Colistin resistance profiles and genotypes of Escherichia coli isolates from dogs and cats

Background: Nowadays, antibiotic resistance has become an important problem, posing a serious threat to both human and animal medicine. Colistin is one of the last-resort drugs for the treatment of particularly caused by multidrug resistant bacteria. The aim of this study was to investigate the resistance of Escherichia coli strains against colistin and the presence of colistin resistance genes (mcr1, mcr2 and mcr3) in them. Antibiotyping and genotyping of all strains was also aimed. Materials, Methods & Results: A total of 75 isolates of Escherichia coli from healthy animals (38 dogs and 37 cats) were screened for colistin resistance by cultivation in a screening agar and then microbroth dilution method was performed. Antibiotic susceptibilities of the isolates were determined by KBDDM. The presences of mcr1, mcr2 and mcr3 genes were investigated by PCR. The colistin resistant strains were genotyped by using RAPD-PCR, and antibiotyped based on resistance profiles. In the screening test, 1 strain in cats and 2 strains in dogs were colistin-resistant. However, 18.6% of strains (from 14 cats and 3 dogs) were found as colistin-resistant in the microdilution test. MDR status was 76.31% and 97.29% in dog and cat strains, respectively. The colistin-resistant strains showed 78-100% and 65-90% similarities with respect to their antibiotypes and genotypes, respectively. mcr1, mcr2 and mcr3 genes were not found in any of the strains. Discussion: There is an increase in infections brought on by Gram negative bacteria with various antibiotic resistances in addition to infections brought on by bacteria that are antibiotic-resistant. In order to cure illnesses caused by resistant bacteria, the repurposing of outdated antibiotics may be on the table. Colistin is a crucial antibiotic in veterinary medicine, according to a number of published perspectives, although it should only be administered with caution. However, the discovery of the plasmid-derived mcr1 gene and subsequent reports that this gene has propagated around the world. Escherichia coli strains isolated from companion animals have been found to carry the mcr1 (colistin resistance gene), and possible human-animal cross-contamination has been looked into. The findings demonstrated that mcr1-carrying E. coli might inhabit pets and spread between people and animals. The cat and dog strains used in this investigation had variable colistin resistance rates, which varied between trials. Although no isolates were found to be positive for the mcr1-3 genes in this study, it is believed that colistin resistance, which is determined phenotypically, should not be ignored in terms of spreading both in cat and dog populations as well as in terms of risk to human health, given the possibility that resistance could occur with other different mechanisms. Epidemiological research still uses in vitro antibacterial susceptibility patterns. Our antibiotyping method, which was based on an analysis of several antibiotic resistances, provided quantitative data. Commercial software was utilized to conduct the evaluation. There are no reports or publications that provide quantitative antibiotyping data for E. coli strains in the literature. A popular technique for genotyping different bacterial species is RAPD-PCR. By determining if certain specific genotypes are similar to those of other resistance strains, RAPD-PCR and other genotyping data can be compared with antibiotic resistance profiles to determine the specific risk of treatment resistance in infectious diseases. All organisms that were colistin resistant exhibited multiple antibiotic resistance, and these findings were also related to RAPD genotypes. The findings indicated that colistin-resistant E. coli bacteria could potentially represent a risk to human health and were thought to be transmitted from cats and dogs to humans and vice versa.

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

Antimicrobial and antibiofilm activity of the essential oil from dried leaves of Eucalyptus staigeriana / Atividade antimicrobiana e antibiofilme do óleo essencial de folhas secas de Eucalyptus staigeriana

In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.(AU)

Nos últimos anos, compostos com propriedades biológicas produzidas por plantas têm recebido atenção como alternativa de controle de micro-organismos. Óleos essenciais extraídos de folhas verdes de Eucalyptus sp. têm demonstrado atividades antimicrobianas. No entanto, até o momento não há nenhum relato de atividade antimicrobiana de óleos essenciais extraídos de folhas secas de Eucalyptus staigeriana. O objetivo deste estudo foi determinar a composição química dos óleos essenciais obtidos de folhas secas de E. staigeriana e avaliar in vitro a sua atividade antimicrobiana e de antibiofilme contra gram-positivas e gram-negativas e também resistentes e multirresistentes de Enterococcus faecalis isolados de amostras de alimentos e clínicas. A caracterização de E. staigeriana foi realizada por CG-EM. Para este estudo foram utilizadas 26 cepas bacterianas, que incluíram 11 cepas referência e 15 cepas de E. faecalis resistentes a antibióticos. A atividade antimicrobiana de E. staigeriana contra gram-positivas e gram-negativas foi determinada utilizando o método de disco-difusão. Os valores da concentração inibitória mínima foram avaliados pela técnica de microdiluição. Os efeitos de antibiofilme foram avaliados pelo método de placa de microtitulação. Como resultado, 21 compostos foram identificados, sendo monoterpenos oxigenados (69,58%) a grande família química. E. staigeriana mostrou apenas atividade antimicrobiana contra cepas gram-positivas. Cepas de E. faecalis resistentes e multirresistentes mostraram a menor concentração inibitória mínima (3,12 para 6,25%) quando comparado com a cepa referência de E. faecalis. E. staigeriana apresentou a capacidade de inibir a formação de biofilme, mas pouca ou nenhuma capacidade de inibir o biofilme pré-formado. Este estudo demonstra que o óleo essencial obtido de folhas secas de E. staigeriana é uma alternativa promissora para controle importante de bactérias gram-positivas resistentes de origem alimentar e clínicas.(AU)

