First Detection of CTX-M-1,CMY-2,and QnrB19 Resistance Mechanisms in Fecal Escherichia coli Isolates from Healthy Pets in Tunisia

Abstract

Our objective was to analyze the carriage rate of extended-spectrum β-lactamase (ESBL)- and plasmidic AmpC β-lactamase (pAmpC)-producing Escherichia coli isolates in fecal samples of healthy pets (dogs and cats) and to characterize the recovered isolates for the presence of other resistance genes and integrons. Eighty fecal samples of healthy pets were inoculated in MacConkey agar plates supplemented with cefotaxime (2 μg/mL) for cefotaxime-resistant (CTX^R) E. coli recovery. CTX^R E. coli isolates were detected in 14 of the 80 fecal samples (17.5%) and the following β-lactamase genes (number of isolates) were detected: bla _CTX-M-1 (8), bla _CTX-M-1+bla _TEM-1b (3)_, bla _CTX-M-1+bla _TEM-1c (1), bla _CTX-M-1+bla _TEM-135 (1), and bla _CMY-2+bla _TEM-1b (1). The 14 E. coli were distributed into the phylogroups B1 (6 isolates), A (5), and D (3). The qnrB19 gene was detected in one CTX-M-1-producing strain of phylogroup D. Five isolates contained class 1 integrons with the following arrangements: dfrA17-aadA5 (2 isolates), dfrA1-aadA1 (1), and dfrA17-aadA5/ dfrA1-aadA1 (2 isolates). The virulence genes fimA and/or aer were detected in all CTX^R strains. In this study, the pet population harbored β-lactamase and quinolone resistance genes of special interest in human health that potentially could be transmitted to humans in close contact with them.

Introduction

E scherichia coli are a common microorganism of the intestinal microbiota of humans and animals and can also be implicated as an opportunistic pathogen in human and animal infections (Pitout, 2010). E. coli isolates harboring extended-spectrum β-lactamases (ESBLs), especially of the CTX-M-class, or plasmidic AmpC β-lactamases (pAmpC), have increasingly been reported worldwide in the last years (Jacoby 2009, Peirano and Pitout 2010, Pitout 2010). ESBL- or pAmpC-producing E. coli isolates frequently contain other resistance genes, and these microorganisms show a multiresistance phenotype that results in difficulties when therapeutic options are sought (Jacoby 2009, Pitout, 2010). In recent years, it has been observed that ESBL-containing E. coli isolates (and also pAmpC-containing isolates) are frequently detected in food-producing animals or food products, and thus health authorities are worried about the potential transmission of these resistant microorganisms to humans through the food chain (EFSA 2011). In addition, some studies have also reported the emergence of these resistance mechanisms among fecal E. coli isolates of healthy pets (Sánchez et al., 2002, Costa et al. 2004, Carattoli et al. 2005, Murphy et al. 2009, Pomba et al. 2009, Gibson et al. 2010, Sun et al. 2010, Ewers et al. 2011). ESBLs or pAmpC-containing E. coli isolates are of concern, especially if they are transferred from animals to the human environment and are implicated in human infections. It is known that pets frequently live in very close contact with their owners, and several publications have reported the occurrence of the same resistance genes in companion animals and in humans and the possible transfer of bacteria from animals to humans (Guardabassi et al. 2004).

In Tunisia the most prevalent CTX-M variant found among ESBL-producing E. coli of food products or the intestinal microbiota of healthy humans is CTX-M-1 (Ben Slama et al. 2010; Ben Sallem et al. 2012), whereas CTX-M-15 is the predominant ESBL among E. coli isolates of the human origin (Ben Slama et al. 2011). There are no data available on the nature and prevalence of ESBLs or pAmpC enzymes in E. coli strains from companion animals in Africa. The objective of this work was to study the presence of ESBL or pAmpC in cefotaxime-resistant (CTX^R) E. coli isolates recovered from the intestinal carriage of healthy pets in Tunisia and to characterize the recovered isolates.

Materials and Methods

Eighty fecal samples were collected during March to July, 2010, from healthy domestic animals (dogs n=41, cats n=39) from a large private veterinary clinic located in Tunis, where animals were admitted for vaccination or grooming. None of the animals included in the study had been exposed to antibiotics during the 3 months prior to the sampling process. This clinic attends approximately 50–75 healthy animals per month, and only animals for which their owner's gave informed consent were included in this study.

