Article pubs.acs.org/est
Pathogenic Escherichia coli Strains Producing Extended-Spectrum β‑Lactamases in the Yeongsan River Basin of South Korea Jeonghwan Jang,†,# Yae-Seul Suh,†,# Doris Y. W. Di,† Tatsuya Unno,§ Michael J. Sadowsky,∥,⊥ and Hor-Gil Hur*,†,‡ †
School of Environmental Science and Engineering and ‡International Environmental Analysis and Education Center, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea § College of Molecular Life Sciences, Jeju National University, Jeju, Republic of Korea ∥ Department of Soil, Water and Climate and ⊥BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108, United States ABSTRACT: A total of 3564 E. coli isolates obtained from Yeongsan River basin of South Korea were investigated for their production of extendedspectrum β-lactamases (ESBLs) and potential pathogenicity to better understand the linkage between antibiotic-resistant pathogens in the environment and their public health risks. Interestingly, 60% (53 of 89) of the screen-positive ESBL producers were determined to be potentially one or both of the diarrheagenic and extraintestinal pathogenic (ExPEC) pathotypes, suggesting that trade-off between resistance and virulence of E. coli may not apply to this study. In addition, 67% (60 of 89) of the screen-positive ESBL producers possessed more than one β-lactamase gene, and most (59 of 63) of the ESBL producers had the CTX-M-14 enzyme, which is the most dominant ESBL and seems to be related to urban anthropogenic activities. About 68% (36 of 53) of the potential pathogenic strains were resistant to more than 2 non-β-lactam antibiotics. Results from this study indicate that the Yeongsan River basin has been contaminated with antibioticresistant and potential pathogenic E. coli strains. While few studies have examined pathogenecity of ESBL-producing bacteria, this study reports the possible public health risk which could be caused by the fecal indicator bacterium itself containing both ESBL genes and virulence factors. This will likely impact the dissemination of potential pathogenic E. coli producing ESBLs in the environment and suggests the need for further investigations of antibiotic-resistant pathogens to prevent public health impacts in the Yeongsan River basin.
■
INTRODUCTION Water environments have been considered as bacterial genetic reactors in which rapid genetic variation results from recombination caused by genetic exchanges among bacterial strains.1 It is widely accepted that antibiotic-resistant bacteria released from humans and animals into water systems introduce their resistance genes into the natural bacterial community, including human pathogenic bacteria.2 In addition, it has been reported that antibiotic-resistant intestinal bacteria, including Escherichia coli, can be persistent in water environments3,4 and have been considered to be potential public health risk.3,4 Thus, people can be infected by antibiotic-resistant pathogens through direct or indirect contact with water bodies. Escherichia coli, which is one of the most frequently used indicator bacteria to examine fecal contamination in environments,5 can easily gain resistance to antibiotics consumed by human and animals.6 In recent reports, E. coli also has been shown to survive for long periods of time outside of the host intestine and may even reproduce in secondary habitats.7−10 Our studies and those of others have shown that many E. coli strains with multiple antibiotic resistance can be isolated from various host animals, including humans, in South Korea,11 © 2012 American Chemical Society
implying the consequence of the excessive use of antibiotics in the livestock industry.12 Interestingly, E. coli strains carrying extended-spectrum βlactamases (ESBLs), which have increased activity against the oxymino-cephalosporins (including cefotaxime, ceftazidime, and ceftriaxone), have been isolated from urban river water.13,14 The ESBL producers in human-related environments might increase public health risks since they could easily infect human through drinking water and direct contact with water bodies like other pathogens causing waterborne diseases.15,16 Like other antibiotic resistances, genes for ESBLs are most often encoded on plasmids, which can readily be transferred between strains.17 According to studies performed in South Korea,18 Klebsiella pneumonia and E. coli strains isolated from hospitals in every region of Korea were found to produce ESBLs, such as TEM-19, TEM-20, TEM-52, TEM-116, SHV-2a, and SHV-12. More importantly, 52% of Received: Revised: Accepted: Published: 1128
September 3, 2012 December 19, 2012 December 20, 2012 December 20, 2012 dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
used were well described in the previous study,31 and sampling locations are briefly summarized in Table 1.
them were reported to transfer ceftazidime resistance via conjugation. Furthermore, TEM-52 and CTX-M-14 containing E. coli strains have been found in the Han River in Seoul, South Korea.13 E. coli was observed to be most often responsible for CTX-M production and seems to be a true community pathogen producing ESBLs.19,20 Many recent studies have reported ESBL-producing bacteria including E. coli isolated from environments such as river water, sediments, and sewage sludge and are concerned with the dissemination and transfer of ESBL-encoding genes to pathogens in the environment.13,14,21−24 However, few studies have investigated the pathogenecity of ESBL-producing bacteria which may be directly linked to public health risk. E. coli includes not only commensal strains but also pathogenic ones causing a broad spectrum of human diseases.25 Among intestinal pathotypes of E. coli, shiga toxin-producing E. coli (STEC) is one of important causes of gastrointestinal disease in humans; infections by this bacterium may lead to hemolytic−uremic syndrome (HUS) and thrombocytopenic purpura.26 The STEC strains producing virulence factors such as intimin (encoded by the eaeA gene) and the plasmidmediated enterohemolysin (encoded by the hlyA gene) are among the most virulent STEC strains.27,28 Moreover, E. coli strains producing type 2 shiga toxin (encoded by stx2 gene) have been reported to be more responsible for serious human diseases than strains producing type 1 shiga toxin (encoded by the stx1 gene). Pathotypes implicated in extraintestinal infections, referred to as extraintestinal pathogenic E. coli (ExPEC), have also been considered to be important pathogens for humans because their infections may cause serious human diseases, such as meningitis and sepsis.25 The ExPEC have been defined as strains having more than two of these five genes: afa/dra, kpsMT II, iutA, pap, and sfa/foc.29,30 Potential pathogenic ESBL-producing E. coli strains in surface water of rivers may cause public health risks because they are persistent in the environment and their plasmid-borne antibiotic resistance genes are easily transferred to other bacteria. Fecal indicator bacteria are generally more prevalent in the environment compared to known zoonotic pathogens and thus are used for fecal contamination monitoring. We examined environmental E. coli isolates, a fecal indicator bacterium, for the presence of ESBL genes and virulence factors to investigate possible public health risks in the environment. Accordingly, 3564 E. coli isolates obtained from the Yeongsan River basin in 2009 were examined for the ESBL production and further evaluated for potential pathogenicity based on the presence and absence of virulence factors indicative of STEC and ExPEC strains. In addition, the distribution of four phylogenetic groups (A, B1, B2, and D) and the genetic relatedness among the ESBL-producing isolates were determined to examine correlations between ESBL production and genetic features of E. coli strains.
