Spread of CTX-Type Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates of Epidemic Clone B2-O25-ST131 Among Dogs and Cats in Japan

Abstract

This study was performed to investigate the carriage rates of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Escherichia coli among ill companion animals in Japan. Among the 178 nonrepetitive E. coli isolates, including 131 from dogs and 47 from cats, collected between September and November 2015, 42 (23.6%) isolates from 29 dogs and 13 cats were identified as ESBL producers. The antimicrobial susceptibility, O serotype, phylogenetic group, β-lactamase genotype, plasmid replicon type, and sequence type (ST) of each isolate were analyzed. The major ESBL types were CTX-M-14 (26.8%), CTX-M-15 (24.4%), CTX-M-27 (19.5%), and CTX-M-55 (19.5%); predominant replicon types of bla_CTX-M-carrying plasmid were IncF group and IncI1-Iγ. The most prevalent STs were ST131 (n = 15, 35.7%), followed by ST38, ST10, and ST410. The 15 isolates of ST131 belonged to B2-O25. E. coli B2-O25-ST131 isolates harboring bla_CTX-M-15 or bla_CTX-M-27 were resistant to ceftazidime and ciprofloxacin. In particular, CTX-M-15 producers showed multidrug resistance. Our results demonstrated that the CTX-M-producing pandemic E. coli clone B2-O25-ST131 has already spread in Japanese companion animals as well. Moreover, the similarity of genotypes, serotypes, phylogenetic groups, and STs of the isolates from companion animals to those from humans suggested probable transmission of resistant bacteria between pets and humans.

Introduction

The production of extended-spectrum β-lactamases (ESBLs) is one of the most commonly acquired cephalosporin resistance mechanisms in Enterobacteriaceae.¹ Since the 2000s, the resistance rate to third-generation cephalosporins, including cefotaxime and ceftriaxone, in Escherichia coli isolates has increased rapidly because of the dissemination of isolates producing CTX-M-type enzymes.² In particular, a specific E. coli lineage that produces CTX-M-15 and belongs to phylogenetic group B2, serotype O25:H4, and sequence type 131 (ST131) has spread globally as a pandemic clone.³ The worldwide spread of B2-O25:H4-ST131 CTX-M-15-producing E. coli clone has become a serious public health concern, since this clone generally shows coresistance to fluoroquinolones (FQs), aminoglycosides, and trimethoprim (TMP)/sulfamethoxazole (SMX).⁴

ESBL-producing E. coli isolates have been frequently recovered from nonhuman sources, including food-producing and companion animals, as well as the environment.^5–9 In particular, the companion animals such as dogs and cats are considered possible reservoirs of antimicrobial-resistant bacteria, given their close contact with humans and the extensive use of antimicrobial agents approved for humans in companion animals. Indeed, the transmission of resistant bacteria between companion animals and humans has been documented.¹⁰ Of greater concern is that the pandemic clone B2-O25:H4-ST131 CTX-M-15-producing E. coli has also been isolated from companion animals in various European countries.¹¹

Japan faces a similar situation because broad-spectrum cephalosporins have been widely used for treatment of bacterial infections in veterinary medicine. However, in Japan, data on carriage rates and characteristics of ESBL-producing E. coli in companion animals are limited.^12,13 A recent investigation found that B2-O25-ST131 CTX-M-15-producing E. coli is increasingly being isolated from human clinical specimens in Japan,¹⁴ leading us to speculate that companion animals may act as a reservoir for these drug-resistant microorganisms in our country also.

To address this issue, this study investigated the prevalence of CTX-M-type ESBL-producing E. coli isolates among ill companion animals. In particular, we focused on the emergence and spread of isolates of the CTX-M-producing E. coli pandemic clone B2-O25-ST131 to evaluate the possibility of their transmission between humans and companion animals.

