Clonal diversity of extended-spectrum beta-lactamase producing Escherichia coli isolates in fecal samples of wild animals

Abstract

The clonal diversity of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates from nine different species of wild animals from distinct regions of Portugal and Spain and their content in replicon plasmids were analyzed. Among the initial 53 ESBL-producing E. coli isolates that were studied (from previous studies), 28 were selected, corresponding to different animal origins with distinct ESBL types and pulsed-field gel electrophoresis (PFGE) patterns. These 28 isolates produced different ESBLs ascribed to the following families: CTX-M, SHV and TEM. The isolates were classified into three phylogenetic groups: B1 (n = 11), A (n = 10) and D (n = 7). The seven E. coli of phylogroup D were then typed by multilocus sequence typing and ascribed to four distinct sequence types: ST117, ST115, ST2001 and ST69. The clonal diversity and relationship between isolates was studied by PFGE. Lastly, the plasmids were analyzed according to their incompatibility group using the PCR-based-replicon-typing scheme. A great diversity of replicon types was identified, with up to five per isolate. Most of the CTX-M-1 and SHV-12 producing E. coli isolates carried IncI1 or IncN replicons. The diversity of ESBL-producing E. coli isolates in wild animals, which can be disseminated in the environment, emphasizes the environmental and health problems that we face nowadays.

wild animals, ESBL-producing Escherichia coli, β-lactamases, pulsed-field gel electrophoresis

INTRODUCTION

The excessive consumption of antibiotics in the last decades, both by humans and animals, has created some of the main and greatest serious public health complications nowadays in society: the development and spread of bacterial antibiotic resistance. Therefore, several reports were conducted in order to comprehend the tools used by bacteria to promote drug resistance, since different bacterial strains resistant to several antibiotics have been detected in companion animals and in humans and the pharmaceutical industry kept developing new drugs to bridge the resistances that bacteria were building up (Duo, Hou and Ren 2008; Roca et al. 2015). Among the furthermost clinical important mechanisms of antibiotic resistance, the one constituted by extended-spectrum β-lactamases (ESBLs) is noticed in both human and veterinary bacteria (Nordmann 1998; Bradford 2001; Goncalves et al. 2010). The detection of ESBLs produced mainly by Escherichia coli isolates has increased substantially and became the focus of many studies due to the location of the encoding genes on mobile elements such as plasmids, allowing the resistance genes to be transported from commensal to pathogenic bacteria (Canton et al. 2008).

The rising number of reported cases of ESBL-producing E. coli detected in healthy animals and the influence in human infections drew attention in diverse countries, including Portugal (Paterson and Bonomo 2005; Radhouani et al. 2013). Over time, our investigation group has concentrated the efforts on wild animals, studying how colonizing bacteria obtain resistance to antimicrobial compounds, even though these animals do not have direct contact with antimicrobials or with humans or animals exposed to them (Costa et al. 2008; Poeta et al. 2008; Radhouani et al. 2010). Therefore, the purpose of our work was to analyze ESBL-producing E. coli isolates, previously obtained from nine different species of wild animals from distinct regions of Portugal and Spain, to analyze the clonal diversity of ESBL isolates using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for selected strains, and to perform plasmid replicon typing of these isolates.

MATERIAL AND METHODS

Samples and Escherichia coli isolates

Fifty-three ESBL-positive E. coli isolates obtained from fecal samples of wild animals of nine different animal species were analyzed and compared. They were obtained in previous studies developed by our research group: 14 isolates from Iberian wolf (Canis lupus signatus) (Goncalves et al. 2012b), 10 isolates from gilthead seabream (Sparus aurata) (Sousa et al. 2011), 9 isolates from Iberian lynx (Lynx pardinus) (Goncalves et al. 2012a), 8 isolates from wild boars (Sus scrofa scrofa) (Poeta et al. 2009), 4 isolates from birds of prey (Costa et al. 2006), 3 isolates from foxes, from which 2 were from red foxes (Vulpes vulpes) (Costa et al. 2006; Radhouani et al. 2013), 2 isolates from wild birds (Sylvia atricapilla) from the Azores Archipelago (Silva et al. 2011), 2 isolates from deer and 1 isolate from an owl (Costa et al. 2006).