Table 1. Sampling Locations in the Yeongsan River Basin of South Korea sampling locations YS1 YS2 YS3 YS4 GJ1, GJ2, GJ3
sub-basin (administrative district) Manbong tributary (Naju) Jangseong tributary (Naju) main stream of the Yeongsan River (Gwangju) Orye tributary (Damyang) Gwangju tributary (Gwangju)
major land use affecting sub-basin agricultural agricultural agricultural and urban agricultural urban
Initial Screening for Putative ESBL Producers and Preparation of Their Genomic DNA. All 3564 E. coli strains were initially screened to screen for phenotypical (screenpositive) ESBL producers by using the agar dilution method.32 Briefly, E. coli strains were inoculated onto Muller Hinton agar plates containing 2 mg/L ceftazidime (CAZ), cefotaxime (CTX), or aztreonam (AZT) and incubated at 37 °C for 22 h. Initial screening analyses indicated that 89 of 3564 E. coli strains (2.5%) were screen-positive ESBL producers, and these strains were used for further experiments. Total genomic and plasmid DNA was extracted from the E. coli isolates by boiling in 0.05 N NaOH.33 Briefly, colonies formed on Luria−Bertani agar plates were harvested by sterilized loops and suspended in 100 μL of 0.05 N NaOH. The cells were lysed at 95 °C for 15 min, and cell debris was precipitated by brief centrifugation. A 1:10 dilution of the supernatant was used as DNA template for the further PCR assays. Detection and Identification of ESBL Genes. All 89 screen-positive ESBL-producing strains were examined for the presence of CTX-M, OXA, SHV, and TEM genes by multiplex PCR by using the universal primers described in Table 2.34 The multiplex PCR assay was performed in 20 μL total volume containing 10 μL of AccuPower Multiplex PCR PreMix (Bioneer, Korea), 0.2 μM of each primer, and 2 μL of DNA template. The temperature and time conditions of the PCR program were as described previously.34 PCR products of the expected band sizes for CTX-M and OXA from the multiplex PCR assay were excised from the agarose gel and purified by a AccuPrep Gel Purification kit (Bioneer, Korea). TEM- and SHV-positive strains were subjected to other PCR assays using single primers to obtain larger DNA fragments of the TEM and SHV genes.35 PCR primers used in the PCR assays are described in Table 2. The PCR mix (20 μL total) contained 16 μL of AccuPower PCR PreMix (Bioneer, Korea), 50 pmol of each primer, and 2 μL of DNA template. The PCR conditions used were described previously.35 DNA amplicons from the TEM and SHV PCR assays were purified by a AccuPrep PCR Purification kit (Bioneer, Korea). DNA sequencing services (Macrogen Inc., Korea) for all purified PCR products were provided by ABI PRISM 3730XL Analyzer (Applied Biosystems, Foster City, CA). Database similarity searches for nucleotide sequences were performed using the BLAST tool at the National Center for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih.gov/ BLAST), and protein sequence analyses were performed using a DNA to protein translation program (http://insilico.ehu.es/
■
MATERIALS AND METHODS Isolation of Escherichia coli Strains from Surface Water of the Yeongsan River Basin. E. coli isolates from the Yeongsan River basin in South Korea were isolated in 2009 as previously described.31 A total of 3564 E. coli strains isolated from the same surface water samples at the same time were used in this study. Among them, 3480 E. coli strains were examined in the previous study for season-specific E. coli genotypes.31 The sampling approach and isolation procedures 1129
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
Table 2. Primer Sets Used for Detection of β-Lactamase Genes target type of βlactamase genes SHV
TEM
CTX-M
OXA
SHV
TEM
primer sequence 5′-CTT TAT CGG CCC TCA CTC AA-3′ 5′-AGG TGC TCA TCA TGG GAA AG-3′ 5′-CGC CGC ATA CAC TAT TCT CAG AAT GA-3′ 5′-ACG CTC ACC GGC TCC AGA TTT AT-3′ 5′-ATG TGC AGY ACC AGT AAR GTK ATG GC-3′ 5′-TGG GTR AAR TAR GTS ACC AGA AYC AGC GG-3′ 5′-ACA CAA TAC ATA TCA ACT TCG C-3′ 5′-AGT GTG TTT AGA ATG GTG ATC-3′ 5′-GCC GGG TTA TTC TTA TTT GTC GC-3′ 5′-ATG CCG CCG CCA GTC A3′ 5′-TCG GGG AAA TGT GCG-3′ 5′-TGC TTA ATC AGT GAG GCA CC-3′
amplicon size (bp)
ref
237
34
cefotaxime (0.25−64 mg/L), and cefotaxime (0.25−64 mg/ L)−clavulanic acid (4 mg/L). Phylogenetic Grouping and Virulence Gene Detection. The phylogenetic grouping of the 89 E. coli strains was performed using the Clermont multiplex PCR method as described previously.37 The presence of virulence genes related to diarrheagenic pathotypes, such as shiga toxigenic E. coli (STEC) (eaeA, hlyA, stx1, and stx2), was determined by using multiplex PCR as previously described,38 and virulence factors of extraintestinal pathogenic E. coli (ExPEC) (afa/dra, kpsMT II, iutA, papA/C, and sfa/foc) were detected by the PCR assays previously described.29 E. coli isolates containing more than two of five ExPEC virulence factors have been considered to be potential ExPEC strains.30 Horizontal Fluorophore-Enhanced rep-PCR (HFERP) DNA Fingerprints of E. coli Strains. The horizontal fluorophore-enhanced rep-PCR (HFERP) method for E. coli was used to determine the genetic relatedness of strains.39 The experimental procedures and computative analysis tools of HFERP DNA fingerprinting for the E. coli strains examined in this study have been well described previously,31 and detailed protocols are also accessible at http://www.ecolirep.umn.edu.
