Abstract
Recently, CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains have emerged worldwide. In particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131), which is often associated with the CTX-M-15 ESBL, has been increasingly reported globally; however, epidemiology reports on ESBL-producing E. coli in Asia are limited. Patients with clinical isolates of ESBL-producing E. coli in the Tribhuvan University teaching hospital in Kathmandu, Nepal, were included in this study. Whole-genome sequencing of the isolates was conducted to analyze multilocus sequence types, phylotypes, virulence genotypes, O25b-ST131 clones, and distribution of acquired drug resistance genes. During the study period, 105 patients with ESBL-producing E. coli isolation were identified, and the majority (90%) of these isolates were CTX-M-15 positive. The most dominant ST was ST131 (n = 54; 51.4%), followed by ST648 (n = 15; 14.3%). All ST131 isolates were identified as O25b-ST131 clones, subclone H30-Rx. Three ST groups (ST131, ST648, and non-ST131/648) were compared in further analyses. ST648 isolates had a proportionally higher resistance to non-β-lactam antibiotics and featured drug-resistant genes more frequently than ST131 or non-ST131/648 isolates. ST131 possessed the most virulence genes, followed by ST648. The clinical characteristics were similar among groups. More than 38% of ESBL-producing E. coli isolates were from the outpatient clinic, and pregnant patients comprised 24% of ESBL-producing E. coli cases. We revealed that the high resistance of ESBL-producing E. coli to multiple classes of antibiotics in Nepal is driven mainly by CTX-M-producing ST131 and ST648. Their immense prevalence in the communities is a matter of great concern.
INTRODUCTION
Escherichia coli is a part of the normal human and animal gastrointestinal flora; it is the most common cause of urinary tract infections and also causes various other infectious conditions, such as intra-abdominal infections, neonatal meningitis, and septicemia (1–3).
Recently, extended-spectrum-beta-lactamase (ESBL) producing E. coli strains, particularly strains producing CTX-M-type ESBLs, have emerged worldwide (4). In particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131) is often associated with the CTX-M-15 ESBL and has been increasingly reported globally. These bacteria are resistant to classes of antibiotics distinct from β-lactams, such as fluoroquinolones and trimethoprim-sulfamethoxazole (5, 6). Epidemiology reports on ESBL-producing E. coli in Asia are limited to date. To the best of our knowledge, there has been no report on the prevalence of pandemic ESBL-producing E. coli ST131, or other potentially dominant ESBL-producing E. coli STs, or clinical and microbiological information pertaining to their isolation in Nepal. Nepal is located in south Asia and adjacent to India, where a high proportion of resistant Gram-negative bacteria has been reported (7); understanding the epidemiology of ESBL-producing E. coli in this region is therefore particularly important. In addition, the patients population and their clinical background in developing countries are different from those in developed countries, where the majority of studies on ESBL-producing E. coli have been conducted. It is thus imperative to reveal the clinical and microbiological characteristics of ESBL-producing E. coli in developing countries in order to better understand the global epidemiology of this pathogen. In this study, we aimed to elucidate the clinical and microbiological characteristics of ESBL-producing E. coli and specifically to reveal the unique aspects of the dominant ESBL-producing E. coli ST in Nepal.
MATERIALS AND METHODS
Study settings and design.
Microbiological investigations and clinical epidemiological analyses of ESBL-producing E. coli were conducted among patients from whom ESBL-producing E. coli was isolated in the Tribhuvan University teaching hospital, which serves as a tertiary referral hospital in Kathmandu, Nepal. Institutional review boards at Tribhuvan University approved the study before its initiation. The study period, including chart reviewing, was from 1 February 2013 to 31 January 2014.
Patients and variables.
Patients with clinical isolation of ESBL-producing E. coli between 1 February 2013 and 31 July 2013 were divided into three groups, i.e., ESBL-producing E. coli ST131, ESBL-producing E. coli ST648, and ESBL-producing E. coli non-ST131/648 (ESBL-producing E. coli isolates of ST other than ST131 and ST648), based on multilocus sequence type (MLST). For patients from whom more than one ESBL-producing E. coli strain was isolated during the study period, only the first episode was analyzed; this study therefore incorporated only unique patient episodes. Parameters retrieved from the patient records included (i) demographics, (ii) background conditions and clinical diagnosis. (iii) duration of hospital stay, and (iv) antimicrobial treatment during the current hospital stay (or at the outpatient clinic for outpatients).
Isolates.
Standard identification and susceptibility testing of E. coli were performed and interpreted in accordance with the Clinical and Laboratory Standard Institute (CLSI) criteria (8), using an automated broth microdilution system (MicroScan; Siemens AG, Germany) unless otherwise stated. To determine the MIC of fosfomycin, an NC6.11J panel (Siemens AG, Germany) was used.
In addition, we tested MICs of flomoxef, cefoperazone-sulbactam, and fosfomycin, as they are potentially active against ESBL-producing E. coli (9–11). The breakpoints for susceptibility were ≤8 μg/ml for flomoxef (12), and ≤16/8 μg/ml for cefoperazone-sulbactam (13). ESBL production was confirmed with disc diffusion tests in accordance with the 2009 CLSI criteria (14).
Molecular analysis.
Molecular analysis was conducted in the Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan. Whole-genome sequences of all 105 isolates were obtained using a MiSeq system with Nextera XT library kits (Illumina, Tokyo, Japan) for the analysis of MLST, phylotypes (15), H30 and H30-Rx subclones of ST131 (16), virulence genotypes (17), O25b-ST131 clones (18), and distribution of acquired drug resistance genes. Approximately one million 301-bp by 2 pair-end reads were obtained. After trimming based on base quality (quality score limit = 0.05; removing reads with more than 2 ambiguous nucleotides or of less than 15 bp in length), the reads were assembled de novo to construct contigs without annotation using the commercial software CLC genomics workbench (CLC Bio, Tokyo, Japan). The contigs were subjected to further analyses with the BLAST algorithm (19) and Resfinder (20). An identification rate of more than 98% was considered positive for each targeted gene. The virulence score was determined as the number of virulence genes detected, with pap elements counting collectively as a single trait (17).
