Identification and characterization of pathogenic and multidrug-resistant bacteria in feral pigeons surrounding a veterinary hospital in Minas Gerais, Brazil / Identificação de bactérias patogênicas e multirresistentes a antimicrobianos em pombos urbanos no entorno de um Hospital Veterinário em Belo Horizonte, Minas Gerais, Brasil

Pigeons are known for their capacity to harbor and spread several zoonotic agents. Studies have suggested that pigeons are also relevant disseminators of multidrug-resistant strains. In this study, pigeons surrounding a veterinary hospital were sampled and tested for the presence of pathogenic Escherichia coli, Salmonella spp., Staphylococcus spp., and Clostridioides (Clostridium) difficile. E. coli isolates from 19 (40.4%) pigeons tested positive for the E. coli heat-stable enterotoxin 1 (EAST1)-encoding gene. The intimin-encoding gene (eae) of enteropathogenicE. coli (EPEC) was found in one isolate (2.1%). Salmonella spp. were found in nine (19.1%) pigeons, all from the first capture event (P < 000.1). S. Typhimurium and S. Heidelberg were isolated from six and three pigeons, respectively. Enterobacterial repetitive intergenic consensus (ERIC-PCR) of the Salmonella spp. isolates suggested that eight of the nine strains had a high genetic similarity, supporting the hypothesis of an outbreak of salmonellosis in these pigeons. Twenty (42.5%) staphylococcal isolates were recovered from 18 (38.3%) pigeons. Eight different species were detected, with S. xylosus being the most frequent. Two (4.3%) C. difficile strains were isolated. Three isolates, one each of S. Typhimurium, S. aureus, and C. difficile, were classified as multidrug-resistant strains. The present research suggested that pigeons residing in urban areas can act as reservoirs and disseminators of pathogenic bacteria, including nosocomial pathogens, such as diarrheagenicE. coli and multidrug-resistant Staphylococcus spp., C. difficile, and Salmonella spp.

Pombos urbanos são conhecidos pela sua capacidade de carrear e disseminar diversos agentes zoonóticos. Estudos tem sugerido que pombos são também relevantes na disseminação de estirpes resistentes a múltiplas drogas. No presente estudo, pombos no ambiente de um hospital veterinário foram amostrados em três diferentes períodos e testados para a presença de Escherichia coli patogênica, Salmonella spp., Staphylococcus spp. e Clostridioides (Clostridium) difficile. Isolados de E. coli de 19 pombos (40.4%) foram positivos para o gene codificador da toxina EAST1. O gene codificador de intimina (eae) do patotipo E. coli enteropatogênica foi encontrada em um isolado (2.1%). Salmonella spp. foi encontrada em nove pombos (19.1%), sendo todos isolados do primeiro período de captura (P < 000.1). S. Typhimurium foi isolado de seis animais e S. Heidelberg de três. A tipagem molecular de isolados de Salmonella spp. por ERIC-PCR demonstrou que oito estirpes possuíam alta similaridade genética entre si, sugerindo a ocorrência de um surto de salmonelose nos animais carreadores. Vinte Staphylococcus (42.5%) foram isolados de 18 animais (38.3%). Oito diferentes espécies foram detectadas, sendo S. xylosus a mais frequente. Duas estirpes de C. difficile não-toxigênica (4.3%) foram isoladas. Uma estirpe de S. Typhimurium, uma de S. aureus e um isolado de C. difficile foram classificados como resistentes a múltiplas drogas antimicrobianas. O presente estudo sugere que pombos capturados no ambiente do hospital veterinário podem atuar como reservatórios e disseminadores de bactérias patogênicas e envolvidas em infecção hospitalar, incluindo E. coli diarreiogênica e Staphylococcus sp., C. difficile e Salmonella spp multirresistente.