445
593
813
1074
35
■
1016
RESULTS Distribution of β-Lactamase Genes among the Escherichia coli Strains. Among the 3564 E. coli strains, 89 (2.5%) were determined to be screen-positive ESBL, and relatively more screen-positive ESBL producers were observed in YS1 (n = 19), YS2 (n = 18), and GJ3 (n = 18) sites. Among the 89 screen-positive ESBL producers, 63 contain ESBL genes, whereas the rest contained only non-ESBL genes such as TEM1 and TEM-171. As shown in Table 3, all of the 89 screenpositive ESBL-producing E. coli isolates possessed TEM genes. The CTX-M genes were also detected in 59 of these E. coli isolates. Only the 3 OXA and 2 SHV genes were found in different strain. Sixty of sixty-three ESBL-producing E. coli strains possess more than 1 β-lactamase gene, and 59 of 60 E. coli strains (98%) producing multiple β-lactamases were also found to contain combinations of TEM and CTX-M genes. Although the TEM-1 gene was the most dominant β-lactamase gene found in this study, the CTX-M-14 gene was the most dominant gene found among those containing the ESBL gene. While the incidence of CTX-M-14 was high at the GJ3 (n = 13), YS1 (n = 9), GJ1 (n = 9), and YS2 (n = 7) sites, greater percentages of E. coli strains carrying CTX-M-14 gene were
translate) and the ClustalW program (http://www.ch.embnet. org/software/ClustalW.html). Antimicrobial Resistance and Susceptibility Test. The resistance of the screen-positive ESBL-producing E. coli strains to nine non-β-lactam antibiotics was tested by the agar dilution method with the minimum inhibitory concentrations (MICs) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.36 The non-β-lactam antibiotics tested included gentamicin (GEN, 16 mg/L), kanamycin (KAN, 64 mg/L), streptomycin (STR, 64 mg/L), tobramycin (TOB, 16 mg/L), tetracycline (TET, 16 mg/L), ciprofloxacin (CIP, 4 mg/L), chloramphenicol (CHL, 32 mg/L), sulfamethoxazole (SUL, 52 mg/L), and trimethoprim (TRI, 16 mg/L). Escherichia coli ATCC-25922 was used as a negative control for all the antibiotic resistance tests. Susceptibility to ceftazidime (CAZ) and cefotaxime (CTX) was tested by the broth microdilution method according to CLSI guidelines,36 with the following concentrations: ceftazidime (0.25−128 mg/ L), ceftazidime (0.25−4 mg/L)−clavulanic acid (4 mg/L),
Table 3. Distribution of β-Lactamases among the 89 Screen-Positive ESBL-Producing Escherichia coli Isolates Collected from the Yeongsan River Basin combinations of β- lactamase type(s) produced (number of isolates)
total nos. 89
TEM
TEM+CTX-M
TEM+CTX-M+OXA
TEM+SHV or TEM+CTX-M+SHV
TEM-1 (13) TEM-22 (2) TEM-52 (1) TEM-171 (13)
TEM-1+CTX-M-3 (2) TEM-1+CTX-M-14 (37) TEM-1+CTX-M-15 (4) TEM-22+CTX-M-3 (1) TEM-22+CTX-M-14 (1) TEM-106+CTX-M-14 (1) TEM-171+CTX-M-3 (1) TEM-171+CTX-M-14 (4) TEM-171+CTX-M-15 (3) TEM-183+CTX-M-14 (1) 55 (61.8%)
TEM-1+CTX-M-14+OXA-1 (1) TEM-1+CTX-M-27+OXA-1 (1) TEM-171+CTX-M-15+OXA-1 (1)
TEM-1+CTX-M-14+SHV-12 (1) TEM-22+SHV-12 (1)
3 (3.4%)
2 (2.2%)
29 (32.6%)
1130
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
Table 4. Prevalence of β-Lactamase Type CTX-M-14 among Sampling Sites sampling locations number of E. coli isolates containing the CTX-M-14 gene number of screen-positive ESBL-producing E. coli isolates percentage of E. coli isolates containing the CTX-M-14 gene among screen-positive ESBL-producing E. coli isolates
Table 5. Distribution of Virulence Genes among Phylogenetic Groups of 89 Screen-Positive ESBL-Producing E. coli Isolates number of traits within phylogenetic group (%)
stx1 stx2 eaeA hlyA papA papC sfa/foc afa/dra iutA kpsMT II EHECa EPECb STECc ExPECd
total number with trait (% of 89) 21 19 22 7 5 7 3 19 29 25 12 10 26 29
(24) (21) (25) (8) (6) (8) (3) (21) (33) (28) (14) (11) (29) (32)
A (n = 24)
B1 (n = 19)
B2 (n = 8)
D (n = 38)
6 (25) 5 (21) 6 (25) 1 (4) 1 (4) 1 (4) 2 (8) 1 (4) 9 (38) 3 (13) 4 (17) 2 (8) 7 (29) 4 (17)
1 (5) 2 (11) 2 (11) 1 (5) 1 (5) 3 (16) 0 (0) 5 (26) 5 (26) 6 (32) 0 (0) 2 (11) 2 (11) 7 (37)
1 (13) 0 (0) 2 (25) 2 (25) 2 (25) 2 (25) 0 (0) 0 (0) 2 (25) 1 (13) 0 (0) 2 (25) 1 (13) 2 (25)
13 (34) 12 (32) 12 (32) 3 (8) 1 (3) 1 (3) 1 (3) 13 (34) 13 (34) 15 (40) 8 (21) 4 (11) 16 (42) 16 (42)
YS2
YS3
YS4
GJ1
GJ2
GJ3
9 19 47.4
7 18 38.9
3 6 50
2 11 18.2
9 13 69.2
3 4 75
13 18 72.2
also found among all of the phylogenetic groups, there were significantly (p < 0.04, t test) more STEC strains in phylogenetic groups A and D (29% and 42%) than in phylogenetic groups B1 and B2 (11% and 13%). Twenty nine of the screen-positive ESBL-producing strains (32%) were found to be ExPEC pathotypes, and the greatest detection rate was observed among E. coli strains belonging to phylogenetic group D (42%), followed by groups B1 (37%), B2 (25%), and A (17%). Distribution of Potential Pathogenic E. coli Strains among ESBL-Producing and Putative ESBL-Producing Isolates. The 53 potential pathogenic E. coli strains were composed of 40 ESBL producers and 13 putative ESBL producers (Tables 6 and 7). Interestingly, 87% of the potential pathogenic strains were also observed to be resistant to