Samples were inoculated onto MacConkey agar plates supplemented with CTX (2 μg/mL). One CTX^R E. coli isolate per sample was selected and screened for ESBL and AmpC phenotypes by a double-disk test. Susceptibility testing to 17 antibiotics was carried out by the disk-diffusion method (CLSI 2010).

The clonal relationship among the CTX^R E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE) using XbaI (Tenover et al. 1995; Sáenz et al. 2004). The isolates were assigned to the phylogenetic groups A, B1, B2, or D as previously reported (Clermont et al. 2000). All isolates were screened for O25b and O157 serotypes and for afa/dra operon (Clermont et al. 2008; Blanco et al. 2009). In addition, the sxt, fimA, papG allele III, hlyA, cnf1, papC, aer, eae, and bfp genes encoding virulence factors were tested by PCR (Ruiz et al. 2002).

The genes encoding TEM, SHV, OXA-1, CMY, and CTX-M type β-lactamases and the genetic environment of bla _CTX-M genes were analyzed by PCR and sequencing (Vinué et al. 2008). In addition, plasmidic AmpC β-lactamases were searched using multiplex PCR (Pérez-Pérez and Hanson 2002). The presence of antibiotic resistance genes [tet(A), tet(B), sul1, sul2, sul3, aac(3)-II, aac(3)-IV, strA, strB, qnr, qepA, and aac(6′)-Ib-cr) was determined by PCR (Ben Slama et al. 2011). The qnrB genetic environment was determined by PCR and sequencing based on previous reports (Jacoby et al. 2011). Mutations in the gyrA and parC genes were studied using PCR and sequencing in strain C3566 (Sáenz et al. 2003).

The presence of the intI1 and intI2 genes (encoding class 1 and class 2 integrases, respectively) and the 3′-conserved segment (3′-CS) (qacEΔ1-sul1 genes) of class 1 integrons was examined by PCR. The variable regions of class 1 integrons were characterized by PCR and sequencing (Sáenz et al. 2004). Non-classic class 1 integrons were characterized using a PCR primer-walking strategy, as previously reported (Sáenz et al. 2010).

Results

CTX^R E. coli isolates were detected in 14 of the 80 fecal samples of healthy pets analyzed (17.5%), and 1 isolate per sample was studied further. Thirteen of these samples (8 from cats and 5 of dogs) contained ESBL-positive E. coli isolates, and all of them harbored the bla _CTX-M-1 gene. Different variants of the bla _TEM gene (bla _TEM-1b, bla _TEM-1c and bla _TEM-135) were also detected in 5 of the 13 bla _CTX-M-1-positive isolates. The remaining CTX^R E. coli isolate (recovered from a dog sample) harbored the bla _CMY-2 gene (encoding the pAmpC β-lactamase CMY-2), which also contained the bla _TEM-1b gene (Table 1).

Table 1.

Characteristics of the 14 CTX-Resistant E. coli Strains Recovered from Healthy Companion Animals in This Study

