Abstract
Objectives
The third-generation cephalosporin (3GC)-resistant E. coli strains have been detected worldwide in humans and animals. Hence, in this study, we evaluated the prevalence and genetic characteristics of 3GC-resistant E. coli in livestock, farmers, and patients to further analyse if livestock serves as a potential reservoir of antimicrobial-resistant bacteria.
Methods
Faecal samples were collected from 330 healthy livestock (216 cattle and 114 swine), 61 healthy livestock farmers (52 cattle farmers and 9 swine farmers), and 68 non-duplicate 3GC-resistant E. coli isolates were also obtained from the clinical specimens of patients in Japan between 2013 and 2015. Genes associated with resistance in 3GC-resistant E. coli were identified using polymerase chain reaction (PCR) and DNA sequencing. Genotypic diversity was determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE).
Results
We obtained 39 and 17 non-duplicated 3GC-resistant E. coli strains from healthy livestock (33 cattle and six swine) and livestock farmers, respectively. All isolates carried either CTX-M-type extended-spectrum β-lactamase or plasmid-mediated AmpC β-lactamase genes, with CTX-M-14 being the most frequent. CTX-M producers from livestock and patients belonged to 22 and 19 different sequence types (STs), respectively, and only three STs were the same. Among the 3GC-resistant E. coli from livestock and farmers, three types of CTX-M producers have shown similar characteristics (CTX-M genotype, ST, PFGE patterns, and antimicrobial susceptibilities) and were identified as clonal isolates shared among their farms.
Conclusions
Our study findings indicate that CTX-M-14 is predominant in Japan. No distinct relationship was observed between the 3GC-resistant E. coli isolated from livestock and patients; however, some clonal relatedness was observed between the isolates from livestock and farmers due to their close contact.
Keywords: CTX-M β-lactamase, Escherichia coli, Genotype, Livestock, Farmers
Graphical abstract
Highlights
-
•
CTX-M-14 is predominantly detected in livestock, farmers, and patients.
-
•
No distinct relationship in 3GC-resistant E. coli isolates of livestock and patients.
-
•
Clonal relatedness between livestock and farmers was due to their close contact.
1. Introduction
Antimicrobial resistance (AMR) is a global threat that contributes to serious adverse consequences such as therapeutic failure, increased morbidity, mortality, and healthcare costs [1,2]. The increase in AMR has become a major public health concern [3]. In an attempt to overcome this global public health challenge, the World Health Organization (WHO) has created a priority list of antibiotic-resistant bacteria to support the research and development of effective drugs [4] and has issued a red alert for carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, and carbapenem-resistant or third-generation cephalosporin (3GC)-resistant Enterobacteriaceae by categorising them as ‘critical priority pathogens’. Among the Enterobacteriaceae isolates, Escherichia coli poses the greatest risk to public health because of its prevalence, wide spectrum of clinical manifestations, multidrug resistance, and rapid spread of antimicrobial resistance to other organisms [5].
Acquired resistance to 3GC is primarily mediated by genes encoding extended-spectrum β-lactamases (ESBLs, commonly coded by TEM-type, SHV-type, and CTX-M-type genes) or plasmid-mediated AmpC β-lactamase (pAmpC, commonly coded by the CMY family gene) and is defined based on their ability to hydrolyse 3GC [[6], [7], [8]]. These genes are typically encoded on plasmids and thus result in widespread dissemination, horizontally or across different bacterial species, through conjugation/mobilization or transformation [9]. Among the ESBLs genes, CTX-M-type has become globally disseminated and is dominant among isolates from hospitals [[10], [11], [12]]. CTX-M-type ESBLs has been divided into several principal groups based on the sequence of amino acid, such as CTX-M-1, −2, −8, −9, and − 25, and over 250 CTX-M-type genotypes have also been identified [13]. CTX-M-15 (CTX-M-1 group) and CTX-M-14 (CTX-M-9 group) are disseminated CTX-M-type ESBLs produced by E. coli and are the predominant genotypes worldwide [14]. The prevalence of CTX-M-15 β-lactamase has increased over time in most countries and presently is dominant in most regions, especially in the Middle East, Asia, and Africa [14], except for China, Southeast Asia, South Korea, Japan, and Spain, where CTX-M-14 β-lactamases are dominant [14]. These CTX-M genotypes have been found mostly in a highly virulent and successful clone belonging to E. coli ST131 in many world regions [14,15].
AMR, including 3GC-resistant E. coli, is on the rise globally, not only in humans but also in animals and the environment, and presents a major threat [16]. 3GC is an important antimicrobial agent used to treat gram-negative infections in humans and food-producing animals [17,18]. The 3GC-resistant E. coli in food-producing animals is suspected to be the source of resistance in humans as the excessive use of 3GC can exert selective pressure favouring resistance in humans [19]. To better understand the relatedness of widespread AMR in humans and animals, monitoring and characterizing AMR pathogens following the One Health approach is needed [16,20,21].
In Japan, the prevalence of ESBL-producing E. coli in patients in hospitals and healthy individuals is 24.3–26.1% and 9.7%, respectively [[22], [23], [24]], and the CTX-M-9 group (CTX-M-14 or CTX-M-27)-producing E. coli ST131 was the most prevalent [22,23]. The ESBL-producing E. coli isolated from domestic chicken meat in Japan predominantly belonged to the CTX-M-1 group (CTX-M-1 and CTX-M-15) [25]. Additionally, the prevalence of ESBL-producing E. coli isolated from cattle and swine were 12.5% and 3.0%, respectively [26]; however, there are few reports on 3GC-resistant E. coli in livestock in Japan and their genetic characteristics and relatedness with those isolated from farmers and patients are unknown. Hence, this study aimed to evaluate the prevalence and genetic relatedness of 3GC-resistant E. coli isolated from livestock, farmers, and patients to further analyse whether livestock serves as a potential reservoir of AMR.
2. Materials and methods
The study protocol was approved by the Ethical Review Committee of the Teikyo University School of Medicine (No. 13–118) and all human participants provided written informed consent to participate in the study.
2.1. Identification of 3GC-resistant E. coli from patient specimens
Sixty-eight non-duplicated 3GC-resistant E. coli isolates were randomly obtained from the clinical specimens of patients (34 inpatients and 34 outpatients) from tertiary hospitals (Nara Medical University Hospital, Teikyo University Hospital, and Tohoku University Hospital) in Japan between January 2013 and August 2015. All isolates were obtained either from infection or colonization/screening, and only one isolate per patient was included in this study. The isolates were identified using matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS; Vitek MS system; bioMérieux, Co., Ltd.), and ESBL production was inferred by analysing β-lactam susceptibility profiles, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [27].