Molecular characterization of Corynebacterium pseudotuberculosis, C. silvaticum, and C. auriscanis by ERIC-PCR / Caracterização molecular de Corynebacterium pseudotuberculosis, C. silvaticum e C. auriscanis pelo ERIC-PCR

The aims of the present study were (i) to genotype Corynebacterium pseudotuberculosis, C. silvaticum, and C. auriscanis strains using enterobacterial repetitive intergenic consensus (ERIC-PCR), and (ii) to analyze the epidemiological relationships among isolates according to biovar (Equi and Ovis), species, host, and geographical origin of the C. pseudotuberculosis strains. Sixty-eight C. pseudotuberculosis, nine C. silvaticum, and one C. auriscanis, C. pseudotuberculosis ATCC® 19410™ strain and the attenuated C. pseudotuberculosis 1002 vaccinal strain were fingerprinted by ERIC 1+2-PCR. Field strains were isolated from various hosts (cattle, buffaloes, sheep, goats, horses, dogs, and pigs) in six countries (Mexico, Portugal, Brazil, Equatorial Guinea, Egypt, and Israel). High genetic diversity was found among the studied Corynebacterium spp. isolates, clustering in 24 genotypes with a Hunter & Gaston diversity index (HGDI) of 0.937. The minimal spanning tree of Corynebacterium spp. revealed three clonal complexes, each associated with one bacterial species. Twenty-two genotypes were observed among C. pseudotuberculosis isolates, with an HGDI of 0.934. Three major clonal complexes were formed at the minimal spanning tree, grouped around the geographic origin of C. pseudotuberculosis isolates. These results reinforce the high typeability, epidemiological concordance, and discriminatory power of ERIC-PCR as a consistent genotyping method for C. pseudotuberculosis, which could be useful as an epidemiological tool to control caseous lymphadenitis. Moreover, our results also indicate the potential of ERIC 1+2-PCR for the genotyping of other species of Corynebacterium other than C. pseudotuberculosis.

Os objetivos do presente estudo foram (i) genotipar amostras de Corynebacterium pseudotuberculosis, C. silvaticum e C. auriscanis usando Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR), bem como (ii) analisar as relações epidemiológicas entre os isolados de acordo com biovar (Equi e Ovis), espécie, hospedeiro e origem geográfica das amostras de C. pseudotuberculosis. Sessenta e oito isolados de C. pseudotuberculosis, nove C. silvaticum, um C. auriscanis, C. pseudotuberculosis ATCC® 19410 ™ e a amostra vacinal atenuada C. pseudotuberculosis 1002 foram tipificadas por ERIC 1 + 2-PCR. As amostras de campo foram isoladas de diferentes hospedeiros (bovinos, búfalos, ovinos, caprinos, equinos, cães e suínos) em seis países (México, Portugal, Brasil, Guiné Equatorial, Egito e Israel). Uma alta diversidade genética foi observada entre os isolados de Corynebacterium spp., agrupados em vinte e quatro genótipos com um índice de diversidade Hunter & Gaston (HGDI) de 0,937. A análise da minimal spanning tree (MST) de Corynebacterium spp. revelou três complexos clonais, cada um associado a uma espécie bacteriana. Vinte e dois genótipos foram observados entre isolados de C. pseudotuberculosis, com um HGDI de 0,934. Na análise da MST, três grandes complexos clonais foram formados, agrupando-se em torno da origem geográfica dos isolados de C. pseudotuberculosis. Esses resultados reforçam a alta tipabilidade, concordância epidemiológica e poder discriminatório do ERIC-PCR como método consistente de genotipagem para C. pseudotuberculosis, podendo ser útil como ferramenta epidemiológica no controle da linfadenite caseosa. Além disso, os resultados também indicam o grande potencial de ERIC 1 + 2-PCR para genotipagem de espécies do gênero Corynebacterium além de C. pseudotuberculosis.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey

ABSTRACT: Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

RESUMO: Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey / Caracterização genômica perfis de resistência antimicrobiana, produção de enterotoxinas e biofilme Staphylococcus aureus resistente à meticilina isolado de origem clínica e de produtos de origem animal no leste da Turquia

Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Outbreak of multidrug resistant Salmonella Typhimurium in calves at a veterinary hospital in Brazil / Surto de Salmonella Typhimurium multirresistante em bezerros em um hospital veterinário do Brasil