Materials and Methods

Bacterial isolates

A total of 1,167 clinical specimens were collected from ill pets across Japan between September and November 2015 and subjected to microbial examination by a private company for diagnostic service, Miroku Medical Laboratory Co., Ltd. (Saku, Nagano, Japan). Among the total of 1,167 specimens, 1,112 bacterial isolates were recovered from 812 specimens because multiple bacterial isolates were obtained from some of these specimens. Finally, a total of 178 nonrepetitive E. coli isolates were obtained from clinical specimens of 131 ill dogs and 47 ill cats. The sources were mainly urine (102 samples), pus (23 samples), and nasal cavity (14 samples); specimens were also acquired from intrauterine liquid (11), otorrhea (8), and other sources (20), including ascites, pleural effusion, and skin. Stool samples were not included.

The phenotype of each isolate was confirmed using the VITEK 2 System (Sysmex bioMérieux, Tokyo, Japan); those showing resistance to extended-spectrum cephalosporins were further evaluated for ESBL production with a phenotypic confirmatory test using both cefotaxime (CTX) and ceftazidime (CAZ) alone and in combination with clavulanic acid according to the Clinical Laboratory Standards Institute (CLSI) guidelines.¹⁵

Characterization of genes encoding β-lactamase

The presence of CTX-M β-lactamase-encoding genes was detected by PCR amplification.¹⁶ Additional consensus primers CTX-M/F (5′-TTT GCG ATG TGC AGT ACC AGT-3′) and CTX-M/R (5′-CTC CGC TGC CGG TTT TAT C-3′) were also used. After classification into CTX-M-1, -2, -8, and -9 groups, the CTX-M-1 and CTX-M-9 groups were subjected to nucleotide sequence analyses. Sequencing of bla_CTX-M-1 group genes was performed using original external PCR primers CTXMGp1F (5′-TCG TCT CTT CCA GAA TAA GGA ATC-3′) and CTXMGp1R (5′-GTT TCC CCA TTC CGT TTC CG-3′) and the following cycling conditions: 95°C for 5 min, 30 cycles of 94°C for 30 sec, 59°C for 30 sec, and 72°C for 60 sec, and a final extension at 72°C for 7 min, which yielded a 925-bp amplicon. Sequencing analyses of bla_CTX-M-9 group genes were performed as previously described¹⁷ using primers CTXGp9F (5′-GAT TGA CCG TAT TGG GAG TTT G-3′) and CTXMGp9R (5′-ATT TAC TTC CAT TAC TTT GCG G-3′), yielding a 1,086-bp amplicon. The bla_TEM and bla_SHV genes were PCR amplified and the genes of positive isolates were sequenced as previously described.¹⁸ The six major plasmid-mediated AmpC (pAmpC) genes were detected by PCR amplification and sequenced as previously described.¹⁹ Sequence analyses and comparisons to known sequences were performed with the BLAST programs on the National Center for Biotechnology Information website (www.ncbi.nlm.nih/gov/BLAST).

Transformation and plasmid replicon typing

Plasmid DNA was prepared using the PureYield Plasmid Midiprep System (Promega, Madison, WI) according to the manufacturer's instructions and then transformed into E. coli DH10B cells by electroporation using a Gene Pulser Xcell system (Bio-Rad, Hercules, CA).²⁰ Transformants were selected on Müller-Hinton agar (Becton Dickinson, Sparks, MD) plates supplemented with 1 mg/L CTX (Wako Pure Chemical Industries, Osaka, Japan) and their acquisition of bla_CTX-M was confirmed by PCR. The plasmid replicon types of the resultant transformants were determined by PCR-based replicon typing using 18 primer pairs.²¹

Phylogenetic grouping, multilocus sequence typing, and H30/H30-Rx subclonal classification within ST131

The phylogenetic group of ESBL-producing E. coli isolates was determined by multiplex PCR as previously described.²² Multilocus sequence typing (MLST) was performed by analyzing seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). In addition, ST131 isolates were assessed for H30 status by subclone-specific PCR.²³ Ciprofloxacin-nonsusceptible H30 isolates were classified as H30R²⁴ and further assessed for H30Rx status by subclone-specific PCR.²⁵

