resistance to some antimicrobial agents (aminoglycoside, sulphanilamides, quinolones, and beta-lactam agents) were observed among integron-positive strains with respect to integron-negative strains (). The fact could be explained by the presence of resistance genes in the conserved or variable region of integrons, or by the inclusion of resistance genes in the same mobile elements that carry integrons.Citation11
The following genes were identified among 51 SXT-resistant isolates (integron-positive/integron-negative isolates): sul1 (3/7), sul2 (1/0), sul1+sul2 (8/0) and sul2+sul3 (1/0). The aphA1 gene was found in 8 of 50 kanamycin-resistant E. coli isolates. Among the 17 chloramphenicol-resistant E. coli isolates, the cmlA and floR genes were found in eight and three isolates, respectively. Among the 29 nalidixie acid-resistant integron-positive isolates, the qnrS gene was found in one isolate. And aadA gene was detected in 8 of 33 streptomycin resistant integron-positive isolates.
In conclusion, this study demonstrates that multidrug resistance genes and integrons are widespread in fecal E. coli strains from healthy people in China. The integrons may contribute to the dissemination of antibiotic resistance in the gut flora. The data of this study provides useful information regarding the dissemination of antibiotic resistance among healthy humans in the community. Continued surveillance of normal flora should be carried out to predicting the antimicrobial resistance trends of clinical Enterobacteriaceae isolates in this region.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant no. 81201328), and the Natural Science Foundation of Fujian Province, China (Grant no. 2012J05147).
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