Introduction

Dogs are perhaps the most favoured do- mestic animal among all  the  pet  animals. In developed countries, companion animals have become an integral part of the house- hold (Chomel and Sun, 2011). Companion animals, especially dogs, suffer from vari- ous bacterial diseases affecting urinary tract leading to malfunctioning of this system, followed by critical medical emergency or death of the animal (Davison et al., 1999). Bacterial urinary tract infections (UTIs) are the most common cause of urinary tract disease in dogs. Approximately 14% of all dogs will acquire bacterial UTIs during their lifetimes with variable age of onset (Os- borne, 1999).

Escherichia coli (E. coli) is the most prevalent and important causative agent of UTIs in dogs and humans, strains of this spe- cies are often abundant in the gastrointes- tinal tract at the time of infection (John- son et al., 2003; Momoh et  al.,  2011; Tramuta et al., 2011 Saraylu et al.,  2012).

However, E. coli that are associated with UTIs are commonly named uropathogenic isolates (UPEC), although there is evidence that different pathotypes may be related to UTIs (Marrs et al., 2005). Urinary tract in- fections are mostly common in dogs, but clinical signs are often minimal, and the infection may go undetected (Ling, 1995). Uropathogenic Escherichia coli is one of the most common bacteria isolated from canine and feline UTIs (Osugui et al., 2014).

Uropathogenic   Escherichia    coli    con- tain  several  virulence  factors  that  facili-  tate its colonization and  invasion  of  host  cells (Ejrnæs, 2011; Kudinha etal, 2012; Agar- wal et al., 2013). Surface virulence factors (ad- hesins) of UPEC are among the most im- portant  virulence  factors (Ni -

cole, 2008; Bien et al., 2012).The main attachment factor, P fimbriae are particularly associated with pyelonephritis and are en- coded by pap genes (Jadhav et al., 2011). Another adhesion that acts as a virulence factor is S fimbrial adhesion, which  is cod- ed by sfa  genes  (Bien et al., 2012; Pobie-  ga et al., 2013). Also, afimbrial adhesions (afa) of E. coli, coded by afa genes, have been  reported  in  cases   of   pyelonephri-  tis (Servin, 2005). Other important  viru- lence factors of UPEC strains are the tox-  ins that act as secretory virulence factors (Bien et al., 2012). The most important secre- tory virulence factor is A-hemolysin (hlyA), which is encoded by the hly gene. Also, cy- totoxic necrotizing factor 1 (cnf1) is reported in a third of pyelonephrogenic strains (John- son, 1991). Other virulence factors also have important roles in the development of UTIs, including serum resistance ability due to the outer membrane protein traT encoded by  traT genes (Mellata et al., 2003; Kawamu- ra-Sato et al., 2010). Aerobactin is a bacterial siderophore encoded by aer genes and has re- cently been documented as a viru- lence factor in UPEC strains (Slaychev et al., 2009). The virulence  factors are  car- ried by pathogenicity islands  (PAIs),  which are mobile  genetic  elements that con- tribute to the horizontal transfer of viru- lence determinants (Hacker et al., 1997; Sa- bate et al., 2006). Phylogenetic analyses showed that E. coli strains and virulence genes were divided into four major phy- logenetic groups (A, BI, B2, and D) based  on  their  genetic  polymorphisms   (Cler- mont et al., 2000). It is known that the ex- pression of virulence genes and phylotypes varies with geographical  location  (Agarw- al et al., 2013). However, phylogenetic  char-

acterization is an important tool to improve the understanding of E. coli populations and their relationship between strains and dis- ease (Coura et al., 2015). Molecular charac- terization of E. coli virulence genes that are associated with UTIs in dogs has been stud- ied in America and most parts of the world. However, in India, there is relatively less documented information on E. coli virulence genes associated with UTIs in dogs. The cur- rent study aimed to determine the virulence genes and their phylogenetic grouping in E. coli  isolates  from  dogs  with  UTIs.

Materials and Methods

Sample Collection

From February  2017  to  January  2018, a total of 103 urine samples were collect-  ed aseptically through cystocentesis from dogs that were diagnosed with urinary tract infection in small animal section of Veteri- nary Clinical Complex (VCC), Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS) Hisar Haryana,  India.