2.2. Identification of 3GC-resistant E. coli from livestock and farmers
Faecal samples from 330 healthy livestock (216 cattle and 114 swine) and 61 healthy livestock farmers (52 cattle farmers and 9 swine farmers) were collected from 78 livestock farms (60 cattle farms and 18 swine farms) located in the southern part of Kyushu region, a major area for cattle and swine farming in Japan, between 2013 and 2015. The faecal samples were directly inoculated onto deoxycholate‑hydrogen sulphide-lactose agar plates and incubated at 37 °C for 24 h. Randomly selected colonies were analysed using MALDI-TOF MS. Only one E. coli isolate was selected for each sample. The prevalence of 3GC-resistant E. coli was calculated by dividing the number of samples containing 3GC-resistant E. coli by the total number of samples tested for each of the four categories (cattle, swine, cattle farmers, and swine farmers).
2.3. Determination of antimicrobial susceptibility and identification of ESBL/pAmpC genes
The antimicrobial susceptibilities of the isolates were determined by the agar dilution method according to CLSI guidelines, and quality control was performed using the reference strain E. coli ATCC 25922 [27]. The presence of ESBL, pAmpC, and carbapenemase genes in the 3GC-resistant E. coli isolates was identified by multiplex PCR, as described previously [[28], [29], [30], [31]]. Gene-specific PCR was performed to identify the genotype, and the amplified products were confirmed by DNA sequencing [32]. Sequence alignment and analysis were performed on the NCBI website using the BLAST tool [33].
2.4. Genotyping of E. coli using multilocus sequence typing and pulsed-field gel electrophoresis
The genotypic diversity of 3GC-resistant E. coli was analysed using multilocus sequence typing (MLST) [34]. Seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) were also sequenced. DNA sequence variations were analysed using the MLST database for E. coli [35] to determine the sequence types (STs).
The similarity of the isolates was compared using pulsed-field gel electrophoresis (PFGE) analysis according to the manufacturer's protocol (Bio-Rad, Hercules, CA, USA). The whole-cell DNA of E. coli isolates embedded in 1.6% low-melt agarose plugs (Bio-Rad, Hercules, CA, USA) was digested with the XbaI restriction enzyme (Takara Bio Inc., Japan) for 18 h at 37 °C [36]. DNA fragments were electrophoresed in 1.0% SeaKem Gold agarose gel (Lonza, USA) on the CHEF-MAPPER system (BioRad Laboratories, Japan) with 0.5× Tris-borate-EDTA running buffer at 14 °C for 20 h at 6 V/cm with a pulse time of 5.3–49.9 s, at an angle of 120°. The PFGE patterns were interpreted according to the criteria described by Tenover et al. [37] as “indistinguishable” isolate if only one band differed; “closely related” isolate if 2–3 bands differed; and “possibly related” isolate if up to six bands differed, from those of the reference strain.
2.5. Characterisation of plasmids using plasmid profiling
Plasmid incompatibility was identified using a PCR-based replicon typing method [38], and conjugation experiments were performed using a broth mating method with E. coli J53 recipient as previously described [22]. CTX-M-encoding plasmids isolated from livestock and farmers were compared using plasmid profiling of the transconjugants. Plasmid DNA from transconjugants was digested with the endonuclease EcoRI or HindIII at 37 °C to determine the restriction fragment length polymorphism (RFLP) profile. The resulting fragments were loaded onto a 0.8% agarose gel and electrophoresed at 50 V for 3 h at 4 °C [39].
3. Results
3.1. Distribution of 3GC-resistant E. coli in livestock and farmers
A total of 391 E. coli isolates selected from faecal samples of 330 livestock and 61 farmers were identified at the species level. Of the analysed E. coli isolates, 3GC-resistant E. coli isolates were detected in 39 samples (11.8%) from livestock and 17 samples (27.9%) from participating farmers (Table 1). The carriage rate of 3GC-resistant E. coli was higher for cattle and cattle farmers than for swine and swine farmers. The susceptibility profiles of 3GC-resistant E. coli from livestock, farmers, and patients are shown in Table 2. The isolates showed similar susceptibility profiles and were highly resistant to cefotaxime; however, they were susceptible to cefmetazole and imipenem. The resistance rates to levofloxacin and gentamicin showed similar trends.
Table 1.
Distribution of β-lactamase genes in 3GC-resistant Escherichia coli isolates from livestock, farmers, and patients.
| Livestock |
Farmers |
Patients |
||||
|---|---|---|---|---|---|---|
| cattle | swine | cattle | swine | outpatients | inpatients | |
| Number of samples | 216 | 114 | 52 | 9 | – | – |
| 3GC-resistant isolates | 33 (15.3%) | 6 (5.3%) | 16 (30.8%) | 1 (11.1%) | 34 | 34 |
| CTX-M-1 group | ||||||
| M-1 | 2 | |||||
| M-3 | 1 | 3 | ||||
| M-15 | 1 | 2 | 3 | |||
| M-22 | 1 | |||||
| M-55 | 2 | 3 | 2 | |||
| M-68 | 1 | |||||
| M-69 | 3 | 1 | 3 | 1 | ||
| M-79 | 1 | |||||
| M-123 | 1 | |||||
| M-156 | 3 | 3 | 2 | 1 | 1 | |
| CTX-M-9 group | ||||||
| M-14 | 17 | 1 | 3 | 1 | 13 | 12 |
| M-24 | 1 | |||||
| M-27 | 2 | 4 | 10 | 7 | ||
| M-65 | 1 | 1 | ||||
| CTX-M-2 group | ||||||
| M-2 | 1 | 1 | ||||
| plasmid-mediated AmpC β-lactamase | ||||||
| CMY-2 | 5 | 3 | ||||
Table 2.
Susceptibility data of 3GC-resistant Escherichia coli isolates from livestock, farmers, and patients.
| Antimicrobial agents | MIC90 (mg/L)a/Resistant %b of the isolates from: |
||
|---|---|---|---|
| Livestock (n = 39) | Farmers (n = 17) | Patients (n = 68) | |
| Cefotaxime | >256/100 | >256/100 | >256/100 |
| Cefmetazole | 16/0 | 2/0 | 8/0 |
| Imipenem | 0.25/0 | 0.25/0 | 0.25/0 |
| Levofloxacin | 16/53.8 | 16/52.9 | 64/63.2 |
| Gentamicin | 128/28.2 | 64/35.3 | 128/39.7 |
MIC90, MIC at which 90% of isolates are inhibited.
Interpreted according to Clinical and Laboratory Standards Institute guidelines [26].
3.2. ESBL/pAmpC genes in livestock, farmers, and patients
Of the 56 E. coli isolates identified in livestock and farmers, 48 were ESBL-type isolates, and 8 were pAmpC-producing isolates. The ESBL-type isolates carried CTX-M genes, including CTX-M-1 (n = 17), CTX-M-9 (n = 29), and CTX-M-2 (n = 2) groups (Table 1). Of the ESBL genes detected in this study, CTX-M-14 was the most prevalent (n = 22), followed by CTX-M-156 (n = 8). The pAmpC genotype belonged to CMY-2 in all the isolates.