We selected sequences of contigs that could contain mobile acquired elements carrying resistance genes in the isolates, based on BLAST (19) and ResFinder (20) analyses. We then conducted BLAST analyses of these sequences using the National Center for Biotechnology Information and identified sequences with close to 100% query cover and identity at the nucleotide level. We then identified isolates that contains the aforementioned sequences with >95% identity at the nucleotide level and >80% coverage level.
Statistical analysis.
All analyses were performed using IBM-SPSS Statistics 20 (2012). Bivariate analyses were performed using Fisher's exact test or the chi-square test for categorical variables and the t test or the Mann-Whitney U test for continuous variables. All P values were two-sided. Throughout, the percentages displayed are the “valid percentage,” which indicates the percentage excluding the missing data from the denominator.
RESULTS
A total of 105 patients with ESBL-producing E. coli isolation were identified during the study period. The STs and phylotypes of the isolated ESBL-producing E. coli are shown in Table 1. The most dominant ST was ST131, which accounted for 54 (51.4%) of all ESBL-producing E. coli isolates, followed by ESBL-producing E. coli ST648 (n = 15; 14.3%). One isolate of ST131 contained a new fumC allele (400), but the other 6 MLST locus sequences and phylotypes were consistent with ST131 and were therefore included in the ST131 group for further analysis. All ESBL-producing E. coli ST131 isolates were identified as the O25b-ST131 clone and the H30-Rx subclone. Among the 15 ST648 isolates, 12 had an identification rate of 97.4%, and 3 had a 96.0% identification rate of the pabB gene, which is reported to be specific for isolates of the O25b-ST131 clone (18). In order to further understand the characteristics of dominant STs, we conducted analyses comparing the three groups of ESBL-producing E. coli isolates (ST131, ST648, and non-ST131/648).
TABLE 1.
Sequence typing and phylotypes of extended-spectrum-β-lactamase-producing Escherichia coli isolates in Nepal
| Sequence type | No. (%) of isolates (total n = 105) in phylotype: |
|||
|---|---|---|---|---|
| A | B1 | D | B2 | |
| ST131 | 54 (51.4) | |||
| ST648 | 15 (14.3) | |||
| ST405 | 5 (4.8) | |||
| ST38 | 3 (2.9) | |||
| ST167 | 2 (1.9) | |||
| ST361 | 2 (1.9) | |||
| ST410 | 2 (1.9) | |||
| ST10 | 1 (1.0) | |||
| ST14 | 1 (1.0) | |||
| ST44 | 1 (1.0) | |||
| ST315 | 1 (1.0) | |||
| ST393 | 1 (1.0) | |||
| ST394 | 1 (1.0) | |||
| ST421 | 1 (1.0) | |||
| ST443 | 1 (1.0) | |||
| ST517 | 1 (1.0) | |||
| ST617 | 1 (1.0) | |||
| ST624 | 1 (1.0) | |||
| ST746 | 1 (1.0) | |||
| ST1312 | 1 (1.0) | |||
| ST2562 | 1 (1.0) | |||
| New ST | 7 (6.7) | 1 (1.0) | ||
| Total | 11 (10.5) | 2 (1.9) | 34 (32.4) | 58 (55.2) |
The comparison between the three groups with regard to their antimicrobial susceptibility is shown in Table 2. ST648 isolates were the most resistant to multiple antibiotics, including non-β-lactams such as levofloxacin, gentamicin, trimethoprim-sulfamethoxazole, and minocycline. More than 70% of ST131 (n = 38; 70%) and ST648 (n = 12; 80%) isolates were resistant to both levofloxacin and trimethoprim-sulfamethoxazole, compared to 14 (39%) of non-ST131/648 isolates (ST131 versus non-ST131/648, P = 0.004; ST648 versus non-ST131/648, P = 0.013).
TABLE 2.
Susceptibility profiles among extended-spectrum-β-lactamase-producing Escherichia coli isolates in Nepal
| Antibiotic and parameter | Value for: |
P valuea |
|||||
|---|---|---|---|---|---|---|---|
| Whole cohort (n = 105) | E. coli ST131 (n = 54) | E. coli ST648 (n = 15) | E. coli non-ST 131/648b (n = 36) | ST131 vs ST648 | ST131 vs non-ST 131/648 | ST648 vs non-ST 131/648 | |
| Levofloxacin | |||||||
| No. (%) of resistant isolatesc | 92 (87.6) | 52 (96.3) | 15 (100) | 25 (69.4) | >0.999 | 0.001 | 0.022 |
| MIC50, MIC90 (μg/ml) | >4, >4 | >4, >4 | >4, >4 | >4, >4 | NA | ||
| Gentamicin | |||||||
| No. (%) of resistant isolates | 41 (39) | 17 (31.5) | 12 (80) | 12 (33.3) | 0.001 | >0.99 | 0.005 |
| MIC50, MIC90 | 2, >8 | 2, >8 | >8, >8 | 2, >8 | NA | ||
| Amikacin | |||||||
| No. (%) of resistant isolates | 11 (10.5) | 5 (9.3) | 3 (20) | 3 (8.3) | 0.358 | >0.99 | 0.343 |
| MIC50, MIC90 | 8, 32 | 8, 16 | 8, 32 | ≤4, 16 | NA | ||
| Trimethoprim-sulfamethoxazole | |||||||
| No. (%) of resistant isolates | 69 (65.7) | 39 (72.2) | 12 (80) | 18 (50) | 0.743 | 0.045 | 0.064 |
| MIC50, MIC90 | >2/38, >2/38 | >2/38, >2/38 | >2/38, >2/38 | ≤2/38, >2/38 | NA | ||
| Minocycline | |||||||
| No. (%) of resistant isolates | 34 (32.4) | 2 (3.7) | 15 (100) | 17 (47.2) | <0.001 | <0.001 | <0.001 |
| MIC50, MIC90 | 4, >8 | 2, 4 | >8, >8 | 4, >8 | NA | ||
| Amoxicillin-clavulanic acid | |||||||