Bacteriological Examination

The fresh urine samples collected asep- tically were inoculated and streaked onto a 5% sheep blood agar (BA) (HiMedia, Mum- bai, India) and MacConkey’s lactose agar (MLA) (HiMedia, Mumbai, India) plates separately with the help of a 4 mm diame- ter platinum loop. The plates were incubat- ed aerobically at 37°C for 24-48h till ad- equate growth was observed. Suspect- ed colonies were streaked onto Eo- sine Methylene Blue agar (EMB), (HiMe- dia, Mumbai, India) which is a selective medium for E. coli and the plates were incubated aerobically at 37°C for 24h. The appearance of blue, green colonies with a metallic luster on EMB was presumptive-  ly considered as indicating the presence   of

Escherichia coli.

Biochemical Examination

Escherichia coli was identified using Gram staining technique. Samples with rod-shaped arrangements  were  subjected  to biochemical tests. (Indole, Methyl Red, Voges Proskauer, Citrates tests, Glucuro- nidase, Nitrate reduction, ONPG, Lysine utilization, Lactose, Glucose, Sucrose and Sorbitol) using commercially KB010 HiE. coliTM Identification Kit (HiMedia Mum- bai, India) following the manufacturer’s in- structions.

Isolation of Bacterial DNA

DNA of Escherichia coli from all the positive isolates was extracted using com- mercial available PureLinkTM Genom- ic DNA mini kit (Invitrogen, USA) fol- lowing the manufacturer’s instructions as shown below.

DNA of Escherichia coli from all the positive isolates was extracted using com- mercial available  PureLinkTM  Genomic DNA mini-kit (Invitrogen,  USA).  Brief-  ly, overnight grown culture in BHI  broth (10 ml) was centrifuged at 10000 rpm for  10 min at 4°C. Supernatant was discarded and pellet was re-suspended in 200μl of di- gestion buffer. Then 20μl of proteinase K was added and vortexed vigorously. The solution was then transferred into 2ml mi- cro centrifuge tube and incubated in water bath (Bench Top Lab System, USA) for 2 h at 55°C. A total 200μl of both lysis/binding buffer and absolute ethanol was then added and each tube was carefully vortexed in or- der to mix the contents of the tube evenly. The solution was then carefully transferred into a PureLinkTM column and then centri- fuged. Five hundred microliters (500μl) of buffer I was added to the samples, mixed and  centrifuged  for  2  min  at  12000 rpm,

the wash trough was discarded. Then 500μl of buffer II was added and the column was centrifuged for 2 min at 12000 rpm, the wash trough was discarded and the empty column was centrifuged for 2 min. The col- umn was then transferred into a fresh 1.5ml micro centrifuge tube. Finally, for eluting the DNA, 30μl of elution buffer was add-  ed at the centre of the column, the column was incubated for 10 min at room tempera- ture and then centrifuged at 12000  rpm for 2 min. The flow through was collected in    a 1.5ml micro centrifuge tube as the DNA. The obtained DNA was stored at -20 °C for further use.

Detection of Virulence Genes

The presence of virulence genes in  E. coli DNA extracts was determined by con- ventional PCR (Manage et al., 2019). Prim- ers sequences, target genes, products size, and references are given in Table 1. The conventional PCR was performed in veriti thermo cycler (ABI, USA) in 25 volume re- action containing 6 µl of template DNA, 1µl of each of the primers (10 pmoles), 12.5 µl Phusion PCR Master-mix (2X) (High Fi- delity, USA), 1µl DMSO and 3.5 µl of nu- clease-free water. Amplification procedure consisted of initial denaturation at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for     30 s, extension at 72°C for 30 s and a final extension at 72°C for 5 min. The PCR prod- ucts were analyzed on 1.5% agarose gel electrophoresis and visualized under UV trans-illuminator GEL-DOC™ (BIO-RAD, India) and documented by photography for further analyses.