All the 68 3GC-resistant E. coli isolates identified in the patients also carried CTX-M genes, including the CTX-M-1 (n = 24) and CTX-M-9 (n = 44) groups' genes, whereas other CTX-M genes, CTX-M-2 group, and CTX-M-8/25 group were not identified. CTX-M-14 was the most prevalent gene (n = 25), followed by CTX-M-27 (n = 17) in patients. CTX-M gene distribution was similar both in outpatients and inpatients.
3.3. Genotypes of ESBL/pAmpC-producing E. coli
The genotype data of the ESBL/pAmpC-producing E. coli isolates are presented (Table 3). The isolates from livestock belonged to 25 different STs with no obvious predominance. The isolates from the patients belonged to 22 different STs and predominantly belonged to ST131 (36/68), which was the same in both outpatients and inpatients. When STs between livestock and patients were compared, only four STs (ST10, ST38, ST101, and ST448) were similar in both groups.
Table 3.
Relationship of genotypes and β-lactamase genes in 3GC-resistant Escherichia coli isolates from livestock, farmers, and patients.
| Sequence type | Livestock and farmers (n = 56) a,b |
Patients (n = 68) c |
Total |
||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CTX-M-1 group n = 13 (5) |
CTX-M-9 group n = 20 (8) |
CTX-M-2 group n = 1 (1) |
pAmpC n = 5 (3) |
CTX-M-1 group n = 24 |
CTX-M-9 group n = 44 |
||||||||||||||||||||||||
| M-3 | M-15 | M-55 | M-69 | M-123 | M-156 | M-14 | M-27 | M-2 | CMY-2 | M-1 | M-3 | M-15 | M-22 | M-55 | M-68 | M-69 | M-79 | M-156 | M-14 | M-24 | M-27 | M-65 | |||||||
| ST10 | (1) | (1) | (2) | 1 | 2 | 1 | 1 (4) 3 | ||||||||||||||||||||||
| ST23 | 1 | 1 | |||||||||||||||||||||||||||
| ST38 | 1 | 1 | 1 1 | ||||||||||||||||||||||||||
| ST43 | 1 | 1 | |||||||||||||||||||||||||||
| ST46 | (1) | (1) | |||||||||||||||||||||||||||
| ST58 | 1 | 1 (1) | 2 (1) | ||||||||||||||||||||||||||
| ST69 | 1 | 1 | |||||||||||||||||||||||||||
| ST83 | 1 | 1 | |||||||||||||||||||||||||||
| ST90 | (1) | (1) | |||||||||||||||||||||||||||
| ST95 | 1 | 1 | 2 | ||||||||||||||||||||||||||
| ST101 | 1 | 1 | 1 1 | ||||||||||||||||||||||||||
| ST117 | 1 | 1 | |||||||||||||||||||||||||||
| ST127 | 1 | 1 | |||||||||||||||||||||||||||
| ST131 | (1) | (1) | 1 | 5 | 1 | 1 | 1 | 2 | 9 | 15 | 1 | (2) 36 | |||||||||||||||||
| ST155 | 1 | 1 | 2 | ||||||||||||||||||||||||||
| ST156 | 1 | 1 | |||||||||||||||||||||||||||
| ST167 | 1 (1) | 1 (1) | |||||||||||||||||||||||||||
| ST189 | 1 (1) | 1 (1) | |||||||||||||||||||||||||||
| ST218 | 1 | 1 | |||||||||||||||||||||||||||
| ST224 | 1 | 1 | |||||||||||||||||||||||||||
| ST297 | 2 (1) | 2 (1) | |||||||||||||||||||||||||||
| ST354 | 1 | 1 | 2 | ||||||||||||||||||||||||||
| ST392 | 3 | 3 | |||||||||||||||||||||||||||
| ST405 | 1 | 1 | 2 | ||||||||||||||||||||||||||
| ST410 | (1) | (1) | |||||||||||||||||||||||||||
| ST446 | 1 | 1 | |||||||||||||||||||||||||||
| ST448 | 1 (1) | 1 | 1 (1) 1 | ||||||||||||||||||||||||||
| ST453 | (1) | (1) | |||||||||||||||||||||||||||
| ST533 | 2 | 2 | |||||||||||||||||||||||||||
| ST537 | 1 | 1 | |||||||||||||||||||||||||||
| ST540 | 1 (1) | 1 (1) | |||||||||||||||||||||||||||
| ST617 | 3 | 3 | |||||||||||||||||||||||||||
| ST636 | 2 | 2 | |||||||||||||||||||||||||||
| ST648 | 1 | 2 | 1 | 1 | 5 | ||||||||||||||||||||||||
| ST654 | 1 | 1 | |||||||||||||||||||||||||||
| ST683 | 2 | 2 | |||||||||||||||||||||||||||
| ST744 | 1 | 1 | 1 | 3 | |||||||||||||||||||||||||
| ST1148 | 1 | 1 | |||||||||||||||||||||||||||
| ST1193 | 1 | 1 | |||||||||||||||||||||||||||
| ST1261 | 3 | 3 | |||||||||||||||||||||||||||
| ST1431 | 1 | 1 | |||||||||||||||||||||||||||
| ST2003 | 1 | 1 | 2 | ||||||||||||||||||||||||||
| ST2929 | 2 (1) | 2 (1) | |||||||||||||||||||||||||||
| ST2973 | 1 | 1 | |||||||||||||||||||||||||||
| ST3268 | 1 | 1 | |||||||||||||||||||||||||||
| ST4248 | 1 | 1 | |||||||||||||||||||||||||||
| ST9528 | 1 | 1 | |||||||||||||||||||||||||||
| Total | 1 | (1) | 2 (1) | 3 (1) | 1 | 6 (2) | 18 (4) | 2 (4) | 1 (1) | 5 (3) | 2 | 3 | 5 | 1 | 5 | 1 | 4 | 1 | 2 | 25 | 1 | 17 | 1 | 39 (17) 68 | |||||
Numbers without parenthesis are of livestock and the numbers within parentheses are of farmers.
Numbers in bold and italic indicate isolates with common characteristics isolated from livestock and farmers on each farm: ST189 (CTX-M-69) isolated from calf and farmer from farm F4, ST297 (CTX-M-14) isolated from calf and cattle from farm F2, ST392 (CTX-M-14) isolated from calf and cattle from farm F1, ST540 (CTX-M-55) isolated from cattle and farmer from farm F5, ST617 (CTX-M-156) isolated from piglet and pig from farm F3, and ST2929 (CTX-M-27) isolated from cattle and farmer from farm F4. The details are shown in Table 5.
Numbers that are underlined represent isolates from patients.
The isolates from the farmers belonged to 13 different STs with no obvious predominance. When STs of isolates from farmers were compared, eight STs (ST10, ST58, ST167, ST189, ST297, ST448, ST540, and ST2929) were the same as isolates from livestock and three STs (ST10, ST131, and ST448) were the same as isolates from patients. Except for the common genotypes ST10 and ST448, the genotype relatedness and their frequency were closer to livestock (six STs with six isolates) than to patients (one ST with two isolates).