The present study aimed to describe and characterize a nosocomial outbreak caused by a multidrug resistant Salmonella Typhimurium in hospitalized calves at a veterinary medical teaching hospital from Brazil. Sixty-three (96.9%) calves showed lethargy, hyperthermia and profuse diarrhea and despite treatment, 26 (41.2%) animals died. Five animals were necropsied and stool samples of six calves were collected. The isolated strains were subjected to antimicrobial susceptibility test by disc-difusion method and were fingerprinted by ERIC-PCR. Macroscopic lesions suggestive of salmonellosis, such as fibrinonecrotic enteritis and hepatosplenomegaly were observed. Salmonellosis was confirmed by isolation of S. Typhimurium from stool samples and organs from seven affected animals. Six out of seven isolates of S. Typhimurium, exhibited 100% of similarity at ERIC-PCR, suggesting occurrence of nosocomial transmission of S. Typhimurium among the hospitalized calves. All but one S. Typhimurium isolated were resistant to marbofloxacin, enrofloxacin, florfenicol, oxytetracycline and trimethoprim/sulfamethoxazole, antimicrobial agents largely used for humans and animal treatment. This is the first study of a nosocomial outbreak of multidrug resistant S. Typhimurium in a veterinary hospital in Brazil and highlighted the need for preventive measures to reduce the risks for inpatients and humans in contact with animals.(AU)

O objetivo do presente estudo é descrever e caracterizar um surto nosocomial provocado por S. Typhimurium multirresistente em bezerros hospitalizados em um hospital escola de medicina veteriária localizado no Brasil. Sessenta e três (96,9%) bezerros apresentaram letargia, hipertermia e diarreia profusa e, apesar do tratamento, vinte e seis animais (41,2%) morreram. Cinco animais foram necropsiados e amostras fecais de seis bezerros foram coletadas. As estirpes isoladas foram submetidas a testes de susceptibilidade a antimicrobianos pelo método de disco-difusão e foram genotipadas pelo ERIC-PCR. Lesões macroscópicas sugestivas de salmonelose, como enterite fibrinonecrótica e hepatoesplenomegalia, foram observadas. Salmonelose foi confirmada pelo isolamento de S. Typhimurium em amostras fecais e órgãos de sete animais. Dos sete isolados, seis apresentaram 100% de similaridade ao ERIC-PCR, sugerindo ocorrẽncia de transmissão nosocomial de S. Typhimurium entre os bezerros hospitalizados. Com excessão de uma estirpe, todas foram resistentes a marbofloxacina, enrofloxacina, florfenicol, oxitetraciclina e trimetoprima/sulfametoxazol, agentes antimicrobianos amplamente utilizados para o tratamento humano e animal. Esse é o primeiro estudo que demonstra um surto nosocomial de estirpes de S. Typhimurium resistentes a múltiplas drogas em um hospital veterinário no Brasil, enfatizando a necessidade de medidas preventivas que reduzam os riscos aos animais hospitalizados e a pessoas que entrarem em contato com esses animais.(AU)

Antimicrobial susceptibility and molecular characterization of Salmonella serovar Ndolo isolated from outbreaks in cattle and horses / Susceptibilidade antimicrobiana e caracterização molecula de isolados de Salmonella sorovar Ndolo de bovinos e equinos

The present study aimed to describe and characterize, for the first time, two outbreaks of salmonellosis caused by Salmonella Ndolo in foals and calves in Brazil and compare the isolated strains with S. Ndolo previously identified in asymptomatic reptiles. The affected calves and foals presented fever, lethargy, and profuse diarrhea. Isolated strains were subjected to antimicrobial susceptibility testing, characterized according to virulence genes, and fingerprinted by ERIC-PCR. Salmonella Ndolo was identified in fecal samples from two foals and four calves. One isolate from a calf was resistant to amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, and florfenicol. Strains from two other calves were resistant to oxytetracycline. All virulence genes tested were present in the isolates, and two major clusters of closely related strains were identified by ERIC-PCR, each per outbreak. This is the first report of Salmonella Ndolo infection in domestic and symptomatic animals. Previously, this serovar had been identified only in human infections. The presence of relevant virulence genes in all Salmonella Ndolo isolates and the detection of antimicrobial multi-resistant strains highlighted the importance of monitoring serovars associated with salmonellosis in domestic animals.(AU)