Nucleotide sequence analysis of the virulence genes and their phylogenetic comparisons

The   PCR   products   obtained   were    pu-

rified using PureLinkTM Quick Gel  Ex- traction   Kit   (Invitrogen,   Germany)   and  the cycle sequencing reactions were per- formed in a total volume of 10µl us-  ing Big Dye Terminator v 3.1 Cycle Sequenc- ing Kit (ABI, USA) in automated DNA se- quencer (Applied Biosystem 3130XL Genetic Analyzer). The DNA sequencing was per- formed in both directions with the PCR prim- ers as sequencing primers (Table 1).

The resulting sequences obtained from  the sequencer were used to make Contigs using SeqMan program in Lasergene suite (version 5). The contigs were then com- pared with those reported in GenBank (NCBI) by a BLAST search online. Clustal W (Bioedit software ver 9.0) was used for multiple sequence alignment, and Mega 6.0 bioinformatics sequence analysis tool was used for phylogenetic studies.

Statistical analysis

Vassarstat was used for the analysis of confidence  interval (proportion).

Results

Examination of a total of 103 urine sam- ples from dogs suffering from UTIs for detec- tion of Escherichia coli revealed 25 (24.3%) positive isolates.

The cultural growth on MacConkey lactose agar was seen as typical purple/pink colonies. However, the positive isolates were further confirmed as E. coli by inoculation on Eo- sine-Methylene blue agar  medium.

Microscopic examination of gram stained colonies shows a gram negative rod-shaped arrangement. The biochemical test showed that E. coli were positive for Indole, Methyl red, Glucuronidase, Nitrate, ONPG, Lysine utilization, Lactose, Glucose, Sucrose, and Sorbitol. On the other hand, all the strains were  negative  for Voges-Proskauer’s  and Ci-

Table 1. Primers for conventional PCR assays

 trate utilization test.

Of the 25 E. coli isolates assayed, 20(80.0%) showed the presence of aer, while 14 (56.0%), 12(48.0%),   8(32.0%)   and  5(20.0%)    were

found to have pap, sfa, afa, cnfI and hly genes respectively. No cnf2 gene was found in any  of the E. coli isolates tested (Table 2).

The aer, afa, cnf1, hly, sfa, and pap gene se-

quences show the highest nucleotide   similar-

ity of 99% with several strains retrieved from the GenBank data base.

hly, sfa, cnf1, and pap gene sequence were highly similar to the sequences that were associated with the phylogenetic group B2 whereas aer gene sequences showed maxi- mum similarity to the one in B2 and D phy- logenetic group and afa sequence grouped within the phylogenetic group A.

Table 2. Distribution of virulence genes of E. coli isolated from 25 dogs diagnosed with UTIs in Hisar Haryana, India.

                        Figure 1. Agarose gel electrophoresis of  aer and  pap

Figure 2. Agarose gel electrophoresis of  sfa and  afa

Figure 3. Agarose gel electrophoresis of  cnf1 and  hly

Figure 4. Agarose gel electrophoresis of  cnf2

Discussion

Distribution of virulence factor gene as- sociated with canine UTIs has not been pre- viously determined and characterized in the study area (Hisar, India). The  presence  of the most common virulence genes such as aer, pap, sfa, afa, and hly detected in E. coli strains isolated in the present study estab- lished the pathogenic potentials of E. coli in dog’s  urogenital infections.

Aerobactic receptor gene, coded by aer, promotes bacterial growth in the limiting iron concentration encountered during in- fection and act as a virulence factor in the pathogenesis of UTIs (Mittals et al., 2014). However, iron is necessary for E. coli metab- olism (Siqueira et al., 2009). Growth under iron-restricted conditions requires bacterial mechanisms that successfully compete with the host for the ion (Emody et al., 2003). Escherichia coli organism uses ion for ox- ygen transport and storage, DNA synthesis, electron transport, and peroxide metabolism (Siqueira et al., 2009). The aer virulent gene may be relevant during bacterial spreading from the urinary tract to the bloodstream (Garcia and Le Bouguenec, 1996). The aer virulent gene was seen in 80.0% of the iso- lates in the present study, this is because   aer

is a virulence factor in UPEC strains. Simi- larly, Slaychev et al. (2009) found compara- ble gene in their study as a virulent factor. In other studies, the detection of aer has been reported to be more than 30.0% and 56.0% (Firoozeh et al., 2014; Munkhdelger et al., 2017), which is in concordance with the current study. Furthermore, Drazenovich  et al. (2004) also found aer in 30.0% from E. coli strains isolated with persistent UTIs in dogs. The present study demonstrated high occurrence of aer genes in uropathogenic E. coli strains suggesting that the frequencies of iron acquisition system genes vary according to the geographical locations.

