Veterinary Microbiology 171 (2014) 242–247

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Short Communication

Virulence genotypes, antibiotic resistance and the phylogenetic background of extraintestinal pathogenic Escherichia coli isolated from urinary tract infections of dogs and cats in Brazil L. Osugui a,b, A.F. Pestana de Castro a, R. Iovine b, K. Irino c, V.M. Carvalho b,* a b c

Department of Microbiology, Biomedical Sciences Institute II, Universidade de Sa˜o Paulo, 05508-900 Sa˜o Paulo, Brazil Laboratory of Molecular and Cellular Biology, Universidade Paulista, 04057-000 Sa˜o Paulo, Brazil Enterobacteria Section, Instituto Adolfo Lutz, 01246-902 Sa˜o Paulo, Brazil

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 December 2013 Received in revised form 19 March 2014 Accepted 23 March 2014

Urinary tract infection (UTI) is a frequent disease of humans and pets and has extraintestinal pathogenic Escherichia coli (ExPEC) strains as one of the main etiologic agent. ExPEC are characterized by specific virulence factors and are related to a heterogeneous group of human and animal disorders, besides to be a relevant participant in the dissemination of antimicrobial resistance. The purpose of this study was to characterize E. coli strains isolated from UTI of dogs and cats for serotypes, virulence markers, phylogenetic groups and sensitivity to antimicrobial drugs. E. coli was identified as the etiologic agent of UTI in urine samples of 43 pets (7 cats and 36 dogs). Serogroups O2, O4 and O6 corresponded to more than one third of the isolates, being 62% of the total strains classified as B2, 18% as D, 16% as B1 and 4% as A. The iucD (22%), fyuA (80%), traT (51%) and cvaC (20%) genes were distributed among the four phylogenetic groups, whereas the papC/ papEF (47%) and malX (67%) genes were found only in groups B2 and D. There were a high number of resistant strains, with 76% of the strains belonging to groups A, B1 and D characterized as multidrug resistant (MDR), whereas only 21% had this phenotype in the group B2. The ExPEC strains isolated in this study displayed pathotypic and phylogenetic similarities with human isolates and high percentages of drug resistance. The finding of MDR ExPEC strains suggests implications for animal and public health and deserves more investigations. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Extraintestinal pathogenic Escherichia coli (ExPEC) Urinary tract infections (UTI) Dogs Cats Virulence factors Phylogenetic groups Antibiotic resistance

1. Introduction ExPEC (extraintestinal pathogenic Escherichia coli) is considered the most common bacteria isolated from urinary tract infections (UTI), from both animals and

* Corresponding author. Tel.: +55 11 55943207. E-mail addresses: [email protected] (L. Osugui), [email protected] (A.F. Pestana de Castro), [email protected] (R. Iovine), [email protected] (K. Irino), [email protected] (V.M. Carvalho). http://dx.doi.org/10.1016/j.vetmic.2014.03.027 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

humans, and is also related to a variety of extraintestinal infections in different species (Smith et al., 2007). A wide variety of virulence factors is associated with isolates from extraintestinal infections. These strains belong mostly to phylogenetic groups B2 and D, differentiating them from commensal strains, which, in addition to being devoid of virulence markers, are predominantly in groups A and B1 (Johnson and Russo, 2005; Maynard et al., 2004; Smith et al., 2007). A number of studies indicate that there may be crosstransmission of ExPEC between animals and humans

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(Johnson and Clabots, 2006; Johnson et al., 2001), suggesting the epidemiological role of pets in the transmission of this agent with zoonotic potential. This phenomenon brings to light another important public health problem: antimicrobial resistance (Smith et al., 2007). Because E. coli is one of the main participants in the dissemination of antimicrobial resistance (Pitout, 2012), the use of antibiotics in veterinary practice can lead to the selection of resistant strains (Guardabassi et al., 2004) that ultimately not only impact animal health but also result in difficult treatments and public health problems. Thus, this study aimed to characterize E. coli strains isolated from UTI of dogs and cats for serotypes, virulence markers, phylogenetic groups and sensitivity to antimicrobial agents used routinely in the clinic.