O objetivo do presente estudo foi descrever e caracterizar, pela primeira vez, dois surtos de salmonelose causados por Salmonella Ndolo em potros e bezerros do Brasil e comparar esses isolados com Salmonella Ndolo previamente identificada em répteis assintomáticos. Os animais infectados apresentaram febre, letargia e diarreia profusa. Os isolados foram submetidos a testes de susceptibilidade a antimicrobianos e foram caracterizados conforme a presença de genes de virulência e diversidade genética, utilizando-se o ERIC-PCR. Salmonella Ndolo foi identificado em amostras fecais de dois potros e quatro bezerros. Um isolado de bezerro foi resistente a amoxicilina/ácido clavulanico, trimethoprima/sulfametoxazol e florfenicol. Estirpes de dois outros bezerros foram resistentes a oxitetraciclina. Todos os genes de virulência testados foram identificados nos isolados e dois grandes grupos de estirpes geneticamente relacionadas foram identificados pelo ERIC-PCR, um para cada surto. Esse é o primeiro relato de Salmonella Ndolo em animais domésticos e sintomáticos. Previamente, esse sorovar foi identificado apenas em infecções humanas. A presença de fatores de virulência relevantes em todos os isolados e a detecção de estirpes multirresistentes a antimicrobianos destaca a importância do monitoramento de sorovares associados a salmonelose em animais domésticos.(AU)

Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks

Background: Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran. Methods and results: Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates. Conclusion: In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.(AU)

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.(AU)

Detection and analysis of different interactions between resistance mechanisms and carbapenems in clinical isolates of Klebsiella pneumoniae

Carbapenems are considered last-line agents for the treatment of serious infections caused by Klebsiella pneumoniae, and this microorganism may exhibit resistance to -lactam antibiotics due to different mechanisms of resistance. We evaluated 27 isolates of K. pneumoniae resistant to carbapenems recovered from inpatients at the University Hospital of Santa Maria-RS from July 2013 to August 2014. We carried out antimicrobial susceptibility, carbapenemase detection, testing for the presence of efflux pump by broth microdilution and loss of porin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Genetic similarity was evaluated by ERIC-PCR. High levels of resistance were verified by the minimum inhibitory concentration for the antimicrobials tested. The blaKPC gene was present in 89% of the clinical isolates. Blue-Carba and combined disk with AFB tests showed 100% concordance, while the combined disk test with EDTA showed a high number of false-positives (48%) compared with the gold-standard genotypic test. Four isolates showed a phenotypic resistance profile consistent with the overexpression of the efflux pump, and all clinical isolates had lost one or both porins. The ERIC-PCR dendrogram demonstrated the presence of nine clusters. The main mechanism of resistance to carbapenems found in the assessed isolates was the presence of the blaKPC gene.(AU)

Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR

PURPOSE:To determine the genetic diversity of MDR P. aeruginosa strains isolated from burn and wound infections in Ahvaz, Iran, by ERIC-PCR.METHODS:From total 99 strains of P. aeruginosa defined as MDR by using drug susceptibility testing, 66 were subjected to ERIC-PCR analysis, comprises 53 strains isolated from burn infection, and 13 randomly selected strains from wound infection with higher resistance to combinations of more numbers of drugs.RESULTS:Eight clusters (I to VIII), and 50 single clones were generated for tested MDR isolates analyzed by ERIC-PCR. The high heterogeneity was observed among the isolates from burn infections including 16 isolates which were categorized in eight clusters and 37 single clones. The isolates in clusters II, III, VI, VIII showed 100% similarity.CONCLUSIONS:The high level of genotypic heterogeneity in P. aeruginosa strains demonstrated no genetic correlation between them. Extremely high drug resistance in isolates from burn, suggests that efficient control measures and proper antibiotic policy should be observed.(AU)

Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water

Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacE1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacE 1 gene at the 3 conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans.(AU)