3.4. Comparing CTX-M-14-producing E. coli
CTX-M-14-producing E. coli isolates were predominantly detected in livestock and patients. As shown in Table 4, almost all the CTX-M-14 β-lactamase-producing E. coli isolates were transferable with high transfer frequencies. Incompatibility groups of the transferred isolate plasmids were analysed, and the isolates from livestock and patients mainly carried IncI1-Iγ (n = 17) and IncF (n = 12), respectively. The constitution of ST isolates from livestock and patients were completely different. In contrast, the constitution of STs from farmers has commonalities.
Table 4.
Phenotypic and genotypic characteristics of CTX-M-14-producing Escherichia coli isolated from livestock, farmers, and patients.
| Livestock | Farmers | Patients | |
|---|---|---|---|
| Number of isolates | 18 | 4 | 25 |
| Number of transferable isolates | 18 | 4 | 24 |
| Transfer frequencies (average) | 6.9 × 10−7 - 1.6 × 10−2 (1.1 × 10−3) | 7.6 × 10−5 - 1.0 × 10−3 (3.6 × 10−3) | 5.6 × 10−8 - 2.8 × 10−1 (2.2 × 10−3) |
| Incompatibility groups of transferred plasmid | |||
| I1-Iγ | 17 | 2 | 3 |
| F | 1 | 2 | 8 |
| F, I1-Iγ | 4 | ||
| NDa | 9 | ||
| Sequence types | |||
| ST131 | 1 | 9 | |
| ST297 | 2 | 1 | |
| ST448 | 1 | 1 | |
| individuals (number of isolates) b | ST58 (1), ST224 (1), ST392 (3), ST446 (1), ST533 (2), ST683 (2), ST744 (1), ST1148 (1), ST1261 (3), | ST46 (1) | ST10 (2), ST38 (1), ST69 (1), ST83(1), ST95 (1), ST218 (1), ST354 (1), ST405 (1), ST537 (1), ST648 (2), ST654 (1), ST1193 (1), ST2003 (1), ST4248 (1) |
ND, not determined.
“Individuals” indicate the STs with no common STs among the isolates from livestock, farmers, and patients.
3.5. Clonal relationship between ESBL carriage in livestock and farmers
The clonal relationship of ESBL producers between livestock and farmers was assessed using resistance genes (CTX-M genotype), genotyping, PFGE, antimicrobial susceptibilities, and plasmid analysis. We identified 17 isolates to be suggested as clonal isolates from Farm 1 (F1) to Farm 6 (F6), as shown in Table 5 and Supplemental Figs. S1 and S2. The ESBL producers from farms F1 and F2 had the same characteristics among calf and parent cattle, respectively. The ESBL producers from farm F3 also had the same characteristics among piglet and parent pig. The ESBL producers from farms F4 and F5 had the same characteristics among cattle (calf) and farmers. They were identified as clonal isolates shared among the isolates on their farms. The ESBL producers from farm F6 had the same resistance gene (CTX-M-14) among calf, cattle, and farmer; however, the genotype and PFGE were different. Analysis of these transconjugants revealed that the replicon type of the plasmid belonged to the same IncI1-Iγ. The transconjugant plasmids were digested with restriction enzymes (EcoRI or HindIII) to confirm whether the plasmids were the same. The RFLP patterns were identical, strongly suggesting that these plasmids were the same (Supplemental Fig. S2). These results show that the CTX-M-14 encoding plasmid was shared among different genotypes of E. coli isolates from calf, cattle, and farmer on farm 6.
Table 5.
Phenotypic and genotypic characteristics of CTX-M-producing Escherichia coli isolates with clonal relatedness among livestock and farmers on their farms.
| Strains | Source a | Resistance gene | Sequence types | PFGE type b | Plasmid replicon type c | MIC (mg/L)c |
Transconjugant profile c |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CTX | CTX/CLA | CAZ | CMZ | AZT | IPM | LEV | GEN | Transfer frequencies | Plasmid replicon type | Plasmid RFLP patternd | ||||||
| TK4934 | F1 calf 1 | CTX-M-14 | ST392 | A | I1-Iγ, FIB, Y | 128 | 0.125 | 1 | 2 | 4 | 0.25 | ≦0.06 | 1 | 2.9 × 10−4 | I1-Iγ | NT |
| TK3027 | F1 calf 2 | CTX-M-14 | ST392 | A | I1-Iγ, FIB | 32 | ≤0.06 | 1 | 2 | 2 | 0.125 | ≦0.06 | 1 | 2.4 × 10−5 | I1-Iγ | NT |
| TK3030 | F1 cattle * | CTX-M-14 | ST392 | A | I1-Iγ, FIB | 32 | ≤0.06 | 1 | 2 | 2 | 0.125 | ≦0.06 | 1 | 6.9 × 10−7 | I1-Iγ | NT |
| TK3016 | F2 calf | CTX-M-14 | ST297 | B | I1-Iγ | 128 | ≤0.06 | 1 | 1 | 4 | 0.125 | 32 | 128 | 1.8 × 10−5 | I1-Iγ | NT |
| TK4763 | F2 cattle * | CTX-M-14 | ST297 | B | I1-Iγ | 64 | ≤0.06 | 1 | 1 | 4 | 0.125 | 32 | 128 | 1.9 × 10−3 | I1-Iγ | NT |
| TK4628 | F3 piglet | CTX-M-156 | ST617 | C | ND | 64 | ≤0.06 | 8 | 1 | 8 | 0.125 | 16 | 0.5 | not | NT | NT |
| TK4639 | F3 pig * | CTX-M-156 | ST617 | C | ND | 64 | ≤0.06 | 8 | 1 | 8 | 0.125 | 16 | 1 | not | NT | NT |
| TK5269 | F4 calf | CTX-M-27 | ST2929 | D | ND | 256 | ≤0.06 | 4 | 2 | 4 | 0.125 | 16 | 64 | not | NT | NT |
| TK5276 | F4 cattle | CTX-M-27 | ST2929 | D | ND | 256 | ≤0.06 | 16 | 1 | 16 | 0.125 | 8 | 0.5 | 4.2 × 10−8 | ND | NT |
| TK5282 | F4 farmer 1 | CTX-M-27 | ST2929 | D | ND | >256 | ≤0.06 | 16 | 1 | 16 | 0.125 | 8 | 0.5 | 1.3 × 10−7 | ND | NT |
| TK5270 | F4 calf | CTX-M-69 | ST189 | E | I1-Iγ, F, HI1, Y | >256 | ≤0.06 | 16 | 1 | 32 | 0.125 | 16 | 64 | 1.6 × 10−7 | I1-Iγ | NT |
| TK5283 | F4 farmer 2 | CTX-M-69 | ST189 | E | I1-Iγ, F, HI1, Y | >256 | ≤0.06 | 16 | 1 | 32 | 0.125 | 16 | 64 | 7.9 × 10−6 | I1-Iγ | NT |
| TK4462 | F5 cattle | CTX-M-55 | ST540 | F | F | >256 | 0.125 | 32 | 2 | 64 | 0.125 | 1 | 128 | 7.7 × 10−5 | F | NT |
| TK4465 | F5 farmer | CTX-M-55 | ST540 | F | F | >256 | 0.125 | 32 | 2 | 64 | 0.125 | 1 | 128 | 1.9 × 10−5 | F | NT |
| TK3887 | F6 calf | CTX-M-14 | ST1148 | G | I1-Iγ, F | 8 | 0.25 | 2 | 4 | 4 | 0.125 | 64 | 64 | 3.9 × 10−5 | I1-Iγ | A |
| TK3893 | F6 cattle * | CTX-M-14 | ST533 | H | I1-Iγ, F | 64 | 0.125 | 1 | 1 | 4 | 0.125 | 16 | 1 | 1.7 × 10−4 | I1-Iγ | A |
| TK3896 | F6 farmer | CTX-M-14 | ST448 | I | I1-Iγ, FIA, Y | 64 | 0.125 | 1 | 1 | 2 | 0.125 | ≦0.06 | 0.5 | 7.6 × 10−5 | I1-Iγ | A |
F1–F6 and farms 1–6, respectively. Parent cattle (pig) of calves (piglets) on their farms (F1, F2, and F6) are indicated by an asterisk.
