Canine Cystitis - Biofilm Formation by Bacterial Isolates

Background: Biofilms have been reported as important virulent markers associated with drug resistance in urinary tract infections (UTIs) in humans and dogs. However, in veterinary medicine, researches involving biofilm formation, treatments and preventions have been limited; yet, it is still possible to find few studies demonstrating biofilm-forming bacteria associated with different comorbidities such as otitis, wound infections, UTIs, and endometritis. These studies generally select dogs with chronic and recurrent infections, which could be an important factor in antibiotic resistance. We aimed to evaluate biofilms in sporadic cystitis regarding prevalence and drug resistance. Materials, Methods & Results: Urine samples were collected by cystocentesis from 36 client-owned dogs under clinical and laboratory suspicion of non-recurrent urinary bladder infection (cystitis). Urine was aseptically plated onto blood agar, MacConkey, and CLED, followed by incubation for 24 to 48 h. Definitive identification of a potential pathogen was made by subculture collected from an isolated colony to obtain a pure culture. The gram staining method and specific biochemical tests (phenol red fermentation, lysine, phenylalanine, citrate, sulfide-indole-motility, and urease) were used to distinguish and classify the bacteria. After identification, the bacteria were tested for antimicrobial susceptibility by a standard disk diffusion method, using the following antimicrobials: amoxicillin with clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, clindamycin, cefazolin, cephalothin, erythromycin, gentamicin, norfloxacin, and sulfamethoxazole-trimethoprim. The biofilm-forming ability was determined based on a culture in Congo red agar (CRA), where biofilm producer strains formed black colonies with a dry crystalline surface, while non-biofilm producer strains formed red colonies with a smooth surface. A crystal violet dye assay was used to confirm the CRA results. Of the 36 urine samples collected from dogs with suspected cystitis, a total of 37 isolates were obtained, from mixed or pure cultures. The most prevalent bacteria were Escherichia coli (11/37), followed by Staphylococcus spp. (8/37), Proteus spp. (7/37), and Enterococcus spp. (5/37). Other less prevalent bacteria were Klebsiella spp., Streptococcus spp., and Enterobacter spp. As for biofilm-forming ability, 67.6% (25/37) of the bacterial isolates had biofilm formation in CRA and 54.05% (20/37) on the microplates containing crystal violet dye. There was no statistical difference in antimicrobial susceptibility between biofilm producer and non-biofilm producer bacteria. Discussion: We found a high proportion (> 54%) of in vitro biofilm-forming ability by different bacteria, which may indicate that biofilms may also be formed in vivo, in simple cystitis. Antimicrobial resistance was not noticed in bacteria capable of forming a biofilm; however, in a future study it is important to evaluate bacterial resistance in vivo, considering the possibility of having a different response than in vitro. In addition, the problem of the presence of a biofilm in vivo is that it can nullify the antimicrobial efficacy of therapeutic agents even with in vitro susceptibility. Besides the possibility of slow or incomplete diffusion of antibiotics through the extracellular matrix of the biofilm, aspects like hydration level, pCO(2), pO(2), pH, pyrimidine, and divalent cation concentration that negatively influence antimicrobial activity in vitro can also cause undesirable effects at the profound layers of the biofilm. In conclusion, all of the genera of bacteria isolated from dog's sporadic cystitis in this study were able to form a biofilm in vitro. The pathogenicity and antibiotic resistance of bacteria appears unrelated to biofilm formation in vitro.