Identification of bacterial pathogens that cause urinary tract infections and detection of their antibiotic susceptibility

Urinary tract infections (UTI) are common clinical problems in companion animals accounting for important use (also overuse and misuse) of antimicrobials. Improper and/or underestimated therapeutical applications may lead to a variety of pet’s health, economic, public health concerns (Weese et al., 2011). Clinically significant infection implies the presence of a clinical abnormality and is characterized by dysuria, pollakiuria and increased urgency of urination along with the presence of bacteria in urine. Coagulase positive Staphylococci are involved in the formation of calculus in dogs. In male dogs, UTI frequently extend to the prostate gland during the infection period. Due to the formation of blood-prostate barrier, it is difficult to eradicate bacteria from the prostate gland, and they may reinfect the urinary tract following appropriate treatment, cause a systemic bacteremia, infect the reproductive tract, or cause a local infection within the prostate and eventually cause an abscess. Antimicrobials are the milestones of UTI therapy, and many patients with recurrent UTI are managed inappropiately with repeated applications of antimicrobial therapy. This approach fails if the underlying pathophysiology predisposing the animal to the UTI is not addressed, and it encourages the developmentof resistant bacteria. Whether clinical signs are present or not, the consequences of untreated UTI include lower urinary tract dysfunction, urolithiasis, prostatitis, pyelonephritis or septicemia accompanying renal failure. Uncomplicated and simple UTI are sporadic bacterial infections of the bladder in an otherwise healthy individual presenting normal urinary tract anatomy and function (Warren et al., 1999).

The purpose of the present research is to present the bacterial involvement in UTI of dogs and cats, in addition, determination of the antibiotic susceptibilities of the identified bacterial agents is aimed also.

Sample collection
 
In this research, 5 ml volume of urine samples were taken by catheter to sterile tubes from 10 dogs and 10 cats which were diagnosed with UTI in clinics of Adnan Menderes University Faculty of Veterinary Medicine Department of Internal Medicine, Aydin province in Turkey. All authors hereby declare that “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable.

After collection of urine samples, they were brought to Adnan Menderes University Faculty of Veterinary Medicine Department of Microbiology Laboratory under the cold chain.
 
Bacterial identification
 
Each urine samples was streaked onto blood agar which contains 5% sheep blood, Eosin Methylene Blue agar and MacConkey agar for bacterial examination. After streaking urine samples, the tubes were centrifuged at 5000 rpm for 10 min, then supernatants were removed. Residual urine sediments were streaked onto blood agar which contains 5% sheep blood, Eosin Methylene Blue agar and MacConkey agar for bacterial examination also. Plates were incubated at 37°C aerobically for 48-72 hours until bacterial growth was present.

After 48 h growth period, the preliminary evaluation of the bacterial colonies were made on the grounds of colony appearance and preparation staining after Gram staining method. Gram positive round shaped and Gram negative rod shaped bacteria were observed. The bacterial cultures were identified with conventional biochemical and carbohydrate fermentation tests as catalase, coagulase, oxidase, urease, indole reaction, mannitol, lactose, glucose fermentation, MR-VP, LDC, Simmon citrate tests, following the standard procedures described before (Holt and Krieg, 1994) and API® 20E,  API® Staph Identification System.
 
Antimicrobial sensitivity of the bacterial strains
 
All the strains were tested for their sensitivity against 8 antimicrobial agents (Weese et al., 2011). The antibiotics used in the study were Amoxicillin-Clavulanic acid, Ceftriaxone, Cefoperazone, Lincomycin, Enrofloxacilin, Erythromycin, Sulfamethoxazole-Trimethoprime and Oxacillin. The antibiogram of all strains was determined on Mueller-Hinton medium. Each strain were inoculated to Brain-Heart Infusion broth and incubated for 18 h. 100 µl of 18 h-old cultures were spread evenly on plates. The cultures were allowed to absorb onto the plates for 5 min and then antimicrobial discs were placed on the plates at an appropriate distance from each other. The plates were then incubated at 37°C for 24 h. The diameters of the inhibition zone were measured and matched with respective standard zone diameters to interpret the isolates as resistant, intermediate or sensitive according to the procedures of Clinical Laboratory Standards Institute (CLSI, 2008).

A total of 9 (22.5%) bacterial strains were isolated through the collected 40 specimen as 20 urine samples and 20 sediment samples. Three of the bacterial strains were isolated from urine samples of dogs and 5 of the bacterial strains were isolated from sediment samples of dogs. One bacterial strain was isolated from sediment sample of a cat. Staphylococcus aureus was identified from urine sample of 1 dog and from sediment samples of 3 dogs, Staphylococcus epidermidis was identified from urine sample of 1 dog and sediment sample of 1 dog, Serratia liquefaciens was identified from urine sample of 1 dog, Plesiomonas shigelloides was identified from sediment sample of 1 dog, Yersinia enterocolitica was identified from sediment sample of 1 cat. There were not observed any bacterial isolation from urine and sediment samples of 2 dogs and 8 cats. Bacterial isolates from urine and sediment samples of dogs and cats are shown on Table 1.