2. Materials and methods 2.1. Samples and bacterial isolates Urine samples were collected from 100 animals with UTI (86 dogs and 14 cats) from different regions of the city of Sa˜o Paulo, at a private veterinary laboratory. All procedures were performed in accordance with the ICB/ USP Ethics Committee of Animal Experimentation (CEEAA) under protocol number 125 (2nd book, p. 38). The samples were inoculated onto MacConkey agar (Difco) and blood agar (Difco) plates and incubated overnight (37 8C). Quantitative analyses, considered the golden standard to confirm UTI, were performed on TSA (Difco). Escherichia coli was identified as the etiologic agent of UTI in 43 animals: seven from cats and 36 from dogs. Three E. coli strains for each positive sample (129 strains in total) were frozen in Brain Heart Infusion Medium (Difco) with 80% glycerol (Invitrogen; 1:1 (v/v)) at 80 8C for further analysis. 2.2. Serotyping Serotyping was performed at a reference center for E. coli serotyping (Instituto Adolfo Lutz, Sa˜o Paulo, SP, Brazil) according to standard international procedures using the tube agglutination test and currently available O (O1O181) and H (H1-H56) antisera. 2.3. Detection of virulence determinants by PCR and phylogenetic group determination For the detection of virulence-related genes (papC, papEF, sfa, afa, cnf1, hlyA, iucD, fyuA, and iha), single PCR reactions were performed as previously described (Johnson et al., 2000; Johnson and Stell, 2000; Yamamoto et al., 1995). The fimH/malX/ibeA and traT/cvaC genes were searched in the triplex and duplex PCR reactions, respectively (Johnson and Stell, 2000). The determination of the major E. coli phylogenetic group (A, B1, B2 and D) was assigned using the multiplex PCR-based method of Clermont et al. (2000). All single reactions were achieved using Taq DNA Polymerase (Invitrogen, Brazil), whereas

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AmpliTaq Gold (Applied Biosystems, USA) was used in the multiplex reactions. As positive E. coli controls, C7, C149, JJ079, BUTI 3-1-4, BUTI 1-7-6 and BUTI 1-5-1 strains were used in all PCR reactions; JJ055 (derived from K12) was used as the negative E. coli control strain (all courtesy of Prof. Dr. James R. Johnson). The controls used for phylogenetic determination were JJ055 (Group A), EC 15 (Group B1), JJ079 (Group B2), and EDL 933 (Group D). 2.4. Antimicrobial susceptibility testing The antimicrobial susceptibility was tested with disk diffusion method, and using E. coli ATCC 25922 as a reference strain. Breakpoints that were not defined according Clinical and Laboratory Standard Institute, document VET01-S2, were determined using document CLSI M100-S23. The following antibiotic disks sourced from Cefar Diagno´stica Ltd. were tested: ampicillin 10 mg (AMP) (only in 38 strains), cephalexin 30 mg (CFL), cefotaxime 30 mg (CTX), ceftiofur 30 mg (CEF), ciprofloxacin 5 mg (CIP), enrofloxacin 5 mg (ENR), chloramphenicol 30 mg (CHL), streptomycin 10 mg (STR), gentamicin 10 mg (GEN), tetracycline 30 mg (TET), trimethoprim-sulfamethoxazole 25 mg (SXT), and nitrofurantoin 300 mg (NIT). A strain was considered multidrug resistant (MDR) when demonstrating resistance to three or more antimicrobial classes (Schwarz et al., 2010). 2.5. Statistical methods For statistical analyses, Fisher’s exact test was used. 3. Results In 42 of the 43 animals (36 dogs and 7 cats) with confirmed UTI, the three strains presented the same profile of virulence genes, serotype and phylogenetic group and were, therefore, regarded as having the same clonal origin. The strains showed three different serotypes in only one feline. Thus, for the purposes of presenting the results, 45 strains will be considered: 36 from dogs and 9 from cats. It was possible to determine the O antigen in 64% (29/45) of these strains. Serogroup O6 was the most prevalent, with a frequency of 20% (9/45), followed by O2 with 15% (7/45) of the typified isolates. A considerable percentage of strains (36%, 16/45) was untypeable with available antisera or were rough strains (Supplementary data, Table 1). Considering the four main phylogenetic groups, 62% (28/ 45) of the strains were classified as B2. The remaining strains were subdivided as follows: 18% as D (8/45), 16% as B1 (7/45) and 4% as A (2/45). The strains isolated from cats concentrated in group B2 (8/9, 89%), and only one (1/9, 11%) was classified as group D. With respect to the 36 strains isolated from dogs, there was a predominance of group B2 (20/36, 56%), with the remaining strains subdivided into D (7/36, 19%), B1 (7/36, 19%) and A (2/36, 6%). The genotypic characterization of the isolates showed that all studied strains had at least one gene encoding VF, with the fimH sequence present in all samples. The iucD (10/45, 22%), fyuA (36/45, 80%), traT (23/45, 51%) and cvaC