non-βlactam antibiotics. The majority of these (73%) showed resistance to tetracycline, while 60% showed resistance to sulfamethoxazole, 51% to streptomycin, 45% to trimethoprim, 38% to each of chloramphenicol and gentamicin, 30% to tobramycin, 19% to kanamycin, and 17% to ciprofloxacin. Furthermore, 68% were resistant to more than two non-βlactam antibiotics. As shown in Table 6, 88% of 40 potential pathogenic ESBLproducers included CTX-M-14 type ESBLs, and all of the strains obtained from urban area (sites GJ1, GJ2, and GJ3) included CTX-M-14 type ESBLs. Interestingly, 23% ESBL producers were also determined to be both pathotypes of diarrheagenic strains and ExPEC. No strains among the 40 ESBL producers belonged to phylogenetic group B2. Thirteen potential pathogenic putative ESBL producers contained TEM1 or TEM-171 type β-lactamases (Table 7) and mainly belonged to phylogenetic groups B2 and D. Two strains, however, belonged to phylogenetic group A and B1. Furthermore, 23% of putative ESBL producers showed traits of both diarrheagenic and ExPEC pathotypes. In addition, we also found that percentages of potential pathogenic E. coli strains among the screen-positive ESBL producers in the Gwangju tributary sites (sites GJ1, GJ2, and GJ3) were generally greater than those of the other sites (Figure 1). Genotypic Relatedness among Potential Pathogenic E. coli Strains. The genetic relatedness among the 53 potential pathogenic E. coli strains was examined by using HFERP DNA fingerprint patterns (Figure 2). As described in a previous study,28 the spatial specificity of E. coli genotypes was not observed because their HFERP DNA fingerprints are clustered without any site specificity (Figure 2), but some groupings of E. coli genotypes clustered in a seasonal-specific manner. The strains obtained from cold months such as October, November, and December are indicated with a bold line (Figure 2). However, neither of the β-lactamases and virulence gene patterns correlated with HFERP DNA fingerprints, indicating that no seasonal specificity was observed for ESBL production and pathogenecity of the E. coli strains. In addition, no
observed from the surface water samples of the Gwangju tributary (Table 4). The detection rate of CTX-M-14 gene among screen-positive ESBL-producing isolates obtained from the Gwangju tributary (the GJ1, GJ2, and GJ3) were significantly (two-sample t test assuming equal variances using Microsoft Excel) greater compared to the Naju region (the YS1 and YS2 sites, p < 0.003), the main stream of the Yeongsan River (the YS3 site, p < 0.02), and the Damyang region (the YS4 site, p < 0.002). Prevalence of Virulence Traits among the 89 Putative ESBL-Positive E. coli and Phylogenetic Groups. Multiplex PCR analyses indicated that the 89 screen-positive ESBLproducing E. coli strains were composed of phylogenetic groups A (n = 24), B1 (n = 19), B2 (n = 8), and D (n = 38). Table 5
bacterial trait or gene
YS1
a
stx1 or/and stx2, eaeA present. beaeA present: without stx1 or stx2. stx1 or/and stx2 present. dMore than or equal to two markers present: papA and/or papC, afa/dra, sfa/foc, iutA, kpsMT II. c
shows the distribution of virulence factors of diarrheagenic and extraintestinal pathogenic E. coli (ExPEC) among the phylogenetic groups among the 89 screen-positive ESBLproducing E. coli strains. On the basis of previously accepted definitions of E. coli pathotypes,30,33,40 the 89 screen-positive ESBL-producing E. coli strains possessing virulence genes were assigned to the pathotypes described in Table 5. Twelve (14%) potential enterohemorhagic E. coli (EHEC) strains were detected among the 89 screen-positive ESBL-producing strains and belonged only to phylogenetic groups A and D. However, 10 (11%) of the potential enteropathogenic E. coli (EPEC) were observed from all of 4 phylogenetic groups. Although 26 of the shiga toxin containing E. coli (STEC) strains (29%) were 1131
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
Table 6. Characteristics of 40 Potential Pathogenic ESBL-Producing E. coli Strains site
strain ID
β-lactamase type
YS1
7-4327
TEM-1/CTXM-3 TEM-171/CTXM-3 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-22 TEM-1/CTX27/OXA-1 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-22
10-4340 7-4326 10-4348 7-4317 7-436 YS2
6-2912 12-2937 10-2946 7-2922 4-2968 4-2967 4-2947 12-2935 5-2915
YS4
4-2351 4-2352
GJ1
6-35-04 5-3517 5-3524 9-356 10-3537 10-3515 5-3570 5-3562
GJ2
7-GJ453 4-GJ452 4-GJ409
GJ3
4-3624 4-3621 4-3625 9-363 9-3610 9-3628 9-361
TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-22/CTXM-14 TEM-106/CTXM-14 TEM-1/CTXM-14 TEM-171/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14/OXA-1 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14
phylogenetic group
diarrheagenic virulence gene
A
stx1, stx2
B1
stx1, stx2, hlyA
B1
eaeA
B1 D
stx1, stx2, eaeA
D
stx2, eaeA
A A
stx1, stx2, eaeA stx2
B1 B1
ExPEC virulence gene
CAZ 128 1
CAZ + CLA
CFT
CFT+ CLA
128
64
64
Tet
64
64
Gen-Tet
64
64
Tet-Sul Gen-Str-Tob-Tet-ChlSul-Tri
0.25
afa/dra
1