O serotyping of E. coli and detection of serotype O25b by PCR

Serotype O25 was identified using E. coli antisera (Denka Seiken, Tokyo, Japan) according to the manufacturer's instructions. Genetic O25b serotyping was also confirmed by PCR.²⁶

Antimicrobial susceptibility testing

CTX, CAZ, imipenem (IPM), gentamicin (GEN), kanamycin (KAN), amikacin (AMK), fosfomycin (FOM), chloramphenicol, tetracycline (TET), ciprofloxacin (CIP), TMP, and nitrofurantoin (NFT) were obtained from Wako Pure Chemical Industries; SMX was from Merck & Co. (Kenilworth, NJ). SMX and TMP were used at a 5:1 ratio. Antimicrobial susceptibility was determined using the agar dilution method in accordance with CLSI guidelines,¹⁵ and minimum inhibitory concentrations were interpreted according to CLSI criteria (document M7-A10).²⁷ E. coli ATCC25922 was used as the control strain.

Detection of plasmid-mediated quinolone resistance gene by PCR

The plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qepA, aac(6′)-Ib-cr, and oqxAB were detected by PCR as previously described.^28–30

Statistical analysis

Data were analyzed using statistical analysis software “R version 3.3.1.” Chi-square tests for the distribution of sequence types, plasmid replicon types, and phylogenetic groups among different bla_CTX-M-harboring isolates or among different sequence types were performed. The Benjamini–Hochberg procedure was used to correct multiple comparison p-values.³¹ A Cochran–Mantel–Haenszel chi-square test was performed to evaluate the dissimilarity in the distribution of antibiotic-resistant isolates between serotypes O25 and non-O25.³² p-Values of <0.05 indicated statistical significance.

Results

Prevalence of ESBL-producing E. coli isolates

The production of ESBLs in 178 E. coli isolates was evaluated with a phenotypic confirmation test. A total of 42 (23.6%) ESBL-positive and distinct E. coli isolates were obtained from sick dogs (n = 29) and cats (n = 13). Many isolates were recovered from animals that had urinary tract infections (UTIs); 25 (59.5%) were obtained from urine specimens, while the remaining isolates were obtained from various specimens including those from the skin, pus, and nasal cavity (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr).

Detection of genes encoding ESBLs and pAmpC

Of 42 ESBL-producing E. coli isolates, 41 harbored the bla_CTX-M gene and one had the bla_SHV-12 gene. There were eight variants of bla_CTX-M, including bla_CTX-M-14 (11/41, 26.8%), bla_CTX-M-15 (10/41, 24.4%), bla_CTX-M-27 (8/41, 19.5%), and bla_CTX-M-55 (8/41, 19.5%) (Table 1). One ESBL producer isolated from a dog urine specimen harbored bla_CTX-M-123, which encodes a hybrid β-lactamase of CTX-M-15 and CTX-M-14. Among the 42 ESBL-producing E. coli isolates, 19 isolates harboring any of the ESBL genes (bla_CTX-15, n = 3; bla_CTX-M-55, n = 5; bla_CTX-2, n = 1; bla_CTX-M-8, n = 1; bla_CTX-M-14, n = 6; bla_CTX-M-27, n = 1; bla_CTX-M-123, n = 1; and bla_SHV-12, n = 1) also coharbored the bla_TEM-1 gene, while no isolate harbored pAmpC genes.

Table 1.

Relationship Among Sequence Types, Extended-Spectrum β-Lactamase Genes, and Replicon Types of Extended-Spectrum β-Lactamase-Encoding Plasmids in 42 Escherichia coli Isolates

	ESBL type
	CTX-M-1group				CTX-M-9 group						Replicon type
ST	CTX-M-15	CTX-M-55	CTX-M-2	CTX-M-8	CTX-M-14	CTX-M-24	CTX-M-27	CTX-M-123	SHV-12	Total	IncF ^a	IncI1-Iγ	ND ^b	No transformants
10					3					3	2			1
38		3			2					5		3	1	1
68		1								1			1
70	2	1								3		3
90		1								1			1
95					1					1			1
117						1				1				1
131	5 (5)^c				2 (2)^b		8 (7)^b			15	10			5
162			1		1				1	3		1	1	1
349								1		1		1
405					1					1				1
410	2 (1)^c			1						3	2			1
602					1					1		1
617	1									1		1
1177		1								1			1
2509		1								1		1
Total	10	8	1	1	11	1	8	1	1	42	14	11	6	11

IncF includes IncFIA, IncFIB, and IncFII.