| No. (%) of resistant isolates | 76 (72.4) | 40 (74.1) | 13 (86.7) | 23 (63.9) | 0.492 | 0.352 | 0.177 |
| MIC50, MIC90 | 16/8, >16/8 | 16/8, >16/8 | >16/8, >16/8 | 16/8, >16/8 | NA | ||
| Cefoperazone-sulbactam | |||||||
| No. (%) of resistant isolates | 32 (30.5) | 19 (35.2) | 2 (13.3) | 11 (30.6) | 0.125 | 0.82 | 0.297 |
| MIC50, MIC90 | ≤16/8, 32/16 | ≤16/8, 32/16 | ≤16/8, ≤16/8 | ≤16/8, 32/16 | NA | ||
| Cefmetazole | |||||||
| No. (%) of resistant isolates | 5 (4.8) | 0 | 0 | 5 (13.9) | NA | 0.009 | 0.305 |
| MIC50, MIC90 | ≤4, 8 | ≤4, ≤4 | ≤4, ≤4 | ≤4, 32 | NA | ||
| Flomoxef | |||||||
| No. (%) of resistant isolates | 2 (1.9) | 0 | 0 | 2 (5.6) | NA | 0.157 | >0.99 |
| MIC50, MIC90 | ≤8, ≤8 | ≤8, ≤8 | ≤8, ≤8 | ≤8, ≤8 | NA | ||
| Fosfomycin | |||||||
| No. (%) of resistant isolates | 0 | 0 | 0 | 0 | >0.999 | 0.532 | >0.999 |
| MIC50, MIC90 | ≤4, 16 | ≤4, 16 | ≤4, 16 | ≤4, 16 | NA | ||
| Both levofloxacin and trimethoprim-sulfamethoxazole | |||||||
| No. (%) of resistant isolates | 64 (61) | 38 (70.4) | 12 (80) | 14 (38.9) | 0.534 | 0.004 | 0.013 |
Bold P values represent statistically significant results. NA, data not available.
ESBL-producing E. coli isolates with ST other than ST131 and ST648.
Including intermediate and resistant isolates, based on 2013 CLSI criteria (M100-S23) unless otherwise noted.
We next analyzed and compared the profiles of resistance genes among the three isolate groups (Table 3). Overall, ST131 and ST 648 isolates had resistance genes more frequently than non-ST131/648. Importantly, aac(3)-IIa was most frequently identified in ST648 isolates, which would explain the higher rates of resistance to gentamicin in ST648 isolates. blaCTX-M-15 was commonly isolated from all 3 groups.
TABLE 3.
Resistance genes among extended-spectrum-β-lactamase-producing Escherichia coli isolates in Nepal
| Drug and resistance gene | No. (%) of isolates |
P valuea |
|||||
|---|---|---|---|---|---|---|---|
| Whole cohort (n = 105) | E. coli ST131 (n = 54) | E. coli ST648 (n = 15) | E. coli non-ST 131/648b (n = 36) | ST131 vs ST648 | ST131 vs non-ST 131/648 | ST648 vs non-ST 131/648 | |
| Aminoglycoside | |||||||
| aac(3)-IIa | 37 (35.2) | 15 (27.8) | 12 (80) | 10 (27.8) | 0.001 | >0.999 | 0.001 |
| aac(3)-IId | 3 (2.9) | 1 (1.9) | 0 | 2 (5.6) | >0.999 | 0.561 | >0.999 |
| aph(3′)-Ia | 2 (1.9) | 2 (3.7) | 0 | 0 | >0.999 | 0.515 | NA |
| aph(3′)-Ic | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| aadA1 | 3 (2.9) | 0 | 0 | 3 (8.3) | NA | 0.061 | 0.54 |
| aadA2 | 10 (9.5) | 8 (14.8) | 0 | 2 (5.6) | 0.186 | 0.305 | >0.999 |
| aadA5 | 59 (56.2) | 31 (57.4) | 12 (80) | 16 (44.4) | 0.14 | 0.283 | 0.03 |
| strA/B | 32 (30.5) | 20 (37) | 1 (6.7) | 11 (30.6) | 0.027 | 0.652 | 0.083 |
| Fluoroquinolone | |||||||
| qnrB4 | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| qnrS1 | 3 (2.9) | 0 | 0 | 3 (8.3) | NA | 0.061 | 0.546 |
| qepA | 2 (1.9) | 0 | 0 | 2 (5.6) | NA | 0.157 | >0.999 |
| Aminoglycoside and fluoroquinolone | |||||||
| aac(6′)Ib-cr | 69 (65.7) | 39 (72.2) | 14 (93.3) | 16 (44.4) | 0.163 | 0.015 | 0.001 |
| Beta-lactams | |||||||
| blaCTX-M-15 | 99 (94.3) | 51 (94.4) | 15 (100) | 33 (91.7) | >0.999 | 0.68 | 0.546 |
| blaCTX-M-27 | 2 (1.9) | 2 (3.7) | 0 | 0 | >0.999 | 0.515 | NA |
| blaOXA-1 | 69 (65.7) | 38 (70.4) | 14 (93.3) | 17 (47.2) | 0.093 | 0.046 | 0.004 |
| blaTEM-1B | 41 (39) | 20 (37) | 12 (80) | 9 (25) | 0.004 | 0.258 | <0.001 |
| blaSHV-12 | 1 (1) | 0 | 1 (6.7) | 0 | 0.217 | NA | 0.294 |
| blaCMY-42 | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| blaDHA-1 | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| Macrolide | |||||||
| mphA | 76 (72.4) | 46 (85.2) | 12 (80) | 18 (50) | 0.694 | 0.001 | 0.064 |
| ermB | 5 (4.8) | 2 (3.7) | 1 (6.7) | 2 (5.6) | 0.527 | >0.999 | >0.999 |
| Chloramphenicol | |||||||
| catA1 | 13 (12.4) | 0 | 9 (60) | 4 (11.1) | <0.001 | 0.023 | 0.001 |
| catB3 | 66 (62.9) | 37 (68.5) | 14 (93.3) | 15 (41.7) | 0.093 | 0.016 | 0.001 |
| Sulfonamide | |||||||
| sul1 | 70 (66.7) | 39 (72.2) | 12 (80) | 19 (52.8) | 0.743 | 0.074 | 0.115 |
| sul2 | 32 (30.5) | 20 (37) | 1 (6.7) | 11 (30.6) | 0.027 | 0.652 | 0.083 |
| Trimethoprim | |||||||
| dfrA12 | 10 (9.5) | 8 (14.8) | 0 | 2 (5.6) | 0.186 | 0.305 | >0.999 |
| dfrA17 | 58 (55.2) | 31 (57.4) | 12 (80) | 15 (41.7) | 0.14 | 0.197 | 0.016 |
| dfrA1 | 2 (1.9) | 0 | 0 | 2 (5.6) | NA | 0.157 | >0.999 |
| dfrA5 | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| Tetracycline | |||||||
| tetA | 50 (47.6) | 40 (74.1) | 2 (13.3) | 8 (22.2) | <0.001 | <0.001 | 0.703 |
| tetB | 31 (29.5) | 1 (1.9) | 14 (93.3) | 16 (44.4) | <0.001 | <0.001 | 0.001 |
| tetD | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
Bold P values represent statistically significant results. NA, data not available.