Antibiotic sensitivity tests revealed that 78% of the isolates were susceptible to Amoxicillin-Clavulanic acid, 67% of the isolates were susceptible to Ceftriaxone and Cefoperazone, 55.5% of the isolates were susceptible to Enrofloxacin, 22.5% of the isolates were susceptible to Lincomycin and 22% of the isolates were susceptible to Erythromycin. The isolates were resistant to Sulfamethoxazole-Trimethoprime and Oxacillin in the ratio of 78% and 67% respectively. The complete antibiotic sensitivities of the isolates were shown in Table 2.

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The urinary pathogens S. aureus and Coagulase Negative Staphylococci are the most frequent agents isolated from dogs with UTI (Norris et al., 2000), however other previous studies in the literature present prevalence of no more than 10% of UTI samples being positive for Staphylococci (Cohn et al., 2003). In this study, Staphylococcus sp. appear to be more prevalent in the present study group than what is usually reported. In previous studies, S. intermedius have been more prevalent than S. aureus, however the dominant strain is S. aureus in this study (Ling et al., 2001; Norris et al., 2000; Ogeer-Gyles et al., 2006; Penna et al., 2002; Rowlands et al., 2011). Ling et al., (2001) reported that Proteus spp. were isolated more frequently from urine specimens collected by catheterization or midstream catch than by cystocentesis, unlikely in our study, Proteus spp. isolation was not present. The Staphylococcal isolates obtained from dogs are in agreement with these findings, however, Pseudomonas spp. prevalence was not observed unlikely in previous studies (Norris et al., 2000; Seguin et al., 2003).

Other gram negative species isolated in this research, which are as S. liquefaciens and P. shigelloides, were isolated only from one urine and one sediment sample. Y. enterocolitica was isolated from one sediment sample of a cat. The results show that, S. aureus was isolated from urine sample of 1 dog and from sediment samples of 3 dogs and S. epidermidis from was isolated from urine sample of 1 dog and from sediment sample of 1 dog. Coagulase positive species were slightly more common than S. epidermidis; this correlates with other studies (Prescott et al., 2002). Among the Coagulase Positive Staphylococci, the dominance of S. pseudintermedius over S. aureus also is in agreement with the vast majority of other relevant studies, since it is well-known that S. pseudintermedius is the most common species of canine infections (Ling, 2000; Lilenbaum, 2000; Ganiere, 2005), including UTI (Hoekstra and Paulton, 2002). However, S. intermedius was not isolated in this study. The research also reveals that sediment the bacterial agents are more prone to be isolated from sediment samples since 6 of the strains were isolated from sediment samples, thus only urine samples are not enough for isolation and identification of bacterial diseases from urinary tract infections. Interestingly, previous studies implied that E. coli was the most dominant strain isolated from UTI in dogs and cats (Balasoiu et al., 1997; Ball et al., 2008; Hall et al., 2013; Kivisto et al., 1977; Thompson et al., 2011), however E. coli isolation was not observed in our study.

Cats with feline lower urinary tract disease usually have bacteriologically sterile urine (Dowling, 1996). This data correlates with the results of our study, since only one bacterial agent, Y. enterocolitica was isolated from feline samples. Ling et al., (2001) reported that Proteus spp. were isolated more frequently from urine specimens collected by catheterization or midstream sample than by cystocentesis, however in this research, Proteus spp. was not isolated. This indicates that there was not any faecal contamination or misinterpretation for sample collection.

Amoxicillin/clavulanic acid has an increased spectrum of activity against gram negative bacteria due to the presence of the “suicide” drug, clavulanic acid. Clavulanic acid irreversibly binds to β-lactamases, allowing the amoxicillin fraction to interact with the bacterial pathogen. This combination usually has excellent bactericidal activity against B-lactamase-producing Staphylococci. Cephalosporins present higher stability in contrast to b-lactamases than do Penicillins, so they have greater activity against Staphylococci and Gram negative bacteria. They have greater activity against Staphylococci also (Dowling, 1996). Correlating with these topics, most effective antimicrobials in this research were detected as Amoxicillin-Clavulanic acid (78%), Ceftriaxone and Cefoperazone (67%). Our findings correlate with previous researchs (Pedersen et al., 2007; Penna et al., 2010).

There was a high resistancy against Sulfamethoxazole-Trimethoprime in the ratio of 78% consistent with data from other countries, such as 74.4% reported in Canada (Hoekstra and Paulton, 2002). This antimicrobial was occasionally used to treat canine UTI in the recent past and resistance towards this class of drugs has increased rapidly; 30 years ago resistance to this drug was reported as only 2% (Rohrich et al., 1983). Due to the widespread and increasing resistance, the use of sulphonamides associated with trimethoprim for UTI cannot be recommended in the absence of antimicrobial susceptibility tests.

Previous studies stated that resistance to Oxacillin, although limited (25.7%), has dramatically increased from 4.6% in a previous study of a similar canine population (Lilenbaum, 2000). Our study is in agreement with this data, since 67% resistancy was obtained in this research.

The present study reported that the antimicrobial resistance of bacteria isolated from canine UTI and highlights the importance of species-spesific differentiations, as in feline urine the bacterial colonization is a minor provision for UTI. It also emphasizes the need for bacterial culture with species identification and antimicrobial susceptibility tests in order to choose appropriate antimicrobial agents to treat companion animal UTI with lower costs and for prevention of recurrent infections.