						Class 1 integron
E. coli isolate	Origin	PFGE Pattern	Phylogroup	β-lactamases detected ^a	bla _CTX-M-1 genetic environment	intI1 / 3′-CS	Integron structure	Resistance phenotype to non-β-lactam antibiotics	Other resistance genes detected outside integron	Virulence factors
C3143	Cat	P1-A	D	bla _CTX-M-1	ISEcp1 — orf477	−/−	—	SUL-TET-NAL	sul2, tet(A)	fimA-aer
C3566	Dog	P1-B	D	bla _CTX-M-1	ISEcp1 — orf477	+/−	dfrA17-aadA5	SXT-SUL-TET-NAL	sul2, tet(A), qnrB19	fimA-aer
C3567	Dog	P2	D	bla _CTX-M-1	ISEcp1 — orf477	−/−	—	SUL-TET	sul2, tet(A)	fimA-aer
C3144	Cat	P3	A	bla _CTX-M-1	ISEcp1^a—orf477	+/−	dfrA17-aadA5/ dfrA1-aadA1	SXT-SUL-TET-CIP-NAL	sul2, sul3, tet(A), tet(B)	aer
C3147	Dog	P4	A	bla _CTX-M-1, bla _TEM-1c	ISEcp1 — orf477	−/−	—	SUL-TET-NAL	sul2, tet(A)	fimA-aer
C3148	Cat	P5	A	bla _CTX-M-1, bla _TEM-135	ISEcp1 — orf477	+/−	—	SXT-SUL-TET	sul2, sul3, tet(A)	fimA
C3149	Dog	P6	A	bla _CTX-M-1, bla _TEM-1b	ISEcp1 — orf477	+/−	—	SXT-SUL-TET-CIP-NAL-STR	sul2, tet(A), strA/strB	fimA-aer
C3150	Cat	P7	A	bla _CTX-M-1	ISEcp1 — orf477	+/+	dfrA1-aadA1	SXT-SUL-TET-STR	sul2,tet(A), strA/strB	fimA
C3151	Cat	P8	B1	bla _CTX-M-1 , bla _TEM-1b	ISEcp1 — orf477	+/−	dfrA17-aadA5/dfrA1-aadA1	SXT-SUL-TET-NAL-KAN-STR	sul2, sul3, tet(A), strA/strB	fimA-aer
C3152	Cat	P9	B1	bla _CTX-M-1, bla _TEM-1b	ISEcp1 — orf477	+/−	—	SXT-SUL-TET-CIP-NAL-STR	sul2, tet(A), strA/strB	fimA-aer
C3565	Cat	P10	B1	bla _CTX-M-1	ISEcp1 — orf477	−/−	—	SUL-TET-CIP-NAL	sul2, tet(A)	fimA
C3145	Dog	P11	B1	bla _CTX-M-1	ISEcp1 — orf477	+/−	dfrA17-aadA5	SXT-SUL-TET-STR	sul2, tet(B), strA/strB	fimA-aer
C3146	Cat	P12	B1	bla _CTX-M-1	ISEcp1 — orf477	−/−	—	SUL-TET-NAL	sul2, tet(A)	fimA-aer
C3564	Dog	P13	B1	bla _CMY-2, bla _TEM-1b	ISEcp1 — blc ^d	+/−	—	SXT-SUL-TET-STR	sul2, tet(A), strA/strB	fimA-aer

SXT, trimethoprim-sulfamethoxazole; SUL, sulfonamides; STR, streptomycin; TET, tetracycline; CHL, chloramphenicol; NAL, nalidixic acid; CIP, ciprofloxacin; KAN, kanamycin.

IS Ecp1 disrupted by the IS 5 element in complementary orientation.

bla cmy-2 genetic environment.

The ISEcp1-bla _CTX-M-1-orf477 structure was found in all 13 ESBL-positive isolates, although the ISEcp1 sequence was truncated in 1 of them by the IS5 element, located in complementary orientation. Additionally, the C3564 isolate presented the ISEcp1-bla _CMY-2-blc structure (Table 1).

Nine of the 14 CTX^R E. coli isolates amplified the intI1 gene, and all but one of them lacked the 3′-CS (qacEΔ1 and sul1 genes) of class 1 integrons. The gene cassette arrangements could be determined in only 5 ESBL-positive isolates: dfrA17-aadA5 (2 isolates), dfrA1-aadA1 (1 isolate), and both arrangements (2 isolates). No class 2 integrons were detected among the studied isolates.

PFGE analysis demonstrated 13 unrelated pulsotypes, 12 of them being among the 13 CTX-M-1–producing strains. Two ESBL-positive isolates presented closely related PFGE patterns (C3143 and C3566), and they were obtained form a dog and a cat from different owners and samples were obtained with a difference of 4 months (March and July, respectively). The studied CTX^R E. coli strains belonged to the phylogroups B1 (6 isolates), A (5 isolates), and D (3 isolates) (Table 1).

A variety of resistance genes located outside integrons were observed among the 14 CTX^R strains (Table 1). The E. coli C3566 amplified the qnrB gene, and after analyzing its immediate genetic environment, the variant detected was the qnrB19. This strain also presented a unique substitution, Ser83Leu, in the QRDR of GyrA protein, whereas none in the ParC protein.

The fimA and aer virulence genes were detected in 13 and 11 isolates of the 14 CTX^R E. coli isolates, respectively, being negative for the remaining virulence or serotype traits tested.

Discussion

This is the first report on the detection and characterization of ESBL and pAmpC β-lactamases among E. coli isolates from healthy companion animals (dogs and cats) in Africa. Our findings show a very high rate of fecal carriage of ESBL-positive E. coli isolates in healthy pets (16.3%) in samples obtained in 2010, this proportion being higher in cat samples (20.5%) than in dog samples (12.2%). Lower percentages were detected in previous studies carried out in Portugal and Italy in pet samples obtained in 2001–2003 (6–7%) (Costa et al. 2004, Carattoli et al. 2005), but a higher rate was identified in China in samples obtained in 2007–2008 (24.5%) (Sun et al. 2010).

