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 37 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, pCO2, pO2, 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.Keywords: sessile bacteria, urine, simple cystitis, antibiotic resistance

Evaluation of biofilm formation by bacteria isolated from urine samples of dogs with cystite

FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas GeraisTrabalho de Conclusão de Curso (Graduação)A cistite é a afecção do trato urinário inferior mais comum em cães e ocorre quando o microrganismo é muito virulento ou há falhas nos mecanismos de defesa do hospedeiro. Sua persistência ou recorrência pode ser causada pela formação de biofilmes, que são agregações de microrganismos com uma arquitetura definida e metabolismo alterado, com melhorar comunicação de célula a célula, e capazes de evadir a resposta imunológica do hospedeiro e dos efeitos dos antimicrobianos. O objetivo dessa pesquisa foi conhecer o perfil de bactérias oriundas de amostras de urina de cães com cistite e analisar a susceptibilidade aos antimicrobianos, além de verificar se essas produzem biofilme. Para a metodologia, avaliou-se 36 amostras de urina de cães com suspeita de cistite. As bactérias foram isoladas por meio de inoculação por técnica de esgotamento em estrias feitas em Ágar Sangue, MacConkey e Cled, e identificadas através de coloração de Gram e provas bioquímicas. Para determinar a suscetibilidade a antimicrobianos utilizou-se o método de difusão em disco em placas de Mueller Hinton. A produção de biofilme foi verificada pelo cultivo em Ágar Vermelho Congo e pelo teste confirmatório de microplacas com o corante cristal violeta. Das 36 amostras, 23 apresentaram crescimento bacteriano e as bactérias isoladas com maior frequência foram Escherichia coli (29.8%), Staphylococcus spp. (21.6%), Proteus mirabilis (18,9%) e Enterococcus spp. (13.5%). Os isolados de E. coli apresentaram 81, 8% de sensibilidade a ceftriaxona e gentamicina, enquanto para cefalotina houve 72,7% de resistência. Staphylococcus spp. demonstram alta resistência a duas das principais drogas de eleição para o tratamento de cistite, ampicilina e sulfametoxazol+trimetropim. Em relação a P. mirabilis, notou-se alta sensibilidade a todos os antibióticos testados. Todos os isolados de Enterococcus spp. apresentaram resistência a sulfametoxazol+trimetropim e a clindamicina. Ao menos um isolado de cada patógeno identificado no presente estudo demonstrou habilidade de formar biofilme in vitro, destacando-se E. coli, Staphylococcus spp. e Enterococcus spp. Torna-se importante a adoção de medidas profiláticas com o intuito de evitar a infecção do trato urinário e a resistência a antimicrobianos

Determinação do período de carência de doramectina a 1% em tecidos bovinos

O presente trabalho teve como objetivo determinar o período de carência da doramectina a 1% em músculo, gordura, rim e fígado de bovinos aplicada na concentração de 1%, por via subcutânea, na dose de 1 ml/50 Kg (200 µg/kg) em bovinos. Foram avaliados no experimento 20 animais, divididos em cinco grupos contendo quatro animais cada e abatidos nos momentos D+7 (grupo A), D+14 (grupo B), D+28 (grupo C), D+35 (grupo D) e D+42 (grupo E). Os Limites Máximos de Resíduos estabelecidos pelo Codex Alimentarius Commission para Doramectina no músculo, fígado, gordura e rins, são 10 µg/kg, 100 µg/kg, 150 µg/kg e 30 µg/kg, respectivamente. A partir de 28 dias após o tratamento, os resultados já se encontravam abaixo desses valores, portanto, de acordo com o método alternativo de determinação de período de carência estabelecido pelo EMEA/CVMP/036/95 Final, definiu-se para a doramectina a 1% o período de carência de 31 dias.

A Ternary Copper (II) Complex with 4-Fluorophenoxyacetic Acid Hydrazide in Combination with Antibiotics Exhibits Positive Synergistic Effect against Salmonella Typhimurium

Salmonella spp. continues to figure prominently in world epidemiological registries as one of the leading causes of bacterial foodborne disease. We characterised 43 Brazilian lineages of Salmonella Typhimurium (ST) strains, characterized drug resistance patterns, tested copper (II) complex as control options, and proposed effective antimicrobial measures. The minimum inhibitory concentration was evaluated for seven antimicrobials, isolated and combined with the copper (II) complex [Cu(4-FH)(phen)(ClO4)2] (4-FH = 4-fluorophenoxyacetic acid hydrazide and phen = 1,10-phenanthroline), known as DRI-12, in planktonic and sessile ST. In parallel, 42 resistance genes were screened (PCR/microarray). All strains were multidrug resistant (MDR). Resistance to carbapenems and polymyxins (86 and 88%, respectively) have drawn attention to the emergence of the problem in Brazil, and resistance is observed also to CIP and CFT (42 and 67%, respectively), the drugs of choice in treatment. Resistance to beta-lactams was associated with the genes blaTEM/blaCTX-M in 39% of the strains. Lower concentrations of DRI-12 (62.7 mg/L, or 100 μM) controlled planktonic and sessile ST in relation to AMP/SUL/TET and AMP/SUL/TET/COL, respectively. The synergistic effect provided by DRI-12 was significant for COL/CFT and COL/AMP in planktonic and sessile ST, respectively, and represents promising alternatives for the control of MDR ST

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 37 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, pCO2, pO2, 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.Keywords: sessile bacteria, urine, simple cystitis, antibiotic resistance