The plasmid replicon type of six transformants was not determined by PCR-based replicon typing.

The numbers of isolates identified as serotype O25 by PCR or antisera are shown in parentheses.

ESBL, extended-spectrum β-lactamase; ST, sequence type.

Relationships among STs, ESBL genes, and plasmid replicon types

MLST analysis revealed 16 STs, including ST131 (n = 15, 35.7%), ST38 (n = 5, 11.9%), ST10, ST70, ST162, and ST410 (n = 3 each, 7.1%) (Table 1). All 15 isolates belonging to ST131 were identified as H30 by fimH-based subclonal typing. All ST131-H30 isolates were ciprofloxacin resistant and, therefore, were classified as H30R. Among them, there were five H30Rx isolates as evidenced by subclone-specific PCR. With respect to relationships between STs and ESBL genes, the STs of the isolates harboring bla_CTX-M-14, bla_CTX-M-15, or bla_CTX-M-55 belonged to different STs, including ST10, ST38, ST70, ST131, and ST410 (Table 1). Although all eight isolates harboring bla_CTX-M-27 were ST131, there was no statistically significant difference in the distribution of ST131 between different ESBL genotypes as evidenced by multiple comparisons with other STs.

Transformation of E. coli DH10B with plasmids that mediate CTX resistance was successful for 31 (73.8%) of the 42 donor isolates. The replicon types of the transformed plasmids were IncF group (n = 14, 33.3%) and IncI1-Iγ (n = 11, 26.2%), whereas those of the remaining six plasmids were undetermined by PCR-based replicon typing using 18 primer pairs. Of the 15 ESBL producers assigned to ST131, 10 harbored the plasmid of replicon types of IncF group (Table 1). ST131 had a tendency of a slight distribution difference compared with other STs (p = 0.0735), although it was not statistically significant.

Among the eight plasmids harboring bla_CTX-M-55, five were of replicon type IncI1-Iγ, while eight plasmids harboring bla_CTX-M-27 showed multiple replicon types of the IncF group. Eleven plasmids harboring bla_CTX-M-14 showed diverse replicon types, including IncI1-Iγ, and multiple replicon types of the IncF group such as IncFIA, FIB, FII, and IncFIA, FII (Table 2). Among different CTX-M types, a statistically significant difference was observed in the distribution of replicon types of the plasmids harboring bla_CTX-M-27 (p < 0.05).

Table 2.

Relationship Between Extended-Spectrum β-Lactamase Genes and Replicon Types of Extended-Spectrum β-Lactamase-Encoding Plasmids in 42 E. coli Isolates

		ESBL type
		CTX-M-1 group		CTX-M-9 group
Replicon type	Number (%)	CTX-M-15	CTX-M-55	CTX-M-14	CTX-M-24	CTX-M-27	CTX-M-2	CTX-M-8	CTX-M-123	SHV-12
IncFIA, FIB, FII	11 (26.2)	2		2		7^a
IncFIA, FIB	1 (2.4)					1
IncFIA, FII	1 (2.4)			1
IncFII	1 (2.4)			1
IncI1-Iγ	11 (28.2)	3	5	2					1
ND^b	6 (14.3)		3	2			1
No transformants	11 (26.2)	5		3	1			1		1
Total (%)	42 (100)	10 (23.8)	8 (19.0)	11 (26.2)	1 (2.4)	8 (19.0)	1 (2.4)	1 (2.4)	1 (2.4)	1 (2.4)

Chi-square tests for the distribution of plasmid replicon types among different CTX-M isolates were performed, and a statistically significant difference was observed in the distribution of replicon types of the plasmids harboring bla_CTX-M-27 (p < 0.05).