ESBL-producing E. coli isolates with ST other than ST131 and ST648.
To further elucidate the microbiological characteristics of ESBL-producing E. coli ST131 and ST648, we investigated the virulence-associated traits (Table 4). Overall, ST131 isolates had a higher prevalence of multiple virulence genes than ST648 and non-ST131/648 isolates, except for hlyD, which was more prevalent in ST648 isolates (n = 8; 53%) than in ST131 (n = 9; 17%) and non-ST131/648 (n = 11; 31%) isolates (ST131 versus ST648, P = 0.007; ST131 versus non-ST131/648, P = 0.131; ST648 versus non-ST131/648, P = 0.203). The median virulence score was highest in the ST131 group (score = 9; interquartile range [IQR], 6 to 10), followed by ST648 (score = 7; IQR, 5 to 8) and non-ST131/648 (score = 5; IQR, 3 to 7).
TABLE 4.
Virulence-associated traits among extended-spectrum-β-lactamase-producing Escherichia coli isolates in Nepal
| Virulence-associated trait and genea | No. (%) of isolates |
P valueb |
|||||
|---|---|---|---|---|---|---|---|
| Whole cohort (n = 105) | E. coli ST131 (n = 54) | E. coli ST648 (n = 15) | E. coli non-ST 131/648c (n = 36) | ST131 vs ST648 | ST131 vs non-ST 131/648 | ST648 vs non-ST 131/648 | |
| Adhesin | |||||||
| papA | 68 (64.8) | 54 (100) | 4 (26.7) | 10 (27.8) | <0.001 | <0.001 | >0.999 |
| papG II | 60 (57.1) | 35 (64.8) | 10 (66.7) | 15 (41.7) | >0.999 | 0.051 | 0.132 |
| sfa/foc | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| focG | 1 (1) | 0 | 0 | 1 (2.8) | NA | 0.4 | >0.999 |
| iha | 70 (66.7) | 53 (98.1) | 4 (26.7) | 13 (36.1) | <0.001 | <0.001 | 0.746 |
| hra | 24 (22.9) | 11 (20.4) | 1 (6.7) | 12 (33.3) | 0.44 | 0.219 | 0.076 |
| Toxin | |||||||
| hlyD | 28 (26.7) | 9 (16.7) | 8 (53.3) | 11 (30.6) | 0.007 | 0.131 | 0.203 |
| cnf1 | 12 (11.4) | 9 (16.7) | 0 | 3 (8.3) | 0.189 | 0.349 | 0.546 |
| sat | 71 (67.6) | 54 (100) | 5 (33.3) | 12 (33.3) | <0.001 | <0.001 | >0.999 |
| vat | 3 (2.9) | 0 | 0 | 3 (8.3) | NA | 0.061 | 0.546 |
| Siderophore | |||||||
| iroN | 6 (5.7) | 0 | 0 | 6 (16.7) | NA | 0.003 | 0.162 |
| fyuA | 92 (87.6) | 53 (98.1) | 14 (93.3) | 25 (69.4) | 0.39 | <0.001 | 0.083 |
| ireA | 6 (5.7) | 2 (3.7) | 0 | 4 (11.1) | >0.999 | 0.213 | 0.307 |
| iutA | 83 (79) | 54 (100) | 14 (93.3) | 15 (41.7) | 0.217 | <0.001 | 0.001 |
| Capsule | |||||||
| kpsM II | 3 (2.9) | 0 | 0 | 3 (8.3) | NA | 0.061 | 0.546 |
| K1 kpsM | 2 (1.9) | 0 | 0 | 2 (5.6) | NA | 0.157 | >0.999 |
| K5 kfiC | 14 (13.3) | 11 (20.4) | 1 (6.7) | 2 (5.6) | 0.44 | 0.067 | >0.999 |
| Miscellaneous | |||||||
| usp | 58 (55.2) | 54 (100) | 0 | 4 (11.1) | <0.001 | <0.001 | 0.307 |
| traT | 87 (82.9) | 47 (87) | 15 (100) | 25 (69.4) | 0.333 | 0.059 | 0.022 |
| ompT | 71 (67.6) | 54 (100) | 11 (73.3) | 6 (16.7) | 0.002 | <0.001 | <0.001 |
| H7 fliC | 2 (1.9) | 0 | 0 | 2 (5.6) | NA | 0.157 | >0.999 |
| malX | 104 (99) | 54 (100) | 15 (100) | 35 (97.2) | NA | 0.4 | 1.0 |
| Median virulence score (IQR)d | 9 (6–10) | 9 (9–10) | 7 (5–8) | 5 (3–7) | <0.001 | <0.001 | 0.074 |
papG III, pic, and ibeA were tested, with no positive isolates detected.
Bold P values represent statistically significant results. NA, data not available.
ESBL-producing E. coli isolates with ST other than ST131 and ST648.
The virulence score represents the number of virulence genes detected, with pap elements counting collectively as a single trait (17).