In the current study, most of the uropatho- genic E. coli was isolated from the urinary tract of dogs. This finding agrees with the findings of Siqueira et al. (2009), in which they documented that adhesion and coloni- zation of the uroepithelium is the most im- portant phenomenon involving uropathogen- ic E. coli. However, these adhesions promote bacterial adherence and are indispensable for the infection to be established as was report- ed by (Johnson, 1991; Emody et al., 2003; Marrs et al., 2005). P fimbria, encoded by pap, could probably contribute to the ad- hesion and colonization of the urinary tract because this virulence gene is always associ-

ated with pyelonephritis (Usein et al., 2001; Chen et al., 2003; Jacobsen et al.,  2008).

In the present study, pap was found in 56.0% E. coli strains isolated from canine urinary tract infections cases indicating that  E .coli isolates from the urine of dogs and cats have greater ability to colonize kidney and generate pyelonephritis (Antao et al., 2009). Shetty et al. (2014) reported that 60.87% of the uropathogenic strains of E. coli obtained from humans showed pap genes. Siquieira et al. (2009) found 23.5% papC positive strains among the E. coli strains isolated from dogs suffering from UTIs. Whereas Tramuta et al. (2014) identified pap in 13.4% E. coli iso- lates obtained from dog urine.

The S fimbriae adhesion (sfa) was found in 48.0% of the isolates obtained from UTIs in- fected dogs. This result is consistent with the occurrence of sfa (45.2%) in clinical isolates of human origin (Rahman and Deka, 2014). Similar findings were recorded in previous studies in dogs (Coogan et al., 2004; Sique- ira et al., 2009). In recent studies involving dogs (Tramuta et al., 2014) and human (Adib et al., 2014), sfa genes were found in more than 20.0% of UTI isolates studied. The demonstration of sfa gene in dog’s urine was observed in 52.0% of the isolates in anoth-  er study (Drazenovich et al., 2004). Like- wise, more than 12.0% of the E. coli strains from cats show sfa genes. Further, Shetty et al. (2014) also observed the presence of sfa gene in 39.1% isolates obtained from human UTIs. Therefore, these findings highlight that sfa gene plays a vital role in both adhesions to the urogenital epithelium and the develop- ment of urinary tract infection in companion animal, especially dogs.

Afimbrial adhesion (afa) gene was found to be implicated in developing nephritis in human (Mulvey, 2002). In the current  study,

afa gene was found in high frequency in  the

E. coli isolates (32.0%) as compared with strains from another study in dogs with UTIs (6.0%) (Drazenovich et al., 2004), but found lower in frequency in E. coli isolates report- ed from humans, 39.1% and 64.0% (Yama- moto et al., 1995; Shetty et al., 2014). On the other hand, Ebadi et al. (2017) reported that 9.3% of the E .coli strains identified from hu- man clinical samples showed fimbrial genes (afa BC). However, Dr adhesion family rec- ognize human receptors on erythrocytes and other tissues (Mulvey, 2002), and this may explain the low incidence of this ‘A’ fimbrial adhesion factor observed in canine isolates as previously reported (Siqueira et al., 2009). Uropathogenic E. coli strains  isolated from a dog with UTI express  the presence of cytotoxic necrotizing factor 1 (cnf1) and Alpha-haemolysin gene (hly). The hly gene is a pore-forming toxin that lyses not only erythrocytes of all mammals and even that of fishes (Johnson, 1991) but also leukocytes, endothelial and renal  epithelial cells (Usein et al., 2001; Emody et al., 2003). Alpha-hae- molysin gene (hly) was found present in 20.0% of the UTI isolates of the current study, similarly, Tramuta et al. (2014), Rah- man and Deka (2014), Liu et al. (2017) and Rashki et al. (2017) also recorded 18.0%, 36.8%, 39.0% and 10.0% of hly gene in UTIs isolates in dogs and humans respec- tively. In another study, Drazenovich et al. (2004) demonstrated that the hly gene was found in 50.0% of the E. coli isolates recov- ered from the urine of dogs. Siqueira et al. (2009) also found that 33.3% of the    canine

isolates contained hly genes.