The plasmid replicon type of six transformants were not determined by PCR-based replicon typing.

Link between phylogenetic groups and STs

The 42 ESBL-producing E. coli isolates were divided into seven phylogenetic groups, including group B2 (n = 16, 38.1%), group D (n = 9, 21.5%), and group A (n = 5, 11.9%) (Table 3). Of the 25 isolates belonging to groups B2 and D, 16 were obtained from urine specimens (group B2, n = 10; group D, n = 6) (Supplementary Table S1). Some STs and phylogenetic groups were linked, such as ST131 and group B2, ST10 and group A, ST162 and group B1, and ST38 and group D (Table 3). Phylogenetic group B2 had a statistically significant link to ST131 (p < 0.01), while group D was significantly associated with ST38 (p < 0.05).

Table 3.

Phylogenetic Groups and Sequence Types of Extended-Spectrum β-Lactamase-Producing E. coli Isolates

Phylogenetic group	ST type (No. of isolates)	Total (%)
A	ST10 (3), ST617 (1), ST2509 (1)	5 (11.9)
B1	ST162 (3), ST602 (1)	4 (9.5)
B2	ST131 (15),^a,b ST95 (1)	16 (38.1)
C	ST410 (3), ST90 (1)	4 (9.5)
D	ST38 (5),^c ST68 (1), ST349 (1), ST405 (1), ST1177 (1)	9 (21.5)
E	ST70 (3)	3 (7.1)
F	ST117 (1)	1 (2.4)

The serotype of 15 isolates belonging to ST131 and phylogenetic group B2 was O25.

Chi-square test was performed, revealing a statistically significant link between ST131 and phylogenetic group B2 compared to other groups (p < 0.01).

Statistically significant relationship was observed between ST38 and group D (p < 0.05).

Serotype O25 was established using E. coli antisera and by PCR. Among the 42 ESBL producers, 14 were determined as O25b by PCR and one isolate was O25 based on the antiserum. All these 15 isolates belonged to phylogenetic group B2-ST131.

Antimicrobial susceptibility profiles

ESBL-producing E. coli isolated from clinical specimens of ill dogs and cats were susceptible to IPM, AMK, FOM, and NFT (Table 4). However, these isolates showed high resistance to CTX (100%) and CIP (88.1%), and tended to be resistant to CAZ, GEN, KAN, TET, and TMP/SMX. In addition, most isolates showed a multidrug-resistant phenotype, with 23/42 (54.8%) resistant to more than four antimicrobial agents (Supplementary Table S1). All the serotype O25 isolates harboring bla_CTX-M-15 (n = 6), bla_CTX-M-14 (n = 2), and bla_CTX-M-27 (n = 7) were resistant to CIP (Table 4). Serotype O25 isolates harboring bla_CTX-M-15 exhibited a multidrug-resistant phenotype, whereas those harboring bla_CTX-M-27 were susceptible to GEN, KAN, and TET. The distribution of drug-resistant isolates between O25 and non-O25 serotypes was analyzed using the Cochran–Mantel–Haenszel chi-square test, indicating a statistically significant difference between the two serotypes (p < 0.01).

Table 4.

Comparison of Antimicrobial Susceptibility Profiles in Extended-Spectrum β-Lactamase Gene Types and O Serotypes