The clinical characteristics of patients with ESBL-producing E. coli isolates were also evaluated as a function of ST (Table 5). The mean age of the study cohort was 40.7 years (±23.2), and 39 patients (37%) were male. With regard to the demographics of the patients, the underlying conditions, the severity of illness, and the duration of hospitalization, there were no statistically significant differences among the ST groups. In female patients, the prevalence of pregnancy was high (n = 16; 24%), and in male patients, benign prostatic hyperplasia (n = 6; 15%) was common. There was a tendency that the ST131 group (n = 4; 8%) received appropriate empirical antimicrobial therapy less frequently than the non-ST131/648 group (n = 8; 23%); however, this trend did not reach statistical significance (P = 0.057).
TABLE 5.
Bivariate analysis of clinical characteristics of patients with isolation of extended-spectrum-β-lactamase producing Escherichia coli as a function of sequence type
| Parameter | Valuea for: |
P value |
|||||
|---|---|---|---|---|---|---|---|
| Whole cohort (n = 105) | E. coli ST131 (n = 54) | E. coli ST648 (n = 15) | E. coli non-ST 131/648b (n = 36) | ST131 vs ST648 | ST131 vs non-ST 131/648 | ST648 vs non-ST 131/648 | |
| Demographics | |||||||
| Mean age, yr (SD) | 40.7 (23.2) | 41.7 (22.8) | 45.7 (27.3) | 37 (22.1) | 0.541 | 0.266 | 0.251 |
| Male patients | 39 (37.1) | 19 (35.2) | 6 (40) | 14 (38.9) | 0.767 | 0.824 | >0.999 |
| Inpatients | 65 (61.9) | 35 (64.8) | 10 (66.7) | 20 (55.6) | >0.999 | 0.388 | 0.543 |
| Departments | |||||||
| Medicine | 40 (38.1) | 20 (37) | 7 (46.7) | 13 (36.1) | 0.558 | >0.999 | 0.539 |
| Surgery | 21 (20) | 12 (22.2) | 1 (6.7) | 8 (22.2) | 0.27 | >0.999 | 0.251 |
| Obstetrics and gynecology | 22 (21) | 13 (24.1) | 2 (13.3) | 7 (19.4) | 0.494 | 0.796 | 0.709 |
| Pediatrics | 11 (10.5) | 6 (11.1) | 2 (13.3) | 3 (8.3) | >0.999 | 0.736 | 0.624 |
| Underlying conditions | |||||||
| Benign prostatic hyperplasia | 6 (15.4) | 4 (21.1) | 1 (6.7) | 1 (7.1) | >0.999 | 0.366 | 0.521 |
| Urolithiasis | 6 (5.7) | 2 (3.7) | 0 | 4 (11.1) | >0.999 | 0.213 | 0.307 |
| Uterine prolapse | 6 (5.7) | 4 (7.4) | 0 | 2 (5.6) | 0.57 | >0.999 | >0.999 |
| Pregnancy | 16 (24.2) | 9 (25.7) | 2 (22.2) | 5 (22.7) | >0.999 | >0.999 | >0.999 |
| Malignancy | 3 (2.9) | 2 (3.7) | 0 | 1 (2.8) | >0.999 | >0.999 | >0.999 |
| Diabetes mellitus | 3 (2.9) | 2 (3.7) | 1 (6.7) | 0 | 0.527 | 0.515 | 0.294 |
| Chronic obstructive pulmonary disease | 8 (7.6) | 2 (3.7) | 2 (13.3) | 4 (11.1) | 0.204 | 0.213 | >0.999 |
| Severity of illness | |||||||
| Sepsis | 10 (9.5) | 6 (11.1) | 2 (13.3) | 2 (5.6) | >0.999 | 0.468 | 0.571 |
| Antimicrobial therapy | |||||||
| Appropriate empirical antimicrobial therapy | 14 (13.6) | 4 (7.5) | 2 (13.3) | 8 (22.9) | 0.607 | 0.057 | 0.702 |
| Median duration of hospitalization, days (IQR) | 10 (8–14) | 10 (8–13) | 10 (6–14) | 11 (9–16) | 0.38 | 0.412 | 0.202 |
Values are number (%) unless otherwise indicated.
ESBL-producing E. coli isolates with ST other than ST131 and ST648.
We also searched for mobile acquired elements carrying resistance genes in the isolates (Tables 6 and 7). We identified five mobile acquired elements carrying multiple resistance genes: pEC958 (E. coli O25b:H4-ST131 strain EC958, plasmid pEC958), pKF3-140 (Klebsiella pneumoniae strain KF3, plasmid pKF3-140), IS26 (E. coli DNA, insertion sequence IS26, insertion sequence ISEcp1, blaCTX-M-27), p6234 (K. pneumoniae strain 6234, plasmid p6234), and pEK499 (E. coli strain A, plasmid pEK499). The pEC958 element was the most common, and was identified in 70 isolates, followed by p6234, which was identified in 64 isolates, and pKF3-140, which was identified in 20 isolates. Eleven isolates carried three mobile acquired elements (pEC958, pKF3-140, and p6234), 10 of which belonged to ST131. All 64 isolates that carried p6234 also carried pEC958. All isolates which had these mobile elements had >95% identity at the nucleotide level and >80% coverage level.
TABLE 6.