afa/dra, kspMT II
0.5
0.25
64
64
4
2
64
32
32
2
32
4
2 1
0.25
32 64
16
afa/dra, iutA, kspMT II afa/dra, kspMT II
1
1
64
64
64
1
afa/dra
2
D
stx1, stx2, eaeA
afa/dra, kspMT II
2
0.5
64
64
D
stx1, stx2
afa/dra, kspMT II
1
0.25
64
8
D
eaeA
afa/dra, kspMT II
2
2
64
64
D
stx1, stx2
1
0.25
1
1
8
4
8
8
PapA, PapC, afa/dra, iutA, kspMT II
D
non-β-lactam resistance
Tet-Sul Tet Tet Gen-Str-Tet-Chl-SulTriGen-Str-Tet-Chl-SulTriGen-Str-Tob-Tet-CipChl-Sul-Tri Gen-Str-Tob-Tet-CipChl-Sul-Tri Gen-Str-Tob-Tet-ChlSul-Tri Gen-Kan-Str-Tob-TetCip-Chl-Sul-Tri Sul-Tri
D
stx1
2
0.5
64
16
D
stx1, stx2
2
0.25
64
4
A
stx1, eaeA
4
0.5
64
32
Gen-Str-Tob-Tet-ChlSul-Tri Gen-Str-Tet-Cip-SulTri Tet
A
eaeA
0.25
B1
eaeA
1
64
Kan-Str-Tob-Tet-SulTri Str-Tet-Sul-Tri
PapA, PapC, iutA, kspMT II
B1
PapA, PapC, iutA, kspMT II PapC, iutA
B1
PapC, iutA
B1
D
A
stx1
A D
eaeA
A
stx1, stx2, eaeA, hlyA stx1, stx2, eaeA
A B1
0.25 32
64 0.25
0.25
64
4
4
64
64
2
0.5
64
64
afa/dra, kspMT II
4
2
64
64
iutA
1
0.25
1
sfa/foc, iutA
2
0.5
64
64
64
4
64
16
iutA, kspMT II
8
1
64
64
iutA, kspMT II
2
0.5
64
2
Gen-Cip
afa/dra, kspMT II
2
0.5
64
4
64
64
Gen-Str-Tob-Tet-ChlSul-Tri Tet
stx2, eaeA, hlyA
D
1
D
stx1, stx2, eaeA
iutA, kspMT II
128
D
stx1
iutA, kspMT II
1
0.25
4
D
stx2
iutA, kspMT II
8
0.25
iutA, kspMT II
8
0.5
D
1132
128
Str-Tet-Sul-Tri
0.25
Str-Tet-Sul-Tri
Gen-Str-Tet-Chl-SulTriGen-Kan-Str-Tob-TetCip-Sul-Tri Kan-Str-Tob-Tet-CipChl-Sul Gen-Kan-Str-Tob-TetCip-Sul-Tri Gen-Tob-Cip
0.25
Tet-Chl-Sul
64
4
Tet-Chl-Sul
64
64
Tet-Chl-Sul
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
Table 6. continued site
strain ID
β-lactamase type
9-362
TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-1/CTXM-14 TEM-183/CTXM-14 TEM-22/CTXM-14
12-3605 12-3666 12-3654 12-3657
phylogenetic group
diarrheagenic virulence gene
ExPEC virulence gene
CAZ
CAZ + CLA
CFT
CFT+ CLA
64
64
D
iutA, kspMT II
8
0.5
D
afa/dra, kspMT II
8
4
8
8
D
afa/dra, iutA
2
1
64
64
D
afa/dra, iutA
2
1
64
64
D
afa/dra, iutA
2
1
8
1
non-β-lactam resistance Tet-Chl-Sul
Gen-Kan-Str-Tob-TetChl-Sul-Tri Gen-Kan-Str-Tob-TetChl-Sul-Tri
Table 7. Characteristics of 13 Potential Pathogenic Putative ESBL-Producing E. coli Strains site
strain ID
β-lactamase type
phylogenetic group
YS1
10-4371 4-4346
TEM-171 TEM-1
10-433 9-4336 4-2953 6-2917 4-2736 6-2350 6-2338 4-3526 9-353 12-3619 4-3645
YS2
YS3 YS4 GJ1 GJ3
diarrheagenic virulence gene
ExPEC virulence gene
CAZ
CAZ + CLA
CFT
B1 B2
stx2 eaeA
kpsMT II papA, papC, kspMT II
0.25 32
0.25 32
8 8
8
TEM-1 TEM-1 TEM-1 TEM-1
B2 D A D
stx1, hlyA stx1, stx2,eaeA eaeA stx1
4 8 0.25 1
0.5 2 0.25 0.25
64 8
4 4
64
8
TEM-171 TEM-171 TEM-171 TEM-1 TEM-1 TEM-171 TEM-1
D B2 D B2 D D D
stx1, stx, eaeA eaeA, hlyA eaeA
64 16 64
2 16 64
Tet Str Gen-Kan-Str-Tob-Cip-Sul Gen-Kan-Str-Tob-TetChl-Sul-Tri Str Tet-Sul-Tri Tet Str-Tet
64 64
64 64
Str-Sul-Tri Gen-Str-Tet-Chl-Sul-Tri
iutA sfa/foc, iutA, kpsMT II iutA
papA, papC, iutA stx1, eaeA eaeA stx1, stx2
sfa/foc, kpsMT II iutA
1 0.5 64 2 0.5 8 8
0.25 0.25 64 0.5 0.5 2 4
CFT+ CLA
non-β-lactam resistance Tet
in clinical studies in South Korea,41−43 suggesting the possible dissemination of β-lactamases, including ESBLs, into the environment. Furthermore, E. coli strains that possess more than one β-lactamase gene possibly increase risks to public health due to therapeutic failures of antibiotics against strains that produce multiple β-lactamases.44 The CTX-M enzymes were previously reported to be the most abundant ESBLs in urban river sediments22 and other urban river environments in Korea13 and the UK.14 Moreover, their appearance has recently increased in hospital settings.42,45 It should be noted that E. coli strains containing CTX-M-14, which was the dominant ESBL enzyme found in this study, were also recovered from sampling sites affected by urban area, suggesting that urban anthropogenic events, such as discharge from hospitals and sewage overflow,21,22,46 may disseminate ESBLs into the environments. This is clearly not a local issue, as CTX-M-type enzymes have rapidly become the dominant ESBL globally.47,48 Likewise, higher frequency of potential pathogenic E. coli strains were recovered in the urban river sites than in the other sites, suggesting that urban anthropogenic events may also disseminate pathogens. Recently, Unno et al. reported that E. coli strains belonging to phylogenetic group B2 were absent from human and livestock animals in the Yeongsan River basin of South Korea but not from geese and the Yeongsan River water.33 The lower prevalence of antibiotic resistance among phylogenetic group B2 strains was also observed in a previous study.49 Several studies have reported that phylogenetic group B2 strains tend to be more virulent than other groups,29,50−52 and virulence genes were most frequently present in phylogenetic group B1 strains while absent in B2 strains.33 However, pathotypes of
Figure 1. Prevalence of potential pathogenic E. coli strains among the screen-positive ESBL producers in each sampling location.
relatedness between phylogenetic groups and HFERP DNA fingerprint types was observed (Figure 2).