PFGE types were assigned as A-I and so on by visual inspection of the macrorestriction profile. Patterns that differed by seven or more bands were considered to represent different profiles.
Abbreviations: CTX, cefotaxime; CLA, clavulanic acid; CAZ, ceftazidime; CMZ, cefmetazole; AZT, aztreonam; IPM, imipenem; LEV, levofloxacin; GEN, gentamicin; not, not transferred; ND, not determined; NT, not tested.
Plasmid RFLP assigned as A was considered to represent the same pattern.
4. Discussion
This study assessed the molecular epidemiology of 3GC-resistant E. coli isolates from livestock, farmers, and human patients. To the best of our knowledge, this is the first study to report the relatedness of CTX-M-producing E. coli among livestock, farmers, and human patients. Previous studies reported the prevalence of ESBL- and pAmpC-producing E. coli in cattle and swine in Japan as 3.1% and 4.6%, respectively [40,41]. For the first time, this study reports the rates of 3GC-resistant E. coli in cattle and swine farmers, and the rates (cattle farmers, 30.8%; swine farmers, 11.1%) were higher even in the farmers, similar to the corresponding livestock. The prevalence of faecal carriage of ESBL-producing E. coli among healthy individuals in Japan was reported to be 9.7% between 2015 and 2019 [22]. Interestingly, the carriage rate was higher for farmers than for healthy individuals in Japan, suggesting a stronger ESBL- and pAmpC-producing E. coli transmission between livestock and farmers.
Almost all 3GC-resistant E. coli strains harboured blaCTX-M in this study. The CTX-M-9 group was predominantly isolated from livestock, farmers, and patients, with CTX-M-14 being the most frequently identified gene. Previous reports also showed that CTX-M-14-producing E. coli is predominantly isolated from patients and individuals in Japan [22,42], China, Southeast Asia, and South Korea [14]. Interestingly, CTX-M-9 group-producing E. coli was also predominantly isolated from livestock in Japan. CTX-M-15 (CTX-M-1 group) incidence has increased over time in most countries and is dominant in most regions; however, it was less frequently detected in this study which corroborates our hypothesis that resistant isolates could be transferred to livestock and patients. Comparing the genotype of isolates, each ST of isolates from livestock and patients was unique and specific. Genotypes of the isolates from livestock belonged to 25 different STs, with no dominant type. The dominant genotype of the isolates from patients was ST131; however, it was not detected in the isolates from livestock. ST131 is most widespread in humans and appears to be associated with the human host. Only four STs (ST10, ST38, ST101, and ST448) were of the same genotype in livestock and patients. Many studies have reported the presence of CTX-M-producing E. coli ST10 and ST38 in humans and animals, suggesting that E. coli isolates belonging to these STs are transmissible between animals and humans [41,[43], [44], [45]]. However, these STs were infrequent, and the characteristics (resistance gene type, plasmid type, and antimicrobial susceptibilities) of the isolates of each STs isolates were different in this study. Thus, it could be hypothesised that the transmission of 3GC-resistant E. coli between livestock and patients was not significant and considered to be low. Previous reports also showed that a high similarity in gene distributions was found in isolates from livestock (swine and poultry) and their farmers; however, the same was not found between livestock and patients [46]. These findings suggest that livestock are not contributing as major reservoirs to 3GC-resistant E. coli in humans.
CTX-M-14-producing E. coli were detected in high frequencies in livestock, farmers, and patients in this study. As previously reported, CTX-M-14 is among the most prevalent ESBLs in companion animals and poultry in Asia (30–33%) and, to a lesser extent, in cattle and pigs (14%) [47]. These isolates were most transmissible and had similar transmission frequencies. Between livestock and patients, Inc. types of both isolates were different, with no common STs. In Japan, CTX-M-14-producing E. coli, mostly ST131, is predominantly detected in patients [42]. CTX-M-14-producing E. coli was also isolated from livestock, but its characteristics were different from those of the isolates from patients. In European countries, CTX-M-15, which has spread like a pandemic in humans, was detected rarely (8%) in cattle and pigs, but CTX-M-1 was frequent (72%) [47,48]. However, the comparison between livestock and farmers revealed some similarities. There were three types of isolates with the same characteristics (CTX-M genotype, STs, PFGE band pattern, antimicrobial susceptibilities, and plasmid characteristics) between calves (cattle) and farmers. Farmers are in close contact daily with their livestock, favouring clonal transfer of ESBL-producing E. coli. Similarly, three other isolates with the same characteristics were identified between calves, cattle, piglets, and pigs on each farm, indicating that ESBL-producing E. coli is transmitted through close contact. Additionally, we identified three isolates with the same plasmid characteristics (transferability, plasmid incompatibility, and plasmid RFLP pattern) but different genotypes (STs and PFGE patterns) from calf, cattle, and farmer in farm F6. Interestingly, there might be a possible transfer of the resistance gene coding plasmid in E. coli between livestock and farmers due to their close contact relationship. Resistance gene coding plasmid transmission across different strains or species in hospital patients has been reported [39,49,50]. To our knowledge, this is the first report describing a transfer of resistance plasmids across different strains of farmers and livestock.