Antimicrobial and antibiofilm activity of the essential oil from dried leaves of Eucalyptus staigeriana / Atividade antimicrobiana e antibiofilme do óleo essencial de folhas secas de Eucalyptus staigeriana

In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.(AU)

Nos últimos anos, compostos com propriedades biológicas produzidas por plantas têm recebido atenção como alternativa de controle de micro-organismos. Óleos essenciais extraídos de folhas verdes de Eucalyptus sp. têm demonstrado atividades antimicrobianas. No entanto, até o momento não há nenhum relato de atividade antimicrobiana de óleos essenciais extraídos de folhas secas de Eucalyptus staigeriana. O objetivo deste estudo foi determinar a composição química dos óleos essenciais obtidos de folhas secas de E. staigeriana e avaliar in vitro a sua atividade antimicrobiana e de antibiofilme contra gram-positivas e gram-negativas e também resistentes e multirresistentes de Enterococcus faecalis isolados de amostras de alimentos e clínicas. A caracterização de E. staigeriana foi realizada por CG-EM. Para este estudo foram utilizadas 26 cepas bacterianas, que incluíram 11 cepas referência e 15 cepas de E. faecalis resistentes a antibióticos. A atividade antimicrobiana de E. staigeriana contra gram-positivas e gram-negativas foi determinada utilizando o método de disco-difusão. Os valores da concentração inibitória mínima foram avaliados pela técnica de microdiluição. Os efeitos de antibiofilme foram avaliados pelo método de placa de microtitulação. Como resultado, 21 compostos foram identificados, sendo monoterpenos oxigenados (69,58%) a grande família química. E. staigeriana mostrou apenas atividade antimicrobiana contra cepas gram-positivas. Cepas de E. faecalis resistentes e multirresistentes mostraram a menor concentração inibitória mínima (3,12 para 6,25%) quando comparado com a cepa referência de E. faecalis. E. staigeriana apresentou a capacidade de inibir a formação de biofilme, mas pouca ou nenhuma capacidade de inibir o biofilme pré-formado. Este estudo demonstra que o óleo essencial obtido de folhas secas de E. staigeriana é uma alternativa promissora para controle importante de bactérias gram-positivas resistentes de origem alimentar e clínicas.(AU)

Antifungal activity against Cryptococcus neoformans strains and genotoxicity assessment in human leukocyte cells of Euphorbia tirucalli L

In the last times, focus on plant research has increased all over the world. Euphorbia tirucalli L., a plant known popularly as Aveloz, and originally used in Africa, has been drawing attention for its use in the United States and Latin America, both for use as an ornamental plant and as a medicinal plant. E. tirucalli L. is a member of the family Euphorbiaceae and contains many diterpenoids and triterpenoids, in particular phorbol esters, apparently the main constituent of this plant, which are assumed to be responsible for their activities in vivo and in vitro. The in vitro antifungal activities of Euphorbia tirucalli (L.) against opportunistic yeasts were studied using microbroth dilution assay. The results showed that aqueous extract and latex preparation were effective against ten clinical strains of Cryptococcus neoformans in vitro (Latex and extract MIC range of 3.2 - > 411 µg/mL). Aiming the safe use in humans, the genotoxic effects of E. tirucalli were evaluated in human leukocytes cells. Our data show that both aqueous extract and latex preparation have no genotoxic effect in human leukocytes cells in vitro. Although the results cannot be extrapolated by itself for use in vivo, they suggest a good perspective for a therapeutic application in future. In conclusion, our results show that the aqueous extract and latex preparation from E. tirucalli L. are antifungal agents effectives against several strains of C. neoformans and do not provoke DNA damage in human leukocyte cells, considering the concentrations tested.

PERFIL DE SUSCETIBILIDADE A ANTIMICROBIANOS DE ISOLADOS HISTÓRICOS E CONTEMPORÂNEOS DE Pasteurella multocida DE SUÍNOS

O primeiro estudo desenvolvido teve como objetivo determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. A concentração inibitória mínima (CIM) de quatro antimicrobianos foi calculada para os isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) pelo teste de microdiluição em caldo segundo recomendações do CLSI. A menor frequência de isolados resistentes, tanto históricos quanto contemporâneos, foi observada para o florfenicol (0% e 6%, respectivamente), enquanto que a maior frequência foi observada para a tetraciclina (20,5% e 34%, respectivamente). Houve aumento do número de isolados resistentes para todos os antimicrobianos testados, devido, provavelmente, à pressão de seleção pelo uso de antimicrobianos para tratamento e prevenção de infecções por P. multocida. Em relação aos perfis de suscetibilidade, os isolados históricos e contemporâneos da P. multocida foram estatisticamente diferentes (p0.05) apenas para amoxicilina e enrofloxacina, que tiveram um aumento na frequência de isolados resistentes de 3,8% e 18%, respectivamente. Um segundo estudo foi desenvolvido com o objetivo de comparar os métodos de tipificação capsular dos sorotipos A e D de isolados de P. multocida de suínos utilizando técnicas fenotípicas (hialuronidase e acriflavina) e genotípica (PCR multiplex). Foram utilizados 44 isolados liofilizados de P. multocida de suínos do Rio Grande do Sul obtidos entre os anos de 1981 e 1997. Pelos testes fenotípicos, dois isolados foram classificados como tipo D (4,55%), 40 isolados como tipo A (90,9%) e dois isolados não foi tipificado (4,55%). No entanto, pelo teste genotípico, 38 isolados foram classificados como tipo A (86,36%) e 6 como tipo D (13,64%). Os resultados mostraram que, em alguns casos, não existe equivalência entre a tipificação fenotípica e genotípica de P. multocida, neste estudo diferindo em 4/44 isolados (9,09%). Foi concluído que a tipificação capsular de P. multocida pela PCR multiplex é mais eficiente e confiável que pelos métodos fenotípicos.

The first study was carried out to determine and to compare antimicrobial susceptibility profile of P. multocida isolated from pigs with lesions of pneumonia or pleuritis in Brazil during two time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were use to assessed the MIC of four antimicrobial agents by microbroth dilution following CLSI recommendations. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Resistance increased in recent isolates for all antimicrobial tested, most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. Regarding to susceptibility profiles, historical and recent P. multocida isolates were statistically different (p0.05) only for amoxicillin and enrofloxacin, wich had an increase in the resistance of 3.8% and 18%, respectively. The second study was performed to compare the capsular identification methods of type A and type D P. multocida isolates from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques. A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State were analyzed. Phenotypic tests showed that two isolates were type D (4.55%), 40 were type A (90.9%) and two (4.55%) were untypable isolates (4.55%), while PCR showed that 38 isolates were type A (86.36%) and six were type D (13.64%). The results show that, in same cases, there is no equivalence between phenotypic and genotypic typification of P. multocida, in this study differing in 4/44 isolates (9.09%). We conclude that the capsular identification of P. multocida by multiplex PCR is more efficient and reliable compared to the phenotypic methods.

In vitro antibacterial activities of ethanol extract of iranian propolis (EEIP) against fish pathogenic bacteria (Aeromonas hydrophila, Yersinia ruckeri & Streptococcus iniae)

The ''in vitro'' antibacterial activity of ethanol extract of propolis (EEIP) from Urmia, Iran was investigated against three prevalent species of fish bacterial pathogens including: Aeromonas hydrophila LMG 3770, Yersinia ruckeri LMG 3279 and Streptococcus iniae LMG 14520. In this study two standard susceptibility testing techniques (Micro-broth dilution method and Agar-well diffusion method) were used to evaluation of the antibacterial activity of EEIP against the mentioned micro-organisms. Also the chemical composition of propolis was determined by the method of Gas chromatography-mass spectrometry (GC-MS). Twenty-six compounds were identified by gas chromatography-mass spectrometry analysis. Results showed Chemical composition of EEIP contained significant amounts of flavonoids, Sesquiterpenes - mainly Eudesmol and Caryophyllene oxide - aromatic acid, and low amounts of aldehydes and triterpens. Furthermore the ethanol extract of propolis inhibited the growth of all examined micro-organisms with the highest antimicrobial activity against Gram-positive bacteria Streptococcus iniae. Ethanol did not influence the antimicrobial effect of EEIP. These antibacterial properties would warrant further studies on the clinical applications of propolis in aquaculture field.