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(9/45, 20%) genes were distributed among the four phylogenetic groups, whereas the papC/papEF (21/45, 47%) and malX (30/45, 67%) genes were found only in groups B2 and D. Sixty one percent (17/28) and 96% (27/28) of the B2 strains were, respectively, papC/papEF+ and malX+. The iha (2/45, 4%), sfa (15/45, 33%), cnf1 (14/45, 31%) and hlyA (12/45, 27%) genes were present only in strains belonging to group B2 (Supplementary data, Fig. 1), and the combination of these last three genes within the phylogenetic group was statistically significant (P = 0.0002, P = 0.0003 and P = 0.0013, respectively). Although the presence of the malX gene was detected in isolates from group D, the association of this marker for PAICFT073 within phylogenetic group B2 was significantly higher (P < 0.0001).

Regarding the susceptibility to the antimicrobial agents tested, tetracycline (16/45, 36%), ampicillin (14/38, 37%), aminoglycosides (18/45, 40%), and sulfa (18/45, 40%) were the antimicrobials with the higher percentages of resistant strains (Supplementary data, Table 2). Considering both antimicrobial agents of classes of aminoglycosides, a larger number of strains were resistant to only streptomycin (13/ 19, 69%), and 26% of the strains (5/19) were resistant to both drugs tested. With respect to fluoroquinolones, 16% (7/45) of the strains were resistant to this antimicrobial agent. Isolates not susceptible to 1st generation cephalosporins or nitrofurantoin presented the same percentage of resistance (6/45, 13%). Among the drugs tested, 3rd generation cephalosporins were the most effective in

Table 1 Profile of Escherichia coli strains isolated from cats (9) and dogs (36) with UTI with regard to the phylogenetic group (PG), serotype, virulence genes, and resistance to antibiotic drugs. PG

Serotype

Virulence Factors

Antibiotic Resistancea

A

O11:H4 ONT:H-

fimH, iucD, fyuA, cvaC fimH, iucD, fyuA, traT

CHL, STR, TET, SXT *b AMP, STR, TET, SXT

B1

O20:H12 O23:H16 ONT:H8 ONT:H21 ONT:H23 ONT:H45 OR:H16

fimH, fimH, fimH, fimH, fimH fimH, fimH

fyuA fyuA, traT traT iucD, traT, cvaC

AMP, CIP, ENR, TET CFL, CTX, CIP, ENR, STR, TET, SXT AMP, CFL, CIP, ENR, TET, SXT CIP, ENR, CHL, SXT STR NIT

O2:H4 O2:H8 O2:HO2:H6 O2:H31 O4:HO6:HO6:HO6:HO6:HO6:HO6:H1 O6:H11 O6:H31 O6:H31 O18:HO21:H14 O21:H14 O25:H11 O25:HNT O83:H31 O83:H31 ONT:H4 ONT:H45 ONT:H45 ONT:HNT ONT:HNT OR:HNT

fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH, fimH,

papC, papEF, iucD, fyuA, traT, cvaC, malX papC, papEF, iucD, fyuA, traT, cvaC, malX papC, papEF, iucD, fyuA, traT, cvaC, malX sfa, ibeA, cnf1, hlyA, fyuA, malX ibeA, fyuA, traT, malX papC, papEF, sfa, cnf1, fyuA, malX papC, papEF, sfa, cnf1, hlyA, fyuA, malX papC, papEF, sfa, cnf1, hlyA, iucD, fyuA, malX papC, papEF, sfa, cnf1, hlyA, fyuA, malX papC, papEF, sfa, cnf1, hlyA, fyuA, traT, malX papC, papEF, sfa, cnf1, hlyA, fyuA, malX sfa, cnf1, hlyA, fyuA, malX papC, papEF, sfa, fyuA, malX papC, papEF, sfa, cnf1, hlyA, fyuA, traT, malX papC, papEF, ibeA, cnf1, fyuA, traT, malX papC, papEF, iha, fyuA, traT, malX papC, papEF, sfa, ibeA, cnf1, hlyA, fyuA, malX papC, papEF, sfa, ibeA, cnf1, hlyA, fyuA, malX papC, papEF, sfa, iha, cnf1, hlyA, fyuA, malX ibeA, fyuA, traT, malX ibeA, fyuA, traT, malX ibeA, fyuA, traT, malX sfa, ibeA, fyuA, traT, malX fyuA, malX fyuA, malX ibeA, iucD, fyuA, traT, cvaC, malX ibeA, fyuA, traT papC, papEF, sfa, ibeA, cnf1, hlyA, fyuA, malX

O2:HNT O2:HNT O15:H45 O153:H34 ONT:H4 ONT:H6 ONT:HNT OR:H-

fimH, fimH, fimH, fimH, fimH, fimH fimH, fimH,

papC, fyuA, traT, cvaC, malX papC, fyuA, traT, cvaC, malX traT ibeA, iucD papC, papEF

B2

D

traT

papC, papEF, iucD, fyuA, traT, cvaC, malX fyuA

*b

CHL, TET TET*b AMP, CHL, STR, SXT AMP, STR, SXT SXT*b STR, TET, SXT, NIT AMP, STR, SXT, NIT STR, SXT

*b

AMP, TET, SXT STR CFL, GEN AMP, CIP, ENR, STR, SXT *b AMP, STR, GEN, TET, SXT AMP, STR, GEN, TET, SXT, NIT AMP CFL, CTX, STR, GEN,TET, NIT AMP, CFL, CIP, ENR, STR, GEN,TET, SXT CFL, ENR, STR, TET, NIT *b AMP, STR, GEN, TET, SXT AMP, STR, TET, SXT

a Drugs tested ampiciillin (AMP), cephalexin (CFL), cefotaxime (CTX), ceftiofur (CEF), ciprofloxacin (CIP), enrofloxacin (ENR), chloramphenicol (CHL), streptomycin (STR), gentamicin (GEN), tetracycline (TET), trimethoprim-sulfamethoxazole (SXT), and nitrofurantoin (NIT). b Ampicillin not tested in this strain.