All of the 13 ESBL-positive isolates detected in our study from healthy dogs and cats were CTX-M-1 producers. This CTX-M-1 type ESBL has been previously detected in E. coli isolates from food and healthy humans, and not in clinical isolates in Tunisia (Jouini et al. 2007, Ben Slama et al. 2010, Ben Sallem et al. 2012). These data may suggest a potential implication of the food chain in the transmission processes and a potential route of transmission in the community. It is also interesting to note that most of the 13 bla _CTX-M-1-containing isolates were clonally unrelated. In this respect, it is the spread of the bla _CTX-M-1 gene in different clones among different animals. This finding suggests that the horizontal transfer of bla _CTX-M genes, mediated by mobile elements, contributes to their dissemination among E. coli isolates from pets instead of the pandemic spread of single clones. Since the first description of CTX-M-1 enzyme in a healthy dog in Portugal (Costa et al. 2004), this type of ESBL has been reported in companion animals in other countries (Carattoli et al. 2005, SVARM 2009, Moreno et al. 2008, Schink et al. 2011), but to our knowledge, E. coli isolates carrying the bla _CTX-M-1 gene have never been described from healthy or diseased companion animals in either Tunisia nor in Africa.

In the same way, the present study constitutes the first description of a bla _CMY-2-positive E. coli isolated from pets in Tunisia (and also in Africa), although CMY-2 has been previously reported in E. coli isolates from food-animals in this country (Ben Slama et al. 2010) and from humans, food animals, and companion animals worldwide (Sánchez et al. 2002, Yan et al. 2004, Briñas et al. 2005, Carattoli et al. 2005, Jacoby 2009, Murphy et al. 2009, Pomba et al. 2009, Gibson et al. 2010b, Mataseje et al. 2010).

The ISEcp1 insertion sequence has been observed upstream of bla _CTX-M-1 and bla _CMY-2 genes in the 14 CTX^R E. coli strains. This ISEcp1 element contains typical −35 and −10 putative promoter regions and could mobilize such genes (Eckert et al. 2006). Interestingly in one of our ESBL-positive strains, this insertion sequence (IS) was found to be disrupted by the IS5 element, which may affect the mobilization and expression of the bla _CTX-M-1 gene. To our knowledge, this organization has never been described before in CTX-M-1–positive E. coli isolates.

Interestingly, a qnrB19 gene was identified in one nalidixic acid–resistant CTX-M-1–producer strain that also exhibited the Ser83-Leu substitution in the GyrA protein. By itself, the qnr gene confers resistance to quinolones at a low level. The clinical importance is linked to its ability to allow the selection of chromosomal mutations of quinolones at concentrations that could have been lethal in the absence of this gene (Bouchakour et al. 2010).

In Tunisia, qnr-carrying E. coli strains have not been described among animal isolates yet. Moreover, to the best of our knowledge, the association of bla _CTX-M-1 and qnrB19 genes in the same organism, from healthy companion animals, has never been reported. However, the simultaneous presence of both genes in clinical E. coli isolates of sick horses has been recently noted (Dolejska et al. 2011). Very recently, it has been reported the presence of qnrB19 gene in a conjugative plasmid of 42.3 kb in an E. coli isolate of horse origin (Schink et al. 2012).

The detection of class 1 integrons lacking the qacEΔ1 and sul1 genes in 8 of the 9 integron-positive E. coli isolates indicates that this unusual structure for gene acquisition, which is also frequently detected in CTX-M-1–producing E. coli isolates of food products and healthy humans in Tunisia (Ben Slama et al., 2010, Ben Sallem et al. 2012), is spreading among the normal microbiota of pets.

Conclusion

Pets represent a community reservoir of antimicrobial-resistant E. coli and resistance genes of special relevance in human medicine, including the bla _CTX-M-1, bla _CMY-2, and qnrB genes that could be transmitted to humans in contact with them, as is the case of pets and their owners, increasing the reservoir in the community. Detailed and careful studies of the genetic support and the plasmids encoding these genes should also be conducted in the future to assess the full extent of sharing between animal and human isolates.

Footnotes

Acknowledgments

This study was financed by an Integrated Action of the Agencia Española de Colaboración Internacional al Desarrollo (AECID) of Spain (A1/038210/11) and from the Tunisian Ministry of Higher Education and Scientific Research. V. Estepa has a predoctoral fellowship from the University of La Rioja (Spain) and N. Porres-Osante has a fellowship from Instituto de Salud Carlos III of Spain.

Author Disclosure Statement

None of the co-authors has conflicts of interest in relation with this manuscript.

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