Mobile acquired elements carrying resistance genes among ESBL-producing E. coli isolates in Nepal
| Sequence type | Isolate no. | Mobile acquired elements carrying resistance genesa |
||||
|---|---|---|---|---|---|---|
| pEC958 | pKF3-140 | IS26 | p6234 | pEK499 | ||
| ST131 | 1 | + | + | |||
| 2 | + | + | ||||
| 3 | + | + | ||||
| 5 | + | + | + | |||
| 8 | + | |||||
| 14 | + | + | ||||
| 16 | + | + | ||||
| 19 | + | + | ||||
| 23 | + | |||||
| 24 | + | + | + | |||
| 25 | + | + | + | |||
| 26 | + | + | ||||
| 28 | + | + | + | |||
| 29 | + | + | + | |||
| 30 | + | + | ||||
| 37 | + | + | ||||
| 38 | + | + | + | |||
| 43 | + | + | + | |||
| 45 | + | + | + | |||
| 47 | + | + | ||||
| 50 | + | + | ||||
| 52 | + | + | ||||
| 59 | + | + | ||||
| 60 | + | |||||
| 61 | + | + | + | |||
| 63 | + | + | ||||
| 64 | + | + | ||||
| 67 | + | |||||
| 68 | + | + | ||||
| 69 | + | + | + | |||
| 70 | + | |||||
| 72 | + | + | ||||
| 75 | + | + | + | |||
| 78 | + | + | ||||
| 79 | + | + | ||||
| 80 | + | + | ||||
| 85 | + | + | ||||
| 86 | + | + | ||||
| 87 | + | + | ||||
| 90 | + | + | ||||
| 92 | + | + | + | |||
| 96 | + | + | ||||
| 99 | + | + | + | |||
| 101 | + | + | ||||
| 102 | + | + | + | |||
| 104 | + | + | + | |||
| ST648 | 7 | + | + | |||
| 15 | + | + | ||||
| 22 | + | + | ||||
| 36 | + | + | ||||
| 40 | + | + | ||||
| 42 | + | + | ||||
| 51 | + | + | ||||
| 57 | + | + | ||||
| 62 | + | + | ||||
| 65 | + | + | ||||
| 76 | + | + | ||||
| 82 | + | + | ||||
| 95 | + | + | ||||
| 98 | + | + | ||||
| ST405 | 12 | + | + | + | ||
| 31 | + | + | + | |||
| 58 | + | + | ||||
| 71 | + | + | ||||
| ST38 | 77 | + | + | |||
| 81 | + | + | ||||
| ST167 | 74 | + | ||||
| ST361 | 105 | + | ||||
| ST410 | 6 | + | + | |||
| 55 | + | + | ||||
| ST44 | 27 | + | + | |||
| ST517 | 97 | + | + | |||
| ST617 | 46 | + | ||||
| ST1312 | 10 | + | + | |||
| New ST | 34 | + | + | |||
| 35 | + | + | ||||
| 39 | + | + | ||||
| 56 | + | + | ||||
| 100 | + | + | + | |||
pEC958, Escherichia coli O25b:H4-ST131 strain EC958 plasmid pEC958; National Center for Biotechnology Information (NCBI) accession no. HG941719.1. pKF3-140, Klebsiella pneumoniae strain KF3 plasmid pKF3-140; NCBI accession no. FJ876827.1. IS26, E. coli DNA, insertion sequence IS26, insertion sequence ISEcp1, blaCTX-M-27; NCBI accession no. AB976590.1. p6234, K. pneumoniae strain 6234 plasmid p6234; NCBI accession no. CP010390.1. pEK499, E. coli strain A plasmid pEK499; NCBI accession no. EU935739.1.
TABLE 7.
Numbers of mobile acquired elements carrying resistance genes in each ST
| Sequence type | No. (%) carrying resistance genesa |
||||
|---|---|---|---|---|---|
| pEC958 | pKF3-140 | IS26 | p6234 | pEK499 | |
| ST131 | 43 (80) | 17 (31) | 2 (4) | 36 (67) | 4 (7) |
| ST648 | 14 (93) | 14 (93) | |||
| ST405 | 4 (80) | 1 (20) | 1 (20) | 3 (60) | 1 (20) |
| ST38 | 2 (67) | 1 (33) | 1 (33) | ||
| ST167 | 1 (50) | ||||
| ST361 | 1 (50) | ||||
| ST410 | 2 (100) | 2 (100) | |||
| ST44 | 1 (100) | 1 (100) | |||
| ST517 | 1 (100) | 1 (100) | |||
| ST617 | 1 (100) | ||||
| ST1312 | 1 (100) | 1 (100) | |||
| New ST | 5 (63) | 1 (13) | 5 (63) | ||
| Total | 76 | 20 | 3 | 64 | 5 |
pEC958, Escherichia coli O25b:H4-ST131 strain EC958 plasmid pEC958; National Center for Biotechnology Information (NCBI) accession no. HG941719.1. pKF3-140, Klebsiella pneumoniae strain KF3 plasmid pKF3-140; NCBI accession no. FJ876827.1. IS26, E. coli DNA, insertion sequence IS26, insertion sequence ISEcp1, blaCTX-M-27; NCBI accession no. AB976590.1. p6234, K. pneumoniae strain 6234 plasmid p6234; NCBI accession no. CP010390.1. pEK499, E. coli strain A plasmid pEK499; NCBI accession no. EU935739.1.
DISCUSSION
To our knowledge, this is the first study to reveal the microbiological and clinical epidemiology of ESBL-producing E. coli in Nepal. We discovered that more than 90% of the ESBL-producing E. coli isolates in Nepal were CTX-M-15 positive, and more than half were ST131 isolates. All 54 ESBL-producing E. coli ST131 isolates were identified as the O25b-ST131 clone, subclone H30-Rx, which have been reported to be more antibiotic resistant and to have virulence profiles (16). These findings correlate with the multiple reports on a global spread of CTX-M-15-positive O25b-ST131 clones (21).
Another key finding of this study is the discovery that ST648 isolates made up 14% of ESBL-producing E. coli in Nepal and that their microbiological characteristics are distinct from those of ST131 isolates. Human isolates of CTX-M-producing E. coli ST648 have been reported from various geographical regions, such as Europe, North and South America, Africa, and Asia; the majority of these isolates belong to phylotype D (22–25). CTX-M-producing E. coli ST648 strains have also been isolated from wild birds in Mongolia and Germany (24, 26–28) and from companion and livestock animals in European countries (24, 29, 30). A recent study on ESBL-producing E. coli isolates (n = 1,152) sampled in Europe, predominantly from dogs, cats, and horses, identified 40 (3.5%) ESBL-producing E. coli strains as ST648 (phylotype D, CTX-M-15 positive; 72.5%), whereas ST131 isolates (phylotype B2, CTX-M-15-positive; 46.9%) occurred less frequently (2.8%) (30). The authors also found that a higher proportion of ST648 strains showed resistance to most non-β-lactam antibiotics, and virulence genes were less abundant in ST648 strains than in ST131strains (30). These findings are concordant with the current study. To our knowledge, this is the first study to systematically evaluate the microbiological and clinical characteristics of human isolates of CTX-M-producing ST648 strains, all of which were positive for CTX-M-15. The fact that ST648 isolates, which show a higher drug resistance than ST131 isolates and contain profuse virulence genes, comprised 14% of all ESBL-producing E. coli isolates in Nepal is a concerning observation. In addition, previous reports have suggested the spread of CTX-M-producing ST648 strains among humans and animals worldwide (24). These findings raise the concern that ST648 strains could present another pandemic clone of ESBL-producing E. coli.