Fecal samples were collected, one sample per animal, in different locations of Portugal and Spain. The fecal samples from Iberian wolf, both wild and in captivity, were obtained in several locations of the north of Portugal, during surveillance studies performed by an organization dedicated to the conversation of the Iberian Wolf (Goncalves et al. 2012b). The fecal samples from Iberian lynx in captivity were obtained in the Centre of Analysis and Diagnosis of the Wildlife in the south of Spain (Doñana National Park), while the samples from wild lynx were obtained from Natural Parks located in south of Spain during the replacement of radio tracking systems (Goncalves et al. 2012a). The fecal samples from red foxes and wild boars were collected during the shooting season in the north of Portugal by diverse organizations of hunters and by the Portuguese Forest Rangers (Poeta et al. 2009; Radhouani et al. 2013). The fecal samples from gilthead seabream and from wild birds from the Azores islands were collected by amateur fishermen in Peniche (Portugal west coast) and by the Research Center from the University of Azores, respectively. For both types of animals, the small intestines were collected and transferred to sterile Stomacher bags (Silva et al. 2011; Sousa et al. 2011). The fecal samples from birds of prey, foxes, deer and owls were collected in Nature Reserves of Portugal (Costa et al. 2006).

The antimicrobial susceptibility of these isolates and the type of β-lactamase genes were previously analyzed for all of them (Costa et al. 2006; Poeta et al. 2009; Silva et al. 2011; Sousa et al. 2011; Goncalves et al. 2012a,b; Radhouani et al. 2013), although susceptibility testing was retested for this study (CLSI 2014). For confirmation purposes, agar plate-based screening for ESBL production was performed by using the double-disk diffusion test (CLSI 2014).

Clonal diversity of ESBL-positive Escherichia coli isolates

Molecular typing of the ESBL-producing isolates was carried out by PFGE and XbaI was used as restriction enzyme (Gautom 1997; Saenz et al. 2004). The patterns obtained were first studied according to formerly described standards (Tenover et al. 1995), and subsequently the dendrogram construction and the pattern comparison was performed in GelCompar (Applied Maths) based on Dice similarity coefficients.

There are four principal phylogenetic groups (A, B1, B2 and D) that can be checked by PCR strategy centered in absence or presence of specific genes (Clermont, Bonacorsi and Bingen 2000). The phylogenetic group of eight of the ESBL-producing E. coli isolates (four from birds of prey, two from deer, one from a fox and one from an owl) was achieved by a multiplex PCR reaction (Clermont, Bonacorsi and Bingen 2000). The phylogenetic group of the remaining isolates was previously determined (Costa et al. 2006; Poeta et al. 2009; Silva et al. 2011; Sousa et al. 2011; Goncalves et al. 2012a,b; Radhouani et al. 2013). Negative and positive controls obtained from the strains collection of the University of La Rioja (Spain) were incorporated in all PCR assays.

Recovered ESBL isolates belonging to phylogenetic group D, due to their clinical importance, were then studied by MLST, analyzing the sequences of seven housekeeping genes (Wirth et al. 2006).

Plasmid replicon typing

PCR-based replicon-typing scheme was used to identify and classify plasmids according to their incompatibility group (Carattoli et al. 2005). The replicon types of the eight ESBL-producing E. coli isolates of wolf origin were previously determined (Goncalves et al. 2012b) and for the remaining isolates it was performed in this study.

RESULTS AND DISCUSSION

Out of the initial 53 ESBL-positive Escherichia coli isolates that were analyzed, 28 showing different PFGE patterns, ESBL types and belonging to distinct animal origins were selected for further characterization. The analysis of these isolates is displayed in Table 1 and their PFGE patterns are presented in Fig. 1.

Figure 1.

Dendrogram of the 28 ESBL-producing E. coli isolates of wild animals from Portugal and Spain.

Table 1.

Characteristics of the 28 ESBL-producing Escherichia coli isolates of wild animals from Portugal and Spain.