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Fig. 1. Distribution of 38 E. coli isolates from cats and dogs with UTI according to their phylogenetic group, number of virulence genes and resistance to eight antibiotic classes. Twenty strains presented MDR and could be divided into two groups, one with isolates from all four phylogenetic groups (left) and another with isolates from the B2 and D phylogenetic groups, predominantly composed of O2 and O6 strains (right).

phenotypic tests against the isolates, all strains were susceptible to ceftiofur and only 4% (2/45) were resistant to cefotaxime (Supplementary data, Table 2). With respect to multidrug resistance, 76% (13/17) of the strains belonging to groups A (2/2), B1 (4/7) and D (7/8) were MDR, whereas only 21% (6/28) had this phenotype in group B2. The MDR profile in groups A, B1 and D was significantly higher when compared to that in group B2 (P = 0.0016). Table 1 shows the complete virulence and resistance pattern profile. When the means of the virulence genes and the means of the resistance to the antimicrobial classes were calculated for each phylogenetic group, an inverse relationship between VF and resistance was observed; this relationship was more significant in group B2 (mean of six VF vs. resistance to one class of antibiotics) than in groups B1 (two VF vs. three classes of antibiotics), A and D (four VF vs. four classes of antibiotics). Nevertheless, by analyzing each isolated strain, it was possible to define two groups of MDR strains. The first group contained representatives of all phylogenetic groups, and the second group contained 18% (8/45) of the strains, most of which were derived from phylogenetic group B2 and belonged to serogroup O6, which concomitantly showed a high incidence of virulence genes and MDR (Fig. 1). 4. Discussion There are few Brazilian studies related to ExPEC strains isolated from extraintestinal infections of animals (Carvalho et al., 2012; Siqueira et al., 2009), and molecular epidemiology studies with strains originating from human infections are also scarce (Ananias and Yano, 2008; Dias et al., 2009; Tiba et al., 2008). In this study, 129 strains collected from 43 pets with confirmed UTI were characterized. Except for strains isolated from one individual, all isolates from the same animal showed the same characteristics, which reinforces the idea that a particular clone

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would be more fit to survive in the urinary tract when presenting competitive advantages (Johnson et al., 2003; Johnson and Russo, 2005). Serogroups such as O2, O4, O6, O11, O18, O25, O75 and O102 are described in the literature as the most frequently related to urogenital infections in pets and humans (Johnson et al., 2001; Yuri et al., 1999), and several of these are also related to more severe diseases such as meningitis and septicemia (Ananias and Yano, 2008; Johnson et al., 2000). In the present study, most of these serogroups were identified, with serogroups O2, O4 and O6 corresponding to more than one third of the isolates. These O antigens, which are typically associated with UTI in human, were prevalent in studies performed by other authors in dogs with UTI (Johnson et al., 2003; Yuri et al., 1999). In Brazil, serogroup O6 is the most prevalent among isolates from birds with omphalitis, salpingitis, chronic respiratory disease, and swollen head syndrome from commercial farms, and it has also been reported to be among the most frequent isolates from human patients with sepsis (Ananias and Yano, 2008; Kno¨bl et al., 2012). Similarly to the others’ findings (Johnson et al., 2003; Maynard et al., 2004), the vast majority of strains with typeable O antigen and that are classically associated with UTI were derived from phylogenetic group B2, followed by group D, especially when only the cats were considered. Some of the markers studied were present in percentages close to 50% of the strains (papC/papEF and traT) or above (fimH, fyuA and malX). There is epidemiological and experimental evidence of the importance of these genes as urovirulence markers (Johnson and Russo, 2005). Genes such as pap and sfa are considered markers that define strains such as ExPEC (Johnson et al., 2003), and genes for P fimbriae have been associated with isolates capable of causing urosepsis in humans and pets (Maynard et al., 2004; Ramos et al., 2010). In addition, the malX gene, a marker of a pathogenicity island of archetypal strain CTF073 (O6:K2:H1) was positive in 27 of the 28 strains derived from B2. This gene has been considered an important virulence marker of strains derived from both canine and human UTI, pyelonephritis, bacteremia and neonatal meningitis (Johnson et al., 2003, 2001). It is interesting to note that strains derived from B2 showed high virulence-associated traits when compared to those from other groups. Because strains with increased molecular virulence are considered potentially more pathogenic and capable of causing more severe syndromes (Johnson and Russo, 2005), it is noteworthy that 32% of the B2 strains were typed as O6 and possessed, on average, eight virulence genes. This serogroup is involved in severe human and animal syndromes (Ananias and Yano, 2008; Kno¨bl et al., 2012), and it has been demonstrated that both humans and their pets could be colonized by similar strains that are potentially pathogenic to both species, which characterizes the zoonotic potential of O6 serogroup (Johnson and Clabots, 2006). The increasing antimicrobial resistances of isolates from the urinary tract of dogs have been a matter of concern (Thompson et al., 2011). There were high percentage of resistant strains to the drug of first choice for the treatment of UTI, such as sulfonamide associated