	Resistant isolates ^a
		CTX-M-1 group				CTX-M-9 group
		CTX-M-15		CTX-M-55		CTX-M-14		CTX-M-27
Antimicrobial agent	Number (%)	O25 ^b	Non-O25	O25	Non-O25	O25	Non-O25	O25	Non-O25	CTX-M-2	CTX-M-8	Others ^c
Cefotaxime	42 (100)	6	4	0	8	2	9	7	1	1	1	2
Ceftazidime	27 (46.3)	6	4	0	8	1	0	5	1	0	0	2
Imipenem	0 (0)	0	0	0	0	0	0	0	0	0	0	0
Gentamicin	15 (35.7)	5	0	0	4	1	3	1	0	0	1	0
Kanamycin	12 (58.6)	6	1	0	1	0	2	0	0	1	1	0
Amikacin	0 (0)	0	0	0	0	0	0	0	0	0	0	0
Ciprofloxacin	37 (88.1)	6	2	0	7	2	8	7	1	1	1	1
Fosfomycin	2 (4.8)	0	0	0	1	0	0	0	0	0	0	1
Chloramphenicol	9 (21.4)	1	2	0	1	1	3	0	0	1	0	0
Tetracycline	18 (42.9)	2	1	0	4	2	4	1	0	1	1	2
Trimethoprim/sulfamethoxazole	15 (35.7)	3	1	0	2	2	3	1	0	1	1	1
Nitrofurantoin	0 (0)	0	0	0	0	0	0	0	0	0	0	0
Total (No. of isolates)		6	4	0	8	2	9	7	1
	42 (100)	10		8		11		8		1	0	2

Serotype O25 was established using E. coli antisera and by PCR. Among the 42 ESBL producers, 14 were determined as O25b by PCR and one isolate was O25 based on the antiserum. Non-O25 isolates were negative for serotype O25 by both analysis using E. coli antisera and PCR.

One isolate was positive for serotype O25 only by the E. coli antiserum.

Other types include one CTX-M-123 and one SHV-12.

PCR screening for detection of PMQR genes revealed the presence of the aac(6′)-Ib-cr gene in seven isolates harboring bla_CTX-M-15 (ST131, n = 5; ST410, n = 2) and one harboring bla_CTX-M-14 (ST10). In addition, the isolate harboring bla_CTX-M-123 was positive for the qnrS gene.

Discussion

Most studies of bacterial infections in animals have focused on Salmonella and Campylobacter species.¹⁰ However, the relationship between companion animals and their owners has changed substantially over the years in Japan and other countries. Specifically, cats and dogs are regarded as actual family members, leading to a high frequency of close physical contact with humans, which may increase the possibility for transmission of antimicrobial-resistant bacteria in community settings. In Japan, CTX-M-15-producing E. coli isolates of pandemic clone B2-O25-ST131 are increasingly detected in hospitals and other settings.¹⁴ This study investigated the role of companion animals as carriers of drug-resistant E. coli to address the reason for the wide dissemination of pandemic E. coli clones in the community.

The rate of isolation of ESBL producers from clinical specimens of sick companion animals was 23.6%, which is significantly higher than those in many countries,⁸ although much lower than that in China (40.4%).³³ This finding may reflect the general trend of using antimicrobials in ill companion animals in Japan. Common use of antimicrobials in pets may account for colonization of ESBL-producing E. coli as a result of drug-resistant E. coli transmission between companion animals and their owners.

The differences in the molecular types of ESBLs have been documented in many reports from various countries and regions; thus, CTX-M-14 and CTX-M-55 are mostly found in China,³³ while CTX-M-1 and CTX-M-15 are observed in Italy and the United States.^34,35 In this study, CTX-M-14-, CTX-M-15-, CTX-M-27-, and CTX-M-55-type β-lactamase producers were detected in almost equal rates. It was previously reported in Japan that CTX-M-27-producing E. coli isolates were predominant, whereas CTX-M-15-type β-lactamase strains were rarely found in companion animals.¹² In this study, we detected B2-O25-ST131 E. coli isolates producing CTX-M-15 (11.9%), CTX-M-14 (2.4%), and CTX-M-27 (19.0%) types of β-lactamases; these results suggest that CTX-M-15-producing B2-O25-ST131 clone has been spreading among companion animals in Japan for several years. Many animals tested in this study had UTIs. E. coli isolates belonging to serotype O25 can readily adhere to human and animal bowels,³⁶ causing UTIs. It was recently reported that O25 was the most frequent O serotype among ESBL producers (CTX-M-14 and CTX-M-27 types) isolated from fecal specimens of healthy Japanese individuals.³⁷ In this study, genetic types, serotypes, phylogenetic groups, and STs were similar between humans and companion animals, suggesting possible transmission of ESBL-producing E. coli between them. Overall, our findings together with previous data suggest that the spread of the ESBL-producing pandemic clone must be more closely monitored in both humans and companion animals.