Other STs found in isolates from Nepal in this study have also been reported to be associated with ESBL production in E. coli and have been found in wildlife, livestock, and humans (24). The spread of ESBL-producing E. coli ST405, ST38, ST315, and ST410 has been reported in animals in Europe, as well as in humans worldwide (24). ESBL-producing E. coli ST393 was isolated from livestock animals in Germany and from humans globally (24). Global spread of ESBL-producing E. coli ST10, ST617, and ST167 has also been reported (24).
Due to the limited information available from medical charts, we could not evaluate detailed clinical variables in the current study. Nevertheless, it is noteworthy that more than 38% of ESBL-producing E. coli strains were isolated from the outpatient clinic, and about a quarter of patients with ESBL-producing E. coli were pregnant women. This would suggest a wide spread of CTX-M-producing E. coli across the communities in Nepal. Combined with the high resistance to orally available antibiotics, these community-isolated ESBL-producing E. coli strains present an emerging challenge for community practitioners and hospitals worldwide.
Data on the effectiveness of treatment using cephamycins and oxacephems for the ESBL-producing organisms are scarce (31, 32), and further studies regarding the optimal clinical uses of these drugs for the treatment of ESBL-producing E. coli are warranted.
In conclusion, we revealed that the high resistance of ESBL-producing E. coli to multiple classes of antibiotics in Nepal is driven mainly by CTX-M-producing ST131 and ST648 strains. Their prevalence in communities is a matter of great concern, and further studies are necessary to identify the epidemiology of CTX-M-ST648 to control the spread of ESBL-producing E. coli.
ACKNOWLEDGMENTS
This study was supported by Grants for International Health Research (24S-5 and 26S-101) from the Ministry of Health, Labor, and Welfare of Japan.
We have no conflict of interest to declare.
REFERENCES
- 1.National Nosocomial Infections Surveillance System. 1996. Data summary from October 1986-April 1996, issued May 1996. A report from the National Nosocomial Infections Surveillance (NNIS) System. Am J Infect Control 24:380–388. [PubMed] [Google Scholar]
- 2.Gupta K, Hooton TM, Naber KG, Wullt B, Colgan R, Miller LG, Moran GJ, Nicolle LE, Raz R, Schaeffer AJ, Soper DE, Infectious Diseases Society of America, European Society for Microbiology and Infectious Diseases . 2011. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis 52:e103–e120. doi: 10.1093/cid/ciq257. [DOI] [PubMed] [Google Scholar]
- 3.Klein JO, Feigin RD, McCracken GH Jr. 1986. Report of the Task Force on Diagnosis and Management of Meningitis. Pediatrics 78:959–982. [PubMed] [Google Scholar]
- 4.Pitout JD, Laupland KB. 2008. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 8:159–166. doi: 10.1016/S1473-3099(08)70041-0. [DOI] [PubMed] [Google Scholar]
- 5.Johnson JR, Urban C, Weissman SJ, Jorgensen JH, Lewis JS II, Hansen G, Edelstein PH, Robicsek A, Cleary T, Adachi J, Paterson D, Quinn J, Hanson ND, Johnston BD, Clabots C, Kuskowski MA, AMERECUS Investigators . 2012. Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and blaCTX-M-15 among extended-spectrum-beta-lactamase-producing E. coli from the United States, 2000 to 2009. Antimicrob Agents Chemother 56:2364–2370. doi: 10.1128/AAC.05824-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Rogers BA, Sidjabat HE, Paterson DL. 2011. Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother 66:1–14. doi: 10.1093/jac/dkq415. [DOI] [PubMed] [Google Scholar]
- 7.Reardon S. 2014. Antibiotic resistance sweeping developing world. Nature 509:141–142. doi: 10.1038/509141a. [DOI] [PubMed] [Google Scholar]
- 8.Clinical and Laboratory Standards Institute. 2010. Performance standards for antimicrobial susceptibility testing; 20th informational supplement. Approved standard M100-S23 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 9.Afridi FI, Farooqi BJ. 2012. Activity of beta-lactam beta-lactamase inhibitor combinations against extended spectrum beta-lactamase producing Enterobacteriaceae in urinary isolates. J Coll Physicians Surgeons Pakistan 22:358–362. [PubMed] [Google Scholar]
- 10.Kresken M, Pfeifer Y, Hafner D, Wresch R, Korber-Irrgang B, Working Party ‘Antimicrobial Resistance’ of the Paul-Ehrlich-Society for Chemotherapy . 2014. Occurrence of multidrug resistance to oral antibiotics among Escherichia coli urine isolates from outpatient departments in Germany: extended-spectrum beta-lactamases and the role of fosfomycin. Int J Antimicrob Agents 44:295–300. doi: 10.1016/j.ijantimicag.2014.05.020. [DOI] [PubMed] [Google Scholar]
- 11.Yang Q, Zhang H, Cheng J, Xu Z, Xu Y, Cao B, Kong H, Ni Y, Yu Y, Sun Z, Hu B, Huang W, Wang Y, Wu A, Feng X, Liao K, Shen D, Hu Z, Chu Y, Lu J, Su J, Gui B, Duan Q, Zhang S, Shao H. 31 December 2014. In vitro activity of flomoxef and comparators against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis producing extended-spectrum beta-lactamases in China. Int J Antimicrob Agents doi: 10.1016/j.ijantimicag.2014.11.012. [DOI] [PubMed] [Google Scholar]
- 12.Grimm H. 1991. Interpretive criteria of antimicrobial disk susceptibility tests with flomoxef. Infection 19(Suppl 5):S258–S263. doi: 10.1007/BF01645537. [DOI] [PubMed] [Google Scholar]