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with trimethoprim (Guardabassi et al., 2004), and a high percentage of isolates were also resistant to ampicillin, streptomycin and tetracycline, all these antimicrobial drugs widely used in clinical practice. The emergence of antibiotic resistance in UTI isolates has also been demonstrated with more recent UTItreating drugs such as third-generation cephalosporins and fluoroquinolones (Johnson et al., 2003; Pitout, 2012; Thompson et al., 2011), with the latter widely used in the treatment of human and animal UTI in Brazil. Despite the low resistance observed with regard to the former, 16% of the strains were resistant to fluoroquinolones, and all were MDR. These antimicrobials are used commonly and are recommended for the treatment of cystitis, pyelonephritis and prostastitis, among other pet infections (Guardabassi et al., 2004). The highest proportion of strains with this characteristic was present in group B1, which had a low urovirulence profile. Other authors studying human and animal strains have found a negative association between resistance to fluoroquinolones and VFs or a B2 background, suggesting that this phenomenon may be related to the fact that strains resistant to these drugs may originate from less virulent populations (Johnson et al., 2003) or that a given population, when exposed to fluoroquinolones, may lose the virulence genes contained in the PAIs (Soto et al., 2006). Moreover, both strains of phylogenetic group A and seven of the eight classified as D were MDR (Table 1). The latter deserve particular attention because they have a higher proportion of virulence genes. In one study, MDR strains from nosocomial infections of hospitalized dogs belonged to these two groups, suggesting that the D strains, in addition to presenting sufficient virulence apparatus to begin an extraintestinal infection, are better adapted for colonizing the gut environment in the presence of high antibiotic pressure (Sidjabat et al., 2009). A significant relevance for public and animal health was demonstrated for 18% of the strains in this study that were classified into phylogenetic groups B2 and D; most of them featured both MDR and a high virulence potential. In recent years, there has been a shift in the paradigm that hypothesizes that MDR strains are less virulent than their sensitive peers, which was proposed after the identification of global clones with an associated high virulence and MDR that affect humans and their pets (Pitout, 2012). In conclusion, the ExPEC canine strains isolated in this study displayed pathotypic and phylogenetic similarities with human isolates and high percentages of drug resistance. The relationship of modern society with pets, which live inside the house in close contact with their owners, enables the mutual transmission of potentially pathogenic strains for humans and pets (Johnson and Clabots, 2006). Furthermore, pets can be a potential source for the spread of resistance to humans due to the extensive use of antimicrobials in these animals (Guardabassi et al., 2004). Thus, the finding of ExPEC strains with MDR characteristics suggests implications for animal and public health. Further studies should be performed to evaluate both the involvement of these findings in more severe diseases in dogs and cats and the epidemiological role of pets in human infections by ExPEC in our environment.

Conflict of interest statement The authors have no financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work. Acknowledgements The authors would like to thank to LAB&VET Diagno´stico e Consultoria Veterina´ria LTDA for kindly providing all animals samples and Dr. James R. Johnson for providing E. coli strains used as positive and negative controls. This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP 2008/56005-2 and 2006/54343-2) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).

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Virulence genotypes, antibiotic resistance and the phylogenetic background of extraintestinal pathogenic Escherichia coli isolated from urinary tract infections of dogs and cats in Brazil.

Urinary tract infection (UTI) is a frequent disease of humans and pets and has extra-intestinal pathogenic Escherichia coli (ExPEC) strains as one of ...
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