■
DISCUSSION Results of this study showed the prevalence of potentially pathogenic ESBL-producing E. coli strains in the Yeongsan River basin of South Korea. While a limited number (89) of screen-positive ESBL-producing E. coli strains were detected in this study, their existence should be of concern, as ESBL genes can be easily transferred to pathogens and other bacteria due to their location on plasmids17 and E. coli strains and other enteric pathogens persist in the environment.7−10 Most of ESBL and non-ESBL genes found in this study were previously reported 1133
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
Figure 2. Cluster analysis of potential pathogenic E. coli strains based on similarity between HFERP DNA fingerprints of each strain. The seasonally clustered strains which were obtained from cold months are indicated with a bold line.
resistance to non-β-lactam antibiotics, and 23% of them were determined to be both pathotypes of diarrheagenic and ExPEC. The previously suggested trade-off between resistance and virulence of E. coli49,53,54 may not apply to the strains examined in this study. These results indicate that rapid transfer of antimicrobial resistance and mobile virulence factors between pathogenic strains in the river water likely occurs and suggests that increased monitoring of antibiotic-resistant pathogens is needed to protect public health in the Yeongsan River basin. Moreover, we have recovered potentially pathogenic ESBLproducing E. coli strains, suggesting that ESBL-encoding genes may be disseminated and transferred to pathogens in the environments.
diarrheagenic and ExPEC were less frequently detected among phylogenetic group B2 strains than the other groups in this study, suggesting that the relationship between virulence and phylogeny could depend on research conditions such as geographical reasons and target E. coli group which is like a ESBL producers group. A trade-off between antibiotic resistance and virulence of E. coli has been reported in several previous studies. For example, E. coli strains resistant to quinolones and fluoroquinolones contain fewer virulence factors than susceptible ones and seem to have reduced invasiveness.53,54 A lack of hemolysin and P fimbriae has also been related to resistance to antibiotics.49 In this current study, however, most of potential pathogenic E. coli strains, including ESBL and non-ESBL types, also showed 1134
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
(7) Byappanahalli, M. N.; Fujioka, R. S. Evidence that tropical soil environment can support the growth of Escherichia coli. Water Sci. Technol. 1998, 38, 171−174. (8) Sadowsky, M. J.; Whitman, R. L. Fecal Bacteria; ASM Press: Washington, DC, 2010. (9) Byappanahalli, M. N.; Whitman, R. L.; Shively, D. A.; Sadowsky, M. J.; Ishii, S. Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lakes watershed. Environ. Microbiol. 2006, 8 (3), 504−513. (10) Ishii, S.; Ksoll, W. B.; Hicks, R. E.; Sadowsky, M. J. Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds. Appl. Environ. Microb. 2006, 72 (1), 612−621. (11) Unno, T.; Han, D.; Jang, J.; Lee, S.-N.; Kim, J. H.; Ko, G.; Kim, B. G.; Ahn, J.-H.; Kanaly, R. A.; Sadowsky, M. J.; Hur, H.-G. High diversity and abundance of antibiotic-resistant Escherichia coli isolated from humans and farm animal hosts in Jeonnam Province, South Korea. Sci. Total Environ. 2010, 408 (17), 3499−3506. (12) Park, S.; Choi, K. Hazard assessment of commonly used agricultural antibiotics on aquatic ecosystems. Ecotoxicology 2008, 17 (6), 526−538. (13) Kim, J.; Kang, H.; Lee, Y. The identification of CTX-M-14, TEM-52, and CMY-1 enzymes in Escherichia coli isolated from the Han River in Korea. J. Microbiol. 2008, 46 (5), 478−481. (14) Dhanji, H.; Murphy, N. M.; Akhigbe, C.; Doumith, M.; Hope, R.; Livermore, D. M.; Woodford, N. Isolation of fluoroquinoloneresistant O25b:H4-ST131 Escherichia coli with CTX-M-14 extendedspectrum β-lactamase from UK river water. J. Antimicrob. Chemother. 2011, 66 (3), 512−516. (15) Akashi, S.; Joh, K.; Mori, T.; Tsuji, A.; Ito, H.; Hoshi, H.; Hayakawa, T.; Ihara, J.; Abe, T.; Hatori, M.; Nakamura, T.; Akashi, S. A severe outbreak of haemorrhagic colitis and haemolytic uraemic syndrome associated with Escherichia coli O157:H7 in Japan. Eur. J. Pediatr. 1994, 153 (9), 650−655. (16) Keene, W. E.; McAnulty, J. M.; Hoesly, F. C.; Williams, L. P.; Hedberg, K.; Oxman, G. L.; Barrett, T. J.; Pfaller, M. A.; Fleming, D. W. A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei. New Engl. J. Med. 1994, 331 (9), 579−584. (17) Bradford, P. A. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 2001, 14 (4), 933− 951. (18) Jeong, S. H.; Bae, I. K.; Lee, J. H.; Sohn, S. G.; Kang, G. H.; Jeon, G. J.; Kim, Y. H.; Jeong, B. C.; Lee, S. H. Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey. J. Clin. Microbiol. 2004, 42 (7), 2902−2906. (19) Pitout, J. D. D.; Nordmann, P.; Laupland, K. B.; Poirel, L. Emergence of Enterobacteriaceae producing extended-spectrum βlactamases (ESBLs) in the community. J. Antimicrob. Chemother. 2005, 56 (1), 52−59. (20) Pitout, J. D. D.; Laupland, K. B. Extended-spectrum βlactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 2008, 8 (3), 159−166. (21) Reinthaler, F. F.; Feierl, G.; Galler, H.; Haas, D.; Leitner, E.; Mascher, F.; Melkes, A.; Posch, J.; Winter, I.; Zarfel, G.; Marth, E. ESBL-producing E. coli in Austrian sewage sludge. Water Res. 2010, 44 (6), 1981−1985. (22) Lu, S.-Y.; Zhang, Y.-L.; Geng, S.-N.; Li, T.-Y.; Ye, Z.-M.; Zhang, D.-S.; Zou, F.; Zhou, H.-W. High Diversity of extended-spectrum betalactamase-producing bacteria in an urban river sediment habitat. Appl. Environ. Microb. 2010, 76 (17), 5972−5976. (23) Sharma, A.; Singh, D. P. TN, The prevalence of extendedspectrum β-lactamase in environmental isolates of Enterobacter. Indian J. Pathol. Microbiol. 2008, 51, 130−136. (24) Chen, H.; Shu, W.; Chang, X.; Chen, J.-a.; Guo, Y.; Tan, Y. The profile of antibiotics resistance and integrons of extended-spectrum beta-lactamase producing thermotolerant coliforms isolated from the