SALMONELOSE EM REBANHO LEITEIRO BOVINO CONFINADO: Caracterização dos isolados, aspectos clínicos, epidemiológicos e resistência antimicrobiana

A salmonelose dos bovinos é uma doença primariamente entérica que pode evoluir para forma sistêmicas. É causada por bactérias gram negativas do filo das proteobactérias e família Enterobacteriacea. Os indivíduos de maior interesse são da espécie Salmonella enterica subp. enterica e é composta por 2.600 sorovariedades. A doença acomete animais de diferentes idades e é mais grave em animais jovens. O objetivo do trabalho foi estudar a distribuição da Salmonella spp. em um rebanho portador nas diferentes categorias animais, caracterizando a sua sorovariedade e o perfil de resistência antimicrobiana. O experimento foi conduzido em propriedade bovina que utiliza sistema de confinamento para criação dos animais e que possui produção diária de 29.000 litros de leite. Foram coletadas amostras de fezes de 782 animais em três momentos de cada animal e distribuídos em todas as categorias da propriedade e seguido de cultura e isolamento de Salmonella spp. com posterior avaliação dos sorotipos presentes, perfil de similaridade pelo ERIC-PCR e o perfil de resistência antimicrobiana de todos os isolados. Foram encontrados 60 isolados de Salmonella spp. sendo que 72% destes eram de S. Dublin e apresentavam grande semelhança genética entre si (ERIC-PCR). Foi observado que 85% dos isolados de Salmonella spp. eram multirresistentes, circulam pelas categorias animais da propriedade e causam doença clínica nos animais jovens

Bovine salmonellosis is a primarily enteric disease that can evolve into systemic forms. It is caused by gram negative bacteria from the phylum of the proteobacteria and Enterobacteriacea family. The individuals of greatest interest are the species Salmonella enterica subp. enterica and is composed of 2,600 serovarieties. The disease affects animals of different ages and is more severe in young animals. The objective of the work was to study the distribution of Salmonella spp. in a dairy herd with different animal categories, characterizing its serovariety and antimicrobial resistance profile. The experiment was carried out on a property that uses a confinement system to raise the animals and has a daily production of 29,000 liters of milk. Stool samples were collected from 782 animals at three times of each animal every 48h and distributed in all categories of the property and followed by culture and isolation of Salmonella spp. with subsequent evaluation of the serotypes present, similarity profile by ERIC-PCR and the antimicrobial resistance profile of all isolates. In 60 isolates of Salmonella spp., 72% of them were from S. Dublin and had great genetic similarity to each other. It was observed that 85% of Salmonella spp. they were multidrug-resistant, circulate in the animal categories of the property and cause clinical disease in young animals

Development and application of an ERIC-PCR method for genotyping and differentiating Actinobacillus pleuropneumoniae isolates

Background: The pleuropneumonia caused by Actinobacillus pleuropneumoniae is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for the diagnosis and characterization of this bacterium. However, in some isolates, confl icting results are found. There are at least 15 serotypes with signifi cant differencesin virulence that have been identifi ed until now. Moreover, cross reactions between serotypes are not uncommon. Theserotype determination from isolates occurring in outbreaks is an important procedure in prophylaxis and control of thedisease. The present work focuses on the application of an ERIC-PCR technique for genotyping and differentiation ofA. pleuropneumoniae isolates.Materials, Methods & Results: Fifteen reference strains for the recognized A. pleuropneumoniae serotypes were analyzedin this work, alongside with 27 fi eld isolates that had been previously characterized regarding biochemical, serological andmolecular features. Total DNA from each sample was purifi ed and subjected to PCR amplifi cation using ERIC-specifi cprimers (ERIC1R and ERIC2). The resulting amplicons were analyzed by agarose gel electrophoresis and their sizes wereestimated from the gel images. Bands with similar sizes were identifi ed and used to construct a binary matrix that tookinto account the presence or absence of individual bands in all lanes. Pair-wise similarity coeffi cients were computed fromthe binary matrix and the similarity matrices obtained were utilized to construct an UPGMA-based dendrogram. The amplicons obtained from the A. pleuