Phenotypic, molecular and genomic characterization of Actinobaculum suis isolated from swine in Brazil.

Urinary tract infections (UTI) are considered one of the most important diseases of sows due to its close relationship with reproductive problems such as reduced litter size, increase in the rate of return to estrous, vulvar discharge, abortion, mastitis and anestrus. Actinobaculum suis is one of the main agents involved in porcine urinary tract infection and is responsible for the most severe and fatal cases in sows. In the present report, 23 A. suis strains isolated from a sow and boars in Brazil were identified by PCR and further characterized by broth microdilution, molecular typing by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and whole-genome sequencing. All strains were sensitive to ceftiofur, linezolid, nitrofurantoin, quinupristin-dalfopristin and vancomycin. Ciprofloxacin, daptomycin, lincomycin, erythromycin and tylosin resistance was observed in 100% of tested strains. Tetracycline and tigecycline also presented high resistance rates (87% and 30.4%, respectively). PFGE with eight different restriction enzymes and three programs did not enable strain characterization; however, all strains were typed by SE-AFLP that clustered strains according to their origin, thus proving an effective tool for A. suis genotyping. Whole-genome sequencing and comparative analysis enabled species differentiation from closely related genus. This is the first report of genomic characterization of A. suis.

Desenvolvimento da reação em cadeia pela polimerase para detecção de Actinobaculum suis e caracterização fenotípica e genotípica dos isolados / Development of polymerase chain reaction for Actinobaculum suis detection and phenotypic and genotypic characterization of isolates

O Actinobaculum suis é um dos principais micro-organismos relacionados a infecções de trato urinário em fêmeas suínas. As características de crescimento deste agente dificultam o isolamento bacteriano tradicional, o que pode tornar a sua prevalência subestimada. Este estudo teve por objetivos desenvolver a reação em cadeia pela polimerase (PCR) para detecção do A. suis, avaliar a sensibilidade e especificidade desta técnica e comparar seu desempenho com o isolamento bacteriano. Além disso, as cepas isoladas foram caracterizadas através do polimorfismo de comprimento de fragmentos amplificados (AFLP) e submetidas à determinação da concentração inibitória mínima para caracterização dos perfis de susceptibilidade antimicrobiana. Foram analisados 45 suabes prepuciais de machos e 192 urinas de fêmeas suínas provenientes de três granjas. Os resultados indicaram que a PCR desenvolvida foi específica para o A. suis e apresentou limiar de detecção entre 1,0 X 101 UF/mL e 1,0 X 102 UFC/mL. A frequência de A. suis encontrada através da PCR foi de 82,2% (37/45) nos suabes prepuciais e de 8,9% (17/192) nas urinas de fêmeas. No que se refere ao isolamento, nenhuma das amostras de urina foi positiva para o agente, enquanto 31,1% (14/45) dos suabes foram positivos. A partir das amostras positivas isoladas dos suabes prepuciais foram selecionadas 20 cepas de A. suis. Os perfis de susceptibilidade entre estas cepas foram semelhantes, no entanto diferiram dos isolados utilizados como controle e provenientes de uma fêmea com infecção urinária. A técnica de PCR foi mais eficiente que o isolamento na identificação de amostras positivas para A. suis. Através do AFLP com uma única enzima foi possível caracterizar todos os isolados e relacionar os dados obtidos com a origem das cepas e o perfil de resistência. Até o presente não há relatos na literatura de caracterização genotípica de A. suis através do AFLP ou detecção do agente através da PCR

Actinobaculum suis is an important agent related to urinary infection in swine females. The growth characteristics of this agent hamper the traditional bacterial isolation, which can make their prevalence underestimated. The purpose of this study was to develop the polymerase chain reaction (PCR) for Actinobaculum suis detection, to evaluate the sensitivity and specificity of this technique and compare the results with bacterial isolation. Moreover, the isolates were characterized by amplified fragment length polymorphism (AFLP) and subjected to determination of minimum inhibitory concentration for characterization of the antimicrobial susceptibility profiles. Forty-five preputial swabs from boars and a hundred and ninety-two urine samples from sows of three herds were analyzed. The results indicate that the developed PCR was specific for A. suis, presenting a limit detection between 1.0 X 101 UFC/ml and 1.0 X 102 UFC/ml. A.suis frequency by PCR was 82.2% (37/45) in male preputial swabs and 8.9% (17/192) in females urine. Through traditional isolation, none of the urine samples were positive, while A.suis growing was observed in 31.1% (14/45) of the swabs. From the positive samples of the preputial swabs were selected 20 A.suis strains. The susceptibility profiles among these strains were similar, but differed from the female isolates used as control. The PCR technique was more effective than isolation for the A.suis detection. The AFLP with a single enzyme was able to characterize all isolates and relate the data obtained with the strains origin and resistance profile. Until present, there are no reports of genotypic characterization of A. suis strains through AFLP or agent detection by PCR

Aspectos físicos químicos e microbiológicos da urina de matrizes suínas descartadas

The present study describes the physical-chemical alterations of the urine and identifies etiological agents of the urine of 35 sows. Out of 35 samples, 16 (45.7%) were positive for nitrite and/or blood, positive for urinary infection (UI); eight (50%) were dark yellow and six (37.5%) had clear yellow coloration; in two (12.5%) samples did not show any alteration. Ten samples had ammonia smell, seven among them were positive for UI. Other findings were turbid urine and turbid with grumes, normal pH values (5.5 - 6.5) and densities higher than 1012. Microorganisms were isolated in 91.5% out of 35 urine samples, with prevalence of Escherichia coli (45.7%). In 11 samples (31.4%) Actinobaculum suis was isolated, four were simultaneously positive for UI and Actinobaculum suis; seven were negative for UI, but it was observed the presence of Actinobaculum suis in the indirect immunofluorescence.

O presente estudo objetivou descrever as alterações físico-químicas da urina, identificando os agentes etiológicos presentes na urina de matrizes descartadas sem causa definida. De 35 amostras examinadas, 16 (45,7%) foram positivas para presença de sangue e/ou nitrito (positivo para infecção urinária, IU); 8 (50%) tiveram coloração amarela escura e 6 (37,5%) coloração amarela clara; 2 (12,5%) amostras não apresentavam alteração. Dez amostras tinham odor amoniacal, das quais 7 foram positivas para IU. Também houve prevalência de urinas turvas e turvas com grumos, bem como de pH normal (5,5 - 6,5) e densidades maiores que 1,012. Presença de microrganismos foi verificada em 91,5% das 35 amostras examinadas, sendo a Escherichia coli (45,7%), a bactéria com maior prevalência. Em 11 amostras (31,4%), verificou-se a presença de Actinobaculum suis; destas, 4 foram simultaneamente positivas para IU e Actinobaculum suis; 7 foram negativas para IU, observando-se porém, a presença de Actinobaculum suis pelo exame de imunofluorescência indireta.

Aspectos físicos químicos e microbiológicos da urina de matrizes suínas descartadas

The present study describes the physical-chemical alterations of the urine and identifies etiological agents of the urine of 35 sows. Out of 35 samples, 16 (45.7%) were positive for nitrite and/or blood, positive for urinary infection (UI); eight (50%) were dark yellow and six (37.5%) had clear yellow coloration; in two (12.5%) samples did not show any alteration. Ten samples had ammonia smell, seven among them were positive for UI. Other findings were turbid urine and turbid with grumes, normal pH values (5.5 - 6.5) and densities higher than 1012. Microorganisms were isolated in 91.5% out of 35 urine samples, with prevalence of Escherichia coli (45.7%). In 11 samples (31.4%) Actinobaculum suis was isolated, four were simultaneously positive for UI and Actinobaculum suis; seven were negative for UI, but it was observed the presence of Actinobaculum suis in the indirect immunofluorescence.

O presente estudo objetivou descrever as alterações físico-químicas da urina, identificando os agentes etiológicos presentes na urina de matrizes descartadas sem causa definida. De 35 amostras examinadas, 16 (45,7%) foram positivas para presença de sangue e/ou nitrito (positivo para infecção urinária, IU); 8 (50%) tiveram coloração amarela escura e 6 (37,5%) coloração amarela clara; 2 (12,5%) amostras não apresentavam alteração. Dez amostras tinham odor amoniacal, das quais 7 foram positivas para IU. Também houve prevalência de urinas turvas e turvas com grumos, bem como de pH normal (5,5 - 6,5) e densidades maiores que 1,012. Presença de microrganismos foi verificada em 91,5% das 35 amostras examinadas, sendo a Escherichia coli (45,7%), a bactéria com maior prevalência. Em 11 amostras (31,4%), verificou-se a presença de Actinobaculum suis; destas, 4 foram simultaneamente positivas para IU e Actinobaculum suis; 7 foram negativas para IU, observando-se porém, a presença de Actinobaculum suis pelo exame de imunofluorescência indireta.