The seasonal specificity of HFERP genotypes of E. coli observed in a previous study31 was also seen with the potential pathogenic E. coli strains possessing ESBLs and non-ESBLs. However, there was no relationship between HFERP DNA fingerprints and the presence of ESBL-production genes. In addition, cluster analysis based on similarity of HFERP DNA fingerprints did not correlate with phylogenetic groupings as previously described,33 and this lack of correlation may be caused by low sensitivity of HFERP DNA fingerprints to detect exact phylogenetic groups of E. coli strains.55 In conclusion, we have shown that the Yeongsan River basin is contaminated with antibiotic-resistant and potentially pathogenic E. coli strains throughout the year. Some strains showed traits of both diarrheagenic and ExPEC pathotypes and high levels of multiple antibiotic resistances. The CTX-M-14 enzyme, which is the most dominant ESBL found in this study, appeared to be more prevalent in urban areas. While the number of screen-positive ESBL producers was relatively small compared to the total number of strains examined, our results suggest the need for greater monitoring of antibiotic-resistant and pathogenic microorganisms which might originate from human activities.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +82-62-715-2437; fax: +82-62-715-2434; e-mail:
[email protected]. Author Contributions #
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF: 2011-0029860), a grant from the Next-Generation BioGreen 21 Program (TAGC, PJ009094) of Rural Development Administration, and the Basic Research Project through a grant provided by GIST. Furthemore, the authors acknowledge a grant-in-aid for research from Gwangju Regional Environmental Technology Development Center (10-2-70-73).
■
REFERENCES
(1) Baquero, F.; Martínez, J.-L.; Cantón, R. Antibiotics and antibiotic resistance in water environments. Curr. Opin. Biotechnol. 2008, 19 (3), 260−265. (2) Alonso, A.; Sánchez, P.; Martínez, J. L. Environmental selection of antibiotic resistance genes. Environ. Microbiol. 2001, 3 (1), 1−9. (3) Sapkota, A. R.; Curriero, F. C.; Gibson, K. E.; Schwab, K. J., Antibiotic-resistant enterococci and fecal indicators in surface water and groundwater impacted by a concentrated swine feeding operation. Environ. Health Perspect. 2007, 115, (7). (4) Xi, C.; Zhang, Y.; Marrs, C. F.; Ye, W.; Simon, C.; Foxman, B.; Nriagu, J. Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Appl. Environ. Microb. 2009, 75 (17), 5714−5718. (5) USEPA. Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified Membrane-Thermotolerant Escherichia coli Agar. U.S. Environmental Protection Agency, Washington, DC, 2006. (6) Cristino, J. M. Correlation between consumption of antimicrobials in humans and development of resistance in bacteria. Int. J. Antimicrob. Agents 1999, 12 (3), 199−202. 1135
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
Environmental Science & Technology
Article
type extended-spectrum β-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea. J. Antimicrob. Chemother. 2005, 56 (4), 698−702. (43) Ko, K. S.; Lee, M. Y.; Song, J.-H.; Lee, H.; Jung, D. S.; Jung, S.I.; Kim, S.-W.; Chang, H.-H.; Yeom, J.-S.; Kim, Y.-S.; Ki, H. K.; Chung, D.-R.; Kwon, K. T.; Peck, K. R.; Lee, N. Y. Prevalence and characterization of extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Korean hospitals. Diagn. Microb. Infect. Dis. 2008, 61 (4), 453−459. (44) Ahmad, M.; Urban, C.; Mariano, N.; Bradford, P. A.; Calcagni, E.; Projan, S. J.; Bush, K.; Rahal, J. J. Clinical characteristics and molecular epidemiology associated with imipenem-resistant Klebsiella pneumoniae. Clin. Infect. Dis. 1999, 29 (2), 352−355. (45) Weill, F.-X.; Perrier-Gros-Claude, J.-D.; Demartin, M.; Coignard, S.; Grimont, P. A. D. Characterization of extendedspectrum-β-lactamase (CTX-M-15)-producing strains of Salmonella enterica isolated in France and Senegal. FEMS Microbiol. Lett. 2004, 238 (2), 353−358. (46) Morris, D.; Galvin, S.; Boyle, F.; Hickey, P.; Mulligan, M.; Cormican, M. Enterococcus faecium of the vanA genotype in rural drinking water, effluent, and the aqueous environment. Appl. Environ. Microb. 2012, 78 (2), 596−598. (47) Suzuki, S.; Shibata, N.; Yamane, K.; Wachino, J.-i.; Ito, K.; Arakawa, Y. Change in the prevalence of extended-spectrum-βlactamase-producing Escherichia coli in Japan by clonal spread. J. Antimicrob. Chemother. 2009, 63 (1), 72−79. (48) Livermore, D. M.; Canton, R.; Gniadkowski, M.; Nordmann, P.; Rossolini, G. M.; Arlet, G.; Ayala, J.; Coque, T. M.; Kern-Zdanowicz, I.; Luzzaro, F.; Poirel, L.; Woodford, N. CTX-M: changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 2007, 59 (2), 165−174. (49) Johnson, J. R.; Goullet, P.; Picard, B.; Moseley, S. L.; Roberts, P. L.; Stamm, W. E. Association of carboxylesterase B electrophoretic pattern with presence and expression of urovirulence factor determinants and antimicrobial resistance among strains of Escherichia coli that cause urosepsis. Infect. Immun. 1991, 59 (7), 2311−2315. (50) Le Gall, T.; Clermont, O.; Gouriou, S.; Picard, B.; Nassif, X.; Denamur, E.; Tenaillon, O. Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group Escherichia coli strains. Mol. Biol. Evol. 2007, 24 (11), 2373−2384. (51) Escobar-Páramo, P.; Grenet, K.; Le Menac’h, A.; Rode, L.; Salgado, E.; Amorin, C.; Gouriou, S.; Picard, B.; Rahimy, M. C.; Andremont, A.; Denamur, E.; Ruimy, R. Large-scale population structure of human commensal Escherichia coli isolates. Appl. Environ. Microb. 2004, 70 (9), 5698−5700. (52) Picard, B.; Garcia, J. S.; Gouriou, S.; Duriez, P.; Brahimi, N.; Bingen, E.; Elion, J.; Denamur, E. The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect. Immun. 1999, 67 (2), 546−553. (53) Vila, J.; Simon, K.; Ruiz, J.; Horcajada, J. P.; Velasco, M.; Barranco, M.; Moreno, A.; Mensa, J. Are quinolone-resistant uropathogenic Escherichia coli less virulent? J. Infect. Dis. 2002, 186 (7), 1039−1042. (54) Velasco, M.; Horcajada, J. P.; Mensa, J.; Martinez, A. M.; Vila, J.; Martinez, J. A.; Ruiz, J.; Barranco, M.; Roig, G.; Soriano, E. Decreased invasive capacity of quinolone-resistant Escherichia coli in patients with urinary tract infections. Clin. Infect. Dis. 2001, 33 (10), 1682−1686. (55) Ishii, S.; Sadowsky, M. J. Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution. Environ. Microbiol. 2009, 11 (4), 733−740.