This study presents limitations. First, samples from swine and the farmers were fewer than those from cattle. To gain more insights, more samples would be necessary. A previous study suggested the possibility of clonal and horizontal dissemination of ESBL-producing E. coli between pigs and pig farmers [51]; by increasing the sample size in the present study, we expect to observe similar relatedness between cattle and cattle farmers. Second, the sampling location for livestock (farmers) and patients was different. Sampling from livestock and farmers was conducted in the southern part of Kyushu region, whereas that from patients was conducted throughout Japan, excluding the Kyushu region. However, previous studies on ESBL-producing E. coli isolated from outpatients of hospitals throughout Japan, including the Kyushu region, reported that the CTX-M-9 group was the most frequently detected type (72.7% of ESBL in throughout Japan [52] and 77% of ESBL in Kyushu region [53]). Furthermore, in a study on ESBL-producing E. coli isolated from diarrhoea patients in Kyushu region, 77.1% of the isolates were CTX-M-14-producing ST131 [54]. Based on the results of these studies, it is assumed that the frequency of ESBL-producing E. coli in Kyushu region is similar to that throughout Japan. More rigorous comparisons could be performed between livestock and a sample of patients from the same area. Lastly, there is no analysis of the environmental pollution caused by livestock faeces. Faecal bacteria can survive for a long time in soil, manure, and water [55]. Faeces of livestock could be a reservoir of AMR dissemination, and more studies of exposure 3GC-resistant E. coli in the surrounding environment of the farms are warranted.
5. Conclusions
Our findings indicate that CTX-M-14 is predominant in 3GC-resistant E. coli; however, a distinct relationship was not observed between the isolates, especially in livestock and patients. Some clonal relatedness was observed between CTX-M-producing E. coli isolated from livestock and farmers. We suggest that the close contact of farmers and their livestock daily is a risk factor for more efficient clonal transfer of ESBL-producing E. coli.
The following are the supplementary data related to this article.
Figure S1. PFGE profiles of XbaI-digested fragments of the genomic DNA of E. coli isolates harbouring CTX-M. M, molecular marker; lane 1, TK4934; lane 2, TK3027; lane 3 TK3030; lane 4 TK3016; lane 5, TK4763; lane 6, TK4628; lane 7, TK4639; lane 8, TK5269; lane 9, TK5276; lane 10, TK5282; lane 11, TK5270; lane 12, TK5283; lane 13, TK4462; lane 14, TK4465; lane 15, TK3887; lane 16, TK3893; lane 17, TK3896
Figure S2. Plasmid patterns after restriction enzyme digestion profiles of transconjugants of the three E. coli strains harbouring CTX-M-14 isolated from Farm 6. Lane 1 to 3, EcoRI digestion fragments of plasmid from their corresponding E. coli transconjugants of TK3887, TK3893, and TK3896. Lane 4 to 6, HindIII digestion fragments of plasmid from their corresponding E. coli transconjugants of TK3887, TK3893, and TK3896.
Funding
This study was supported by JSPS KAKENHI (Grant Nos. 21K10403 and 20K10433). The funding agency had no role in the study design, data collection, analysis and interpretation, writing of the manuscript, and in the decision to submit the article for publication.
CRediT authorship contribution statement
Ryuichi Nakano: Conceptualization, Data curation, Funding acquisition, Investigation, Writing – original draft. Akiyo Nakano: Conceptualization, Data curation, Funding acquisition, Investigation, Writing – original draft. Ryuji Nishisouzu: Resources, Investigation, Data curation. Kenji Hikosaka: Investigation, Data curation. Yuki Suzuki: Investigation, Data curation. Go Kamoshida: Investigation, Data curation. Shigeru Tansho-Nagakawa: Resources, Data curation. Shiro Endo: Resources, Data curation. Kei Kasahara: Resources, Data curation. Yasuo Ono: Project administration, Supervision. Hisakazu Yano: Project administration, Supervision.
Declaration of Competing Interest
None.
Acknowledgements
The authors would like to thank Takako Nakano, Keimi Nakano, Kaori Nishisouzu, Kazuyuki Makiguchi, Shigehiro Shimokariya, Miwa Asahara, and Tomoko Asada for their contribution to the sample collection. The authors also thank all farmers, family members, and employees for their participation.
Data availability
No data was used for the research described in the article.
References
- 1.de Kraker M.E., Davey P.G., Grundmann H., B.S. Group Mortality and hospital stay associated with resistant Staphylococcus aureus and Escherichia coli bacteremia: estimating the burden of antibiotic resistance in Europe. PLoS Med. 2011;8 doi: 10.1371/journal.pmed.1001104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Mauldin P.D., Salgado C.D., Hansen I.S., Durup D.T., Bosso J.A. Attributable hospital cost and length of stay associated with health care-associated infections caused by antibiotic-resistant gram-negative bacteria. Antimicrob. Agents Chemother. 2010;54:109–115. doi: 10.1128/AAC.01041-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Woerther P.L., Burdet C., Chachaty E., Andremont A. Trends in human fecal carriage of extended-spectrum beta-lactamases in the community: toward the globalization of CTX-M. Clin. Microbiol. Rev. 2013;26:744–758. doi: 10.1128/CMR.00023-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Tacconelli E., Carrara E., Savoldi A., Harbarth S., Mendelson M., Monnet D.L., et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 2018;18:318–327. doi: 10.1016/S1473-3099(17)30753-3. [DOI] [PubMed] [Google Scholar]
- 5.Bush K. Alarming beta-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr. Opin. Microbiol. 2010;13:558–564. doi: 10.1016/j.mib.2010.09.006. [DOI] [PubMed] [Google Scholar]
- 6.Pfeifer Y., Cullik A., Witte W. Resistance to cephalosporins and carbapenems in gram-negative bacterial pathogens. Int. J. Med. Microbiol. 2010;300:371–379. doi: 10.1016/j.ijmm.2010.04.005. [DOI] [PubMed] [Google Scholar]
- 7.Tamma P.D., Doi Y., Bonomo R.A., Johnson J.K., Simner P.J. G. Antibacterial resistance leadership, a primer on AmpC beta-lactamases: necessary knowledge for an increasingly multidrug-resistant world. Clin. Infect. Dis. 2019;69:1446–1455. doi: 10.1093/cid/ciz173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Nakano R., Okamoto R., Nagano N., Inoue M. Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC beta-lactamase blaCMY-4 promoted by insertion sequence ISEcp1. J. Infect. Chemother. 2007;13:18–23. doi: 10.1007/s10156-006-0483-6. [DOI] [PubMed] [Google Scholar]