- 13.Jones RN, Barry AL, Packer RR, Gregory WW, Thornsberry C. 1987. In vitro antimicrobial spectrum, occurrence of synergy, and recommendations for dilution susceptibility testing concentrations of the cefoperazone-sulbactam combination. J Clin Microbiol 25:1725–1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Clinical and Laboratory Standards Institute. 2009. Performance standards for antimicrobial susceptibility testing; 19th informational supplement. Approved standard M100-S19 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 15.Clermont O, Bonacorsi S, Bingen E. 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 66:4555–4558. doi: 10.1128/AEM.66.10.4555-4558.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Banerjee R, Robicsek A, Kuskowski MA, Porter S, Johnston BD, Sokurenko E, Tchesnokova V, Price LB, Johnson JR. 2013. Molecular epidemiology of Escherichia coli sequence type 131 and its H30 and H30-Rx subclones among extended-spectrum-beta-lactamase-positive and -negative E. coli clinical isolates from the Chicago region, 2007 to 2010. Antimicrob Agents Chemother 57:6385–6388. doi: 10.1128/AAC.01604-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Johnson JR, Menard M, Johnston B, Kuskowski MA, Nichol K, Zhanel GG. 2009. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. Antimicrob Agents Chemother 53:2733–2739. doi: 10.1128/AAC.00297-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Clermont O, Dhanji H, Upton M, Gibreel T, Fox A, Boyd D, Mulvey MR, Nordmann P, Ruppe E, Sarthou JL, Frank T, Vimont S, Arlet G, Branger C, Woodford N, Denamur E. 2009. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J Antimicrob Chemother 64:274–277. doi: 10.1093/jac/dkp194. [DOI] [PubMed] [Google Scholar]
- 19.Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. doi: 10.1186/1471-2105-10-421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640–2644. doi: 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Oteo J, Perez-Vazquez M, Campos J. 2010. Extended-spectrum β-lactamase producing Escherichia coli: changing epidemiology and clinical impact. Curr Opin Infect Dis 23:320–326. doi: 10.1097/QCO.0b013e3283398dc1. [DOI] [PubMed] [Google Scholar]
- 22.Sidjabat HE, Paterson DL, Adams-Haduch JM, Ewan L, Pasculle AW, Muto CA, Tian GB, Doi Y. 2009. Molecular epidemiology of CTX-M-producing Escherichia coli isolates at a tertiary medical center in western Pennsylvania. Antimicrob Agents Chemother 53:4733–4739. doi: 10.1128/AAC.00533-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Zong Z, Yu R. 2010. Escherichia coli carrying the blaCTX-M-15 gene of ST648. J Med Microbiol 59:1536–1537. doi: 10.1099/jmm.0.022459-0. [DOI] [PubMed] [Google Scholar]
- 24.Ewers C, Bethe A, Semmler T, Guenther S, Wieler LH. 2012. Extended-spectrum beta-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect 18:646–655. doi: 10.1111/j.1469-0691.2012.03850.x. [DOI] [PubMed] [Google Scholar]
- 25.Mshana SE, Imirzalioglu C, Hain T, Domann E, Lyamuya EF, Chakraborty T. 2011. Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX-M-15 in a tertiary hospital in Tanzania. Clin Microbiol Infect 17:1279–1282. doi: 10.1111/j.1469-0691.2011.03518.x. [DOI] [PubMed] [Google Scholar]
- 26.Guenther S, Aschenbrenner K, Stamm I, Bethe A, Semmler T, Stubbe A, Stubbe M, Batsajkhan N, Glupczynski Y, Wieler LH, Ewers C. 2012. Comparable high rates of extended-spectrum-beta-lactamase-producing Escherichia coli in birds of prey from Germany and Mongolia. PLoS One 7:e53039. doi: 10.1371/journal.pone.0053039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Guenther S, Ewers C, Wieler LH. 2011. Extended-spectrum beta-lactamase producing E. coli in wildlife, yet another form of environmental pollution? Front Microbiol 2:246. doi: 10.3389/fmicb.2011.00246. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Guenther S, Grobbel M, Beutlich J, Bethe A, Friedrich ND, Goedecke A, Lubke-Becker A, Guerra B, Wieler LH, Ewers C. 2010. CTX-M-15-type extended-spectrum beta-lactamases-producing Escherichia coli from wild birds in Germany. Environ Microbiol Rep 2:641–645. doi: 10.1111/j.1758-2229.2010.00148.x. [DOI] [PubMed] [Google Scholar]
- 29.Cortes P, Blanc V, Mora A, Dahbi G, Blanco JE, Blanco M, Lopez C, Andreu A, Navarro F, Alonso MP, Bou G, Blanco J, Llagostera M. 2010. Isolation and characterization of potentially pathogenic antimicrobial-resistant Escherichia coli strains from chicken and pig farms in Spain. Appl Environ Microbiol 76:2799–2805. doi: 10.1128/AEM.02421-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Ewers C, Bethe A, Stamm I, Grobbel M, Kopp PA, Guerra B, Stubbe M, Doi Y, Zong Z, Kola A, Schaufler K, Semmler T, Fruth A, Wieler LH, Guenther S. 2014. CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J Antimicrob Chemother 69:1224–1230. doi: 10.1093/jac/dkt516. [DOI] [PubMed] [Google Scholar]
- 31.Doi A, Shimada T, Harada S, Iwata K, Kamiya T. 2013. The efficacy of cefmetazole against pyelonephritis caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae. Int J Infect Dis 17:e159–163. doi: 10.1016/j.ijid.2012.09.010. [DOI] [PubMed] [Google Scholar]
- 32.Lee CH, Su LH, Tang YF, Liu JW. 2006. Treatment of ESBL-producing Klebsiella pneumoniae bacteraemia with carbapenems or flomoxef: a retrospective study and laboratory analysis of the isolates. J Antimicrob Chemother 58:1074–1077. doi: 10.1093/jac/dkl381. [DOI] [PubMed] [Google Scholar]