Isolate	Animal	Origin	Phylogenetic group	Phenotypeofresistance for non-β-lactamsa	β-Lactamases detected	Other resistance genes	Virulence factors	ST	Plasmid replicon typesb
C1422	Bird of prey	Portugal	A	NA, CI, TE, ST, SXT, GE	CTX-M-14 + TEM-1	aadA, tet(A), sul1, sul2
C1732	Wild boar	Portugal	A	NA, TE, ST, SXT	CTX-M-1 + TEM-1b	aadA, tet(A), sul2
C2515	Gilthead seabream	Portugal	A	ST, SXT, CHL	SHV-12	tet(A), sul3, cmlA			I1, F, K
C2517	Gilthead seabream	Portugal	A	Susceptible	TEM-52				I1, K
C3064	Wolf	Portugal	A	Susceptible	CTX-M-1		fimA		N,K,
C3066	Wolf	Portugal	A	TE	CTX-M-14a	tet(A)			K, H12, W
C3072	Wolf	Portugal	A	NA, TE, ST, CHL	SHV-12	aadA, tet(B), cmlA	fimA, aer		I1, FIB, T, K
C3077	Lynx	Spain	A	TE, ST	CTX-M-14a	aadA, tet(A)	fimA, aer		K, I1, FIB
C5478	Red fox	Portugal	A	NA, TE, ST, SXT, CI, CHL	SHV-12 + TEM-1b	tet(A), sul2			I1, FIB, F, K
C6831	Bird Azores	Portugal	A	TE	CTX-M-14 + SHV-12	tet(A)			K, I1, X, P
C1417	Deer	Portugal	B1	Susceptible	TEM-52				FIB, K
C1419	Owl	Portugal	B1	Susceptible	TEM-52				K
C1420	Bird of prey	Portugal	B1	NA, TE	CTX-M-14 + TEM-52	tet(B)
C1425	Bird of prey	Portugal	B1	NA, TE, ST, SXT, GE, TB	SHV-12	aadA, tet(B), sul3			N, F, K
C1735	Wild boar	Portugal	B1	SXT	CTX-M-1	sul3			I1, FIB, F, K
C2509	Gilthead seabream	Portugal	B1	TE, ST, SXT, CHL	TEM-52	tet(A), sul1, sul2, sul3, cmlA			I1, FIC, K
C3061	Wolf	Portugal	B1	NA, TE	CTX-M-1	tet(A)	fimA, aer		I1, FIB, K
C3067	Wolf	Portugal	B1	NA, TE, CI	CTX-M-14a + TEM-1b	tet(A)	fimA, aer		K, H12
C3074	Wolf	Portugal	B1	TE	SHV-12 + TEM-1b	tet(A)	fimA, aer		I1, FIA, F
C3079	Lynx	Spain	B1	NA, TE, ST, CI	CTX-M-14a	aadA, tet(B)	fimA, aer		K, FIB, Y, F
C3082	Lynx	Spain	B1	NA, TE, ST, SXT, CI, CHL	SHV-12 + TEM-1b	aadA, tet(A), sul1, sul2, sul3, cmlA	fimA, aer
C1421	Fox	Portugal	D	NA, TE, ST, CI	CTX-M-14 + TEM-1	aadA, tet(B)		ST117
C1737	Wild boar	Portugal	D	NA, SXT	CTX-M-1	sul3		ST115	I1, FIB, T, F, K
C1740	Wild boar	Portugal	D	NA, TE, SXT, CI	CTX-M-1	tet(A), tet(B), sul3		ST117	I1, FIB, P, F, K
C3065	Wolf	Portugal	D	TE, ST, SXT, CHL, GE, TB	CTX-M-1	aadA, tet(A), sul1, sul2, cmlA, aac(3)-II	fimA, aer	ST69	N, FIA, FIB, P, F
C3068	Wolf	Portugal	D	NA, TE, ST, SXT	CTX-M-14a + TEM-1b	aadA, tet(A), sul1	fimA	ST2001	I1, P, T
C3080	Lynx	Spain	D	NA, TE, ST, CI	CTX-M-14a	aadA, tet(A)	fimA, aer	ST117
C3085	Lynx	Spain	D	NA, ST, SXT	SHV-12	aadA, sul3	fimA, aer	ST117	I1, FIB, F

a

NA, nalidixic acid; TE, tetracycline; ST, streptomycin; SXT, sulfamethoxazole/trimethoprim; GE, gentamicin; CI, ciprofloxacin; TB, tobramycin; CHL, chloramphenicol.

b

The plasmid replicon typing of wolf isolates was previously performed (Goncalves et al. 2012b).

The PFGE patterns were analyzed and 28 different profiles were obtained showing an elevated clonal diversity of ESBL-producing E. coli isolates and allowing us to investigate the clonal relationship between all of them (Fig. 1).

The 28 E. coli isolates showed the ESBL encoding genes (number of isolates): bla_CTX-M-1 (7, in 1 case with bla_TEM-1b), bla_CTX-M-14 (8, in 4 cases with bla_TEM-1b), bla_SHV-12 (7, in 3 cases with bla_TEM-1b), bla_TEM-52 (4), bla_CTX-M-14 + bla_SHV-12 (1) and bla_CTX-M-14 + bla_TEM-52 (1). Of these ESBL-producing E. coli isolates, 10 were categorized into the phylogenetic group A, 11 isolates in phylogenetic group B1 and 7 isolates into the phylogroup D (Table 1). As B2 and D group strains have higher probability of existence in clinical settings and are more frequently related to extraintestinal infections, we analyzed the seven E. coli isolates with phylogenetic group D by MLST (no strain of B2 groups was detected) (Clermont, Bonacorsi and Bingen 2000). The seven isolates were grouped into four distinct STs (Table 1) according to the MLST website database (mlst.warwick.ac.uk). Specifically, four isolates were assigned to ST117, one isolate to ST115, one isolate to ST2001 and one isolate to ST69. Previous studies have reported the E. coli ST69 as highly virulent in some animal models with high content in resistance determinants (Tartof et al. 2005; Alghoribi et al. 2014). In our case, the ST69 E. coli isolate harbored the CTX-M-1 β-lactamase and presented resistance to aminoglycosides (gentamicin, streptomycin and tobramycin), sulfonamides (sulfamethoxazole-trimethoprim), tetracycline and chloramphenicol.