- 9.Partridge S.R., Kwong S.M., Firth N., Jensen S.O. Mobile genetic elements associated with antimicrobial resistance. Clin. Microbiol. Rev. 2018;31 doi: 10.1128/CMR.00088-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Bush K., Bradford P.A. Epidemiology of beta-lactamase-producing pathogens. Clin. Microbiol. Rev. 2020;33 doi: 10.1128/CMR.00047-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Nakano R., Nakano A., Abe M., Inoue M., Okamoto R. Regional outbreak of CTX-M-2 beta-lactamase-producing Proteus mirabilis in Japan. J. Med. Microbiol. 2012;61:1727–1735. doi: 10.1099/jmm.0.049726-0. [DOI] [PubMed] [Google Scholar]
- 12.Kakuta N., Nakano R., Nakano A., Suzuki Y., Masui T., Horiuchi S., et al. Molecular characteristics of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in Japan: predominance of CTX-M-15 and emergence of hypervirulent clones. Int. J. Infect. Dis. 2020;98:281–286. doi: 10.1016/j.ijid.2020.06.083. [DOI] [PubMed] [Google Scholar]
- 13.Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 2004;48:1–14. doi: 10.1128/AAC.48.1.1-14.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Bevan E.R., Jones A.M., Hawkey P.M. Global epidemiology of CTX-M beta-lactamases: temporal and geographical shifts in genotype. J. Antimicrob. Chemother. 2017;72:2145–2155. doi: 10.1093/jac/dkx146. [DOI] [PubMed] [Google Scholar]
- 15.Castanheira M., Simner P.J., Bradford P.A. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC Antimicrob. Resist. 2021;3:dlab092. doi: 10.1093/jacamr/dlab092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Rousham E.K., Unicomb L., Islam M.A. Human, animal and environmental contributors to antibiotic resistance in low-resource settings: integrating behavioural, epidemiological and one health approaches. Proc. Biol. Sci. 2018;285 doi: 10.1098/rspb.2018.0332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Hayer S.S., Rovira A., Olsen K., Johnson T.J., Vannucci F., Rendahl A., et al. Prevalence and time trend analysis of antimicrobial resistance in respiratory bacterial pathogens collected from diseased pigs in USA between 2006-2016. Res. Vet. Sci. 2020;128:135–144. doi: 10.1016/j.rvsc.2019.11.010. [DOI] [PubMed] [Google Scholar]
- 18.Hayer S.S., Casanova-Higes A., Paladino E., Elnekave E., Nault A., Johnson T., et al. Global distribution of extended Spectrum cephalosporin and Carbapenem resistance and associated resistance markers in Escherichia coli of swine origin - a systematic review and Meta-analysis. Front. Microbiol. 2022;13 doi: 10.3389/fmicb.2022.853810. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Canton R., Ruiz-Garbajosa P. Co-resistance: an opportunity for the bacteria and resistance genes. Curr. Opin. Pharmacol. 2011;11:477–485. doi: 10.1016/j.coph.2011.07.007. [DOI] [PubMed] [Google Scholar]
- 20.Hernando-Amado S., Coque T.M., Baquero F., Martinez J.L. Defining and combating antibiotic resistance from one health and Global Health perspectives. Nat. Microbiol. 2019;4:1432–1442. doi: 10.1038/s41564-019-0503-9. [DOI] [PubMed] [Google Scholar]
- 21.Nakano A., Nakano R., Nishisouzu R., Suzuki Y., Horiuchi S., Kikuchi-Ueda T., et al. Prevalence and relatedness of mcr-1-mediated Colistin-resistant Escherichia coli isolated from livestock and farmers in Japan. Front. Microbiol. 2021;12 doi: 10.3389/fmicb.2021.664931. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Masui T., Nakano R., Nakano A., Saito K., Suzuki Y., Kakuta N., et al. Predominance of CTX-M-9 group among ESBL-producing Escherichia coli isolated from healthy individuals in Japan. Microb. Drug Resist. 2022;28:355–360. doi: 10.1089/mdr.2021.0062. [DOI] [PubMed] [Google Scholar]
- 23.Komatsu Y., Kasahara K., Inoue T., Lee S.T., Muratani T., Yano H., et al. Molecular epidemiology and clinical features of extended-spectrum beta-lactamase- or carbapenemase-producing Escherichia coli bacteremia in Japan. PLoS One. 2018;13 doi: 10.1371/journal.pone.0202276. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Kosai K., Yamagishi Y., Hashinaga K., Nakajima K., Mikamo H., Hiramatsu K., et al. Multicenter surveillance of the epidemiology of gram-negative bacteremia in Japan. J. Infect. Chemother. 2020;26:193–198. doi: 10.1016/j.jiac.2019.11.003. [DOI] [PubMed] [Google Scholar]
- 25.Kawamura K., Goto K., Nakane K., Arakawa Y. Molecular epidemiology of extended-spectrum beta-lactamases and Escherichia coli isolated from retail foods including chicken meat in Japan. Foodborne Pathog. Dis. 2014;11:104–110. doi: 10.1089/fpd.2013.1608. [DOI] [PubMed] [Google Scholar]
- 26.Hiroi M., Yamazaki F., Harada T., Takahashi N., Iida N., Noda Y., et al. Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals. J. Vet. Med. Sci. 2012;74:189–195. doi: 10.1292/jvms.11-0372. [DOI] [PubMed] [Google Scholar]
- 27.CLSI . 28th ed. 2018. Performance Standards for Antimicrobial Susceptibility Testing. [Google Scholar]
- 28.Perez-Perez F.J., Hanson N.D. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 2002;40:2153–2162. doi: 10.1128/JCM.40.6.2153-2162.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Poirel L., Walsh T.R., Cuvillier V., Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn. Microbiol. Infect. Dis. 2011;70:119–123. doi: 10.1016/j.diagmicrobio.2010.12.002. [DOI] [PubMed] [Google Scholar]
- 30.Dallenne C., Da Costa A., Decré D., Favier C., Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 2010;65:490–495. doi: 10.1093/jac/dkp498. [DOI] [PubMed] [Google Scholar]
- 31.Nakano A., Nakano R., Suzuki Y., Saito K., Kasahara K., Endo S., et al. Rapid identification of bla(IMP-1) and bla(IMP-6) by multiplex amplification refractory mutation system PCR. Ann. Lab. Med. 2018;38:378–380. doi: 10.3343/alm.2018.38.4.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Ohnishi M., Okatani A.T., Harada K., Sawada T., Marumo K., Murakami M., et al. Genetic characteristics of CTX-M-type extended-spectrum-beta-lactamase (ESBL)-producing enterobacteriaceae involved in mastitis cases on Japanese dairy farms, 2007 to 2011. J. Clin. Microbiol. 2013;51:3117–3122. doi: 10.1128/JCM.00920-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi
- 34.Wirth T., Falush D., Lan R., Colles F., Mensa P., Wieler L.H., et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 2006;60:1136–1151. doi: 10.1111/j.1365-2958.2006.05172.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.MLST https://pubmlst.org/bigsdb?db=pubmlst_mlst_seqdef