The predominant replicon types observed in this study were IncI1-Iγ and the IncF group. The plasmid carrying bla_CTX-M-27 had multiple replicon types of the IncF group, whereas that carrying bla_CTX-M-15 had the IncF group and IncI1-Iγ types, which is consistent with earlier reports.^38,39 It was previously found that STs and Inc types existed in different combinations; thus, a human ST131 isolate harbored a plasmid carrying multiple replication origins of the IncF group, while in animal ST10, ST58, and ST117 isolates, IncI1-carrying plasmids were detected.³⁹ These findings are consisted with our result that ST131 was associated with plasmids carrying multiple replication origins belonging to the IncF group, although combinations between ST and Inc type differed in the other STs. Nonetheless, the results of this study demonstrate that E. coli isolates from companion animals are very similar to those from humans.

Phylogenetic lineages of antimicrobial resistance and virulence in ESBL-producing E. coli have been widely studied. ST131 isolates have disseminated to various animal species, including poultry, pigs, and companion animals, as well as humans, and their ST was found to be closely linked to phylogenetic group B2.^40,41 Moreover, the association between different STs and phylogenetic groups, for example, group D (STC38, ST405, and STC69), A (ST10, ST167, and ST617), and B1 (ST410), has been identified.⁸ Our data revealed similar links between STs and phylogenetic groups, confirming that CTX-M-producing E. coli B2-ST131 isolates are predominant in companion animals. Moreover, we identified CIP-resistant D-ST405, which has been reported as the second most prevalent ESBL-producing clonal group among clinical isolates in Japan.¹⁴ These results would suggest a probable transmission of ESBL-producing E. coli isolates between humans and companion animals.

Of greater concern is the fact that ESBL producers are frequently resistant not only to β-lactams but also to FQs, TMP/SMX, and aminoglycosides. Our results showed that resistance rates to these three agents were >35%, whereas that to CIP was remarkably high (88.1%). The proportion of ESBL-producing E. coli isolates resistant to FQs appeared to have increased in parallel with plasmid-mediated resistance mechanisms such as Qnr proteins (qnrA, qnrB, or qnrS), aminoglycoside acetyltransferase variant enzyme [aac(6′)-Ib-cr], or efflux pumps (qepA or oqxAB).⁴² We found, in this study, that CTX-M-15-producing E. coli isolates harbored a variant of the aac(6′)-Ib-cr gene, which is in agreement with a previous study.¹¹ PMQR determinants are still rare⁴³; however, CTX-M-15-producing E. coli ST131 isolates have spread among companion animal populations and may well serve as a reservoir of plasmids conferring resistance to FQs and aminoglycosides. Interestingly, most of the CTX-M-15 and CTX-M-27 producers belonged to serotype O25 and showed multiple resistance profiles. On the other hand, most of the CTX-M-55 and CTX-M-14 producers did not belong to serotype O25, but nevertheless exhibited multiple drug resistance. Thus, differences among pet isolates remain unclear; the relationship between the O serotype and CTX-M type should be clarified in future studies.

In conclusion, we found that CTX-M-producing E. coli isolates derived from a pandemic clone B2-O25-ST131 have already widely spread across companion animal populations in Japan. The CTX-M genes, phylogenetic groups, plasmid replicon types, and antimicrobial susceptibility profiles were highly similar to those of human isolates. These results strongly suggest that drug-resistant bacteria are being transmitted between companion animals and humans in Japan, which may be promoted by close physical contact between humans and household pets, as well as by the use of the same classes of antimicrobial agents.

Continuous studies are required to confirm a potential zoonotic link among ESBL-producing E. coli isolates from companion animals and human cases of infections.

Footnotes

Acknowledgments

We thank Miroku Laboratory diagnostic service for collecting the E. coli isolates.

This research was partly supported by the Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (grant control number: 16fk0108207h0402).

Disclosure Statement

No competing financial interests exist.

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