The antimicrobial resistance genes for non-β-lactams, previously identified by PCR in these 28 ESBL-positive E. coli isolates, were as follows: tet(A) and tet(B) (tetracycline resistance); aadA and strA-strB (streptomycin resistance); aac(3)-II and aac(3)-IV (gentamicin resistance); sul1, sul2 and sul3 (sulfamethoxazole/trimethoprim resistance); and cmlA and floR (chloramphenicol resistance) (Table 1).

Dissimilar plasmid replicon types were recognized among our 28 ESBL-producing PFGE-unrelated E. coli isolates (Table 1). Most of the bla_CTX-M-1 and bla_SHV-12 positive isolates supported the IncI1 or IncN replicon types, and most of bla_CTX-M-14 and bla_TEM-52 isolates carried an IncK replicon.

In this report, it is likely to analyze ESBL-producing E. coli isolates from distinct wild species from Portugal and Spain, most of them belonging to phylogenetic groups A and B1, associated with commensal strains, a smaller number classified into group D, and to underline the absence of isolates classified into phylogenetic group B2. This distribution of ESBL-producing strains into phylogenetic groups could be explained by the fecal source of the isolates.

It is possible to conceive a potential route of colonization based on the origin of the isolates. The Iberian wolf is a predator and can travel long distances in search of food. Thus, the wolf is exposed to fecal material of other animals, wild or farm animals that can carry ESBL-producing bacteria. Another factor influencing the acquisition of ESBL by these animals is the fact that they feed themselves with other animals, such as wild boars, which have been detected with ESBL-producing E. coli strains in other studies. Furthermore, the administration of antibiotics to Iberian wolf and also to Iberian lynx by humans, with management purposes in order to monitor the species, can create some selective pressure (Goncalves et al. 2012a,b). The aquatic environment is often contaminated with the fecal material from animals and humans, which could have contributed to the acquisition and dissemination ESBL-producing E. coli in gilthead seabream (Sousa et al. 2011). Similar to the Iberian wolf, red foxes are also predatory animals and hunt several animals such as small rodents, wild birds and rabbits which can explain the acquisition of ESBL-producing bacteria by these animals (Radhouani et al. 2013). The acquisition of ESBL-producing E. coli by wild boars can be explained by their proximity to human and other animals’ habitats, which may give the possibility of these animals to eat the rests of human food (Poeta et al. 2009). A possible route of ESBL-producing E. coli dissemination is the interaction of several different migratory birds that come from North America, Europe and North Africa with the Azores Archipelago wild birds (Silva et al. 2011). Various outdoor activities practiced by humans, like hiking or bird watching, can contribute to the dissemination of ESBL-producing E. coli leading to their acquisition by several wild animals such as birds, deer, foxes and owls (Costa et al. 2006; Silva et al. 2011).

Since the Iberian wolf and the Iberian lynx are endangered species, it is important to study the virulence genes that are often found in pathogenic E. coli. Previous studies have determined the virulence genes by PCR (Goncalves et al. 2012a,b). The virulence gene fimA was detected in 13 isolates of these animal species, and the aer gene in 3 isolates (Table 1).

This study highlights the important fact that ESBLs can be detected in ecosystems apart from those associated with humans or covering selective resistance stress. It is important to emphasize the high clonal diversity of the ESBL-producing E. coli isolates obtained from diverse wild animal species of diverse ecosystems. It seems clear that wild animals can be vehicles of antimicrobial-resistant bacteria, helping their spread to other species and ecosystems. The emergence of ESBL producers might be explained by the increasing proximity of the wild species to human ecosystems, whether to seek for food or because their habitats are being invaded by humans. In this study, it is shown that a high diversity of E. coli clones are implicated in the dissemination of ESBL in wild animals in Portugal and Spain that carry diverse replicon plasmids. More studies should be achieved in the future to investigate the prevalence and evolution of this kind of resistant bacteria in several species and ecosystems.

FUNDING

Margarida Sousa has her Ph.D. fellowship granted by FCT (Fundação para a Ciência e Tecnologia, Portugal) with the reference SFRH/BD/87302/2012 co-financed by the Social European Union Found and the Operational Program for Human Potential and National Board for Reference and Strategic Programs (POPH/QREN), Portugal. Part of this work was supported by Project SAF2012-35474 from the Ministerio de Economía y Competitividad of Spain and the Fondo Europeo de Desarrollo Regional (FEDER). This work was also supported by the Unidade de Ciências Biomoleculares Aplicadas (UCIBIO) which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and cofinanced by the European Regional Development Fund (ERDF) under the PT2020 Partnership Agreement (POCI-01–0145-FEDER-007728).

Conflict of interest. None declared.