Cytotoxic necrotizing factor 1 (cnf1) is a toxin that can cause reorganization of actin microfilament in eukaryotic cells. The cy- totoxin  may facilitate  the bacterial  invasion

of the bloodstream as a result of  interfer- ence with polymorpho-nuclear phagocyto- sis and even evokes apoptotic death of epi- thelial cells in the bladder (Oelschlaeger et al., 2002; Emody et al., 2003). In the pres- ent study, cnf1 was found in 20.0% of the UTIs strains. This was less than what was reported by Rahman and Deka (2014) who found 61.9% of the isolates from humans UTIs produce cnf1 genes. Drazonavich et al. (2004) reported cnf1 genes in 50. 0% of   the

E. coli isolates from dogs.

Furthermore, Johnson et al. (2003) report- ed that 41.0% of E. coli isolates obtained from canine UTIs produce cnf1. Liu et al. (2017) found 46.6% of the isolates contained cnf1. However, the results of the current study show less occurrence of cnf1 in the E. coli isolates. This could probably be attribut- ed to the mutation of this gene or could be due to variation in the geographical distribu- tion of these genes.

In the present study, none of these E. coli isolates harbored the gene that encodes cy- totoxic necrotizing factor 2 (cnf2), which is also related to UTIs. In contrast,  Rahman and Deka (2014) found cnf2 in E. coli iso- lates obtained from humans suffering from UTIs. These results indicate that cnf2 may probably be of less significant in the uro- pathogenic E. coli pathogenesis in  canine.

The phylogenetic analysis revealed that afa, aer, sfa, cnf1, hly, and pap genes exhib- ited 99% identity with the different variants in several geographical regions of the world. The E. coli falls into four leading phylogenet- ic groups: A, B1, B2, and D (Clermont et al., (2000). In the present study, the phylogenet- ic comparisons of virulence gene sequenc-  es show that sfa, cnf1, hly and pap genes matched group B2 and, these results are con- sistent with the findings noted by other inves-

tigators (Tramuta et al., 2011; Munkhdelger et al., 2017; Coura et al., 2018).  However, aer virulence gene was found to match both B2 and D phylogenetic group. This is consis- tent with the previous study that demonstrat- ed that aer sequence belongs to B2 and D phylogenetic group (ZhuGe et al., 2014; Liu et al., 2015). Moreover, only afa virulence gene sequence was found to fall within phy- logenetic group A. This is also in accordance to recent studies, where it has been shown that afa gene sequence was observed to be associated to phylogenetic group A (Marti et al., 2017; Munkhdelger et al.,  2017).

In the current study, majority of the viru- lence gene sequences were found to be as- sociated with the phylogenetic group B2, suggesting that these strains were more vir- ulent than others. Similar outcomes were observed in a recent study conducted by Pi- atti et al. (2008) who found an association between virulence factors and phylogenetic group in urinary E. coli strains isolated in hu- mans. Furthermore, most of the extraintes- tinal pathogenic E. coli (ExPEC), including those with the most robust virulence factors repertories and those  which  are  best  able  to infect non-compromised hosts, are de- rived from phylogenetic group B2 (Johnson, 2002). Group D contains the second-highest number of ExPEC, extraintestinal isolates. This group typically have somewhat fewer virulence factors and a different mix of viru- lence factors than group B2 isolates. E. coli strains belonging to group A and B1 do not frequently cause extraintestinal infection. These strains that are not highly virulent cause disease only in immunocompromised hosts, and could be pathogenic in healthy hosts only if they were to acquire sufficient extraintestinal virulence factors (Johnson, 2002).