- 36.Yano H., Uemura M., Endo S., Kanamori H., Inomata S., Kakuta R., et al. Molecular characteristics of extended-spectrum β-lactamases in clinical isolates from Escherichia coli at a Japanese tertiary hospital. PLoS One. 2013;8 doi: 10.1371/journal.pone.0064359. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Tenover F.C., Arbeit R.D., Goering R.V., Mickelsen P.A., Murray B.E., Persing D.H., et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 1995;33:2233–2239. doi: 10.1128/jcm.33.9.2233-2239.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Carattoli A., Bertini A., Villa L., Falbo V., Hopkins K.L., Threlfall E.J. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods. 2005;63:219–228. doi: 10.1016/j.mimet.2005.03.018. [DOI] [PubMed] [Google Scholar]
- 39.Eda R., Nakamura M., Takayama Y., Maehana S., Nakano R., Yano H., et al. Trends and molecular characteristics of carbapenemase-producing Enterobacteriaceae in Japanese hospital from 2006 to 2015. J. Infect. Chemother. 2020;26(7):667–671. doi: 10.1016/j.jiac.2020.02.002. [DOI] [PubMed] [Google Scholar]
- 40.Ohnishi M., Okatani A.T., Esaki H., Harada K., Sawada T., Murakami M., et al. Herd prevalence of Enterobacteriaceae producing CTX-M-type and CMY-2 beta-lactamases among Japanese dairy farms. J. Appl. Microbiol. 2013;115(1):282–289. doi: 10.1111/jam.12211. [DOI] [PubMed] [Google Scholar]
- 41.Norizuki C., Kawamura K., Wachino J.I., Suzuki M., Nagano N., Kondo T., et al. Detection of Escherichia coli producing CTX-M-1-group extended-spectrum beta-lactamases from pigs in Aichi prefecture, Japan, between 2015 and 2016. Jpn. J. Infect. Dis. 2018;71:33–38. doi: 10.7883/yoken.JJID.2017.206. [DOI] [PubMed] [Google Scholar]
- 42.Matsumura Y., Johnson J.R., Yamamoto M., Nagao M., Tanaka M., Takakura S., et al. CTX-M-27- and CTX-M-14-producing, ciprofloxacin-resistant Escherichia coli of the H30 subclonal group within ST131 drive a Japanese regional ESBL epidemic. J. Antimicrob. Chemother. 2015;70:1639–1649. doi: 10.1093/jac/dkv017. [DOI] [PubMed] [Google Scholar]
- 43.Blaak H., Hamidjaja R.A., van Hoek A.H., de Heer L., de Roda Husman A.M., Schets F.M. Detection of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli on flies at poultry farms. Appl. Environ. Microbiol. 2014;80:239–246. doi: 10.1128/AEM.02616-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Araujo S., S.A. T, Tacão M., Patinha C., Alves A., Henriques I. Characterization of antibiotic resistant and pathogenic Escherichia coli in irrigation water and vegetables in household farms. Int. J. Food Microbiol. 2017;257:192–200. doi: 10.1016/j.ijfoodmicro.2017.06.020. [DOI] [PubMed] [Google Scholar]
- 45.Kawamura K., Nagano N., Suzuki M., Wachino J.I., Kimura K., Arakawa Y. ESBL-producing Escherichia coli and its rapid rise among healthy people. Food Saf. (Tokyo) 2017;5:122–150. doi: 10.14252/foodsafetyfscj.2017011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Dorado-Garcia A., Smid J.H., van Pelt W., Bonten M.J.M., Fluit A.C., van den Bunt G., et al. Molecular relatedness of ESBL/AmpC-producing Escherichia coli from humans, animals, food and the environment: a pooled analysis. J. Antimicrob. Chemother. 2018;73(2):339–347. doi: 10.1093/jac/dkx397. [DOI] [PubMed] [Google Scholar]
- 47.Ewers C., Bethe A., Semmler T., Guenther S., Wieler L.H. 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. 2012;18:646–655. doi: 10.1111/j.1469-0691.2012.03850.x. [DOI] [PubMed] [Google Scholar]
- 48.Cantón R., Novais A., Valverde A., Machado E., Peixe L., Baquero F., et al. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 2008;14(Suppl. 1):144–153. doi: 10.1111/j.1469-0691.2007.01850.x. [DOI] [PubMed] [Google Scholar]
- 49.Conlan S., Park M., Deming C., Thomas P.J., Young A.C., Coleman H., et al. Plasmid dynamics in KPC-Positive Klebsiella pneumoniae during long-term patient colonization. mBio. 2016;7(3) doi: 10.1128/mBio.00742-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Nakano R., Nakano A., Abe M., Inoue M., Okamoto R. Regional outbreak of CTX-M-2 beta-lactamase-producing Proteus mirabilis in Japan. J. Med. Microbiol. 2012;61(Pt 12):1727–1735. doi: 10.1099/jmm.0.049726-0. [DOI] [PubMed] [Google Scholar]
- 51.Dohmen W., Liakopoulos A., Bonten M.J.M., Mevius D.J., Heederik D.J.J. Longitudinal study of dynamic epidemiology of extended-Spectrum Beta-lactamase-producing Escherichia coli in pigs and humans living and/or working on pig farms. Microbiol. Spectr. 2023;11(1) doi: 10.1128/spectrum.02947-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Shibasaki M., Komatsu M., Sueyoshi N., Maeda M., Uchida T., Yonezawa H., et al. Community spread of extended-spectrum beta-lactamase-producing bacteria detected in social insurance hospitals throughout Japan. J. Infect. Chemother. 2016;22(6):395–399. doi: 10.1016/j.jiac.2016.03.001. [DOI] [PubMed] [Google Scholar]
- 53.Chong Y., Shimoda S., Yakushiji H., Ito Y., Miyamoto T., Kamimura T., et al. Community spread of extended-spectrum beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis: a long-term study in Japan. J. Med. Microbiol. 2013;62(Pt 7):1038–1043. doi: 10.1099/jmm.0.059279-0. [DOI] [PubMed] [Google Scholar]
- 54.Imuta N., Ooka T., Seto K., Kawahara R., Koriyama T., Kojyo T., et al. Phylogenetic analysis of Enteroaggregative Escherichia coli (EAEC) isolates from Japan reveals emergence of CTX-M-14-producing EAEC O25:H4 clones related to sequence type 131. J. Clin. Microbiol. 2016;54(8):2128–2134. doi: 10.1128/JCM.00711-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Holvoet K., Sampers I., Callens B., Dewulf J., Uyttendaele M. Moderate prevalence of antimicrobial resistance in Escherichia coli isolates from lettuce, irrigation water, and soil. Appl. Environ. Microbiol. 2013;79(21):6677–6683. doi: 10.1128/AEM.01995-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1. PFGE profiles of XbaI-digested fragments of the genomic DNA of E. coli isolates harbouring CTX-M. M, molecular marker; lane 1, TK4934; lane 2, TK3027; lane 3 TK3030; lane 4 TK3016; lane 5, TK4763; lane 6, TK4628; lane 7, TK4639; lane 8, TK5269; lane 9, TK5276; lane 10, TK5282; lane 11, TK5270; lane 12, TK5283; lane 13, TK4462; lane 14, TK4465; lane 15, TK3887; lane 16, TK3893; lane 17, TK3896
Figure S2. Plasmid patterns after restriction enzyme digestion profiles of transconjugants of the three E. coli strains harbouring CTX-M-14 isolated from Farm 6. Lane 1 to 3, EcoRI digestion fragments of plasmid from their corresponding E. coli transconjugants of TK3887, TK3893, and TK3896. Lane 4 to 6, HindIII digestion fragments of plasmid from their corresponding E. coli transconjugants of TK3887, TK3893, and TK3896.
Data Availability Statement
No data was used for the research described in the article.