Yangtze River basin in Chongqing. Environ. Pollut. 2010, 158 (7), 2459−2464. (25) Kaper, J. B.; Nataro, J. P.; Mobley, H. L. T. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2004, 2 (2), 123−140. (26) Karmali, M. Infection by shiga toxin-producing Escherichia coli. Mol. Biotechnol. 2004, 26 (2), 117−122. (27) Schmidt, H.; Karch, H. Enterohemolytic phenotypes and genotypes of shiga toxin-producing Escherichia coli O111 strains from patients with diarrhea and hemolytic-uremic syndrome. J. Clin. Microbiol. 1996, 34 (10), 2364−7. (28) Barrett, T. J.; Kaper, J. B.; Jerse, A. E.; Wachsmuth, I. K. Virulence factors in shiga-like toxin-producing Escherichia coli isolated from humans and cattle. J. Infect. Dis. 1992, 165 (5), 979−980. (29) Johnson, J. R.; Stell, A. L. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J. Infect. Dis. 2000, 181 (1), 261− 272. (30) Johnson, J. R.; Murray, A. C.; Gajewski, A.; Sullivan, M.; Snippes, P.; Kuskowski, M. A.; Smith, K. E. Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chicken products. Antimicrob. Agents Chemother. 2003, 47 (7), 2161−2168. (31) Jang, J.; Unno, T.; Lee, S. W.; Cho, K. H.; Sadowsky, M. J.; Ko, G.; Kim, J. H.; Hur, H.-G. Prevalence of season-specific Escherichia coli strains in the Yeongsan River Basin of South Korea. Environ. Microbiol. 2011, 13 (12), 3103−3113. (32) Thomson, K. S.; Sanders, C. C. A simple and reliable method to screen isolates of Escherichia coli and Klebsiella pneumoniae for the production of TEM- and SHV-derived extended-spectrum βlactamases. Clin. Microbiol. Infect. 1997, 3 (5), 549−554. (33) Unno, T.; Han, D.; Jang, J.; Lee, S.-N.; Ko, G.; Choi, H. Y.; Kim, J. H.; Sadowsky, M. J.; Hur, H.-G. Absence of Escherichia coli phylogenetic group B2 strains in humans and domesticated animals from Jeonnam province, Republic of Korea. Appl. Environ. Microb. 2009, 75 (17), 5659−5666. (34) Fang, H.; Ataker, F.; Hedin, G.; Dornbusch, K. Molecular epidemiology of extended-spectrum β-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006. J. Clin. Microbiol. 2008, 46 (2), 707− 712. (35) Rayamajhi, N.; Kang, S. G.; Lee, D. Y.; Kang, M. L.; Lee, S. I.; Park, K. Y.; Lee, H. S.; Yoo, H. S. Characterization of TEM-, SHV- and AmpC-type β-lactamases from cephalosporin-resistant Enterobacteriaceae isolated from swine. Int. J. Food Microbiol. 2008, 124 (2), 183− 187. (36) CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement; CLSI: Wayne, PA, 2010. (37) Clermont, O.; Bonacorsi, S.; Bingen, E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microb. 2000, 66 (10), 4555−4558. (38) Paton, A. W.; Paton, J. C. Detection and characterization of shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfb O111, and rfb O157. J. Clin. Microbiol. 1998, 36 (2), 598−602. (39) Johnson, L. K.; Brown, M. B.; Carruthers, E. A.; Ferguson, J. A.; Dombek, P. E.; Sadowsky, M. J. Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution. Appl. Environ. Microb. 2004, 70, 4478−4485. (40) Tarr, P. I.; Gordon, C. A.; Chandler, W. L. Shiga-toxinproducing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005, 365 (9464), 1073−1086. (41) Kim, J.; Lim, Y.-M.; Rheem, I.; Lee, Y.; Lee, J.-C.; Seol, S.-Y.; Lee, Y.-C.; Cho, D.-T. CTX-M and SHV-12 β-lactamases are the most common extended-spectrum enzymes in clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 3 university hospitals within Korea. FEMS Microbiol. Lett. 2005, 245 (1), 93−98. (42) Ryoo, N. H.; Kim, E.-C.; Hong, S. G.; Park, Y. J.; Lee, K.; Bae, I. K.; Song, E. H.; Jeong, S. H. Dissemination of SHV-12 and CTX-M1136
dx.doi.org/10.1021/es303577u | Environ. Sci. Technol. 2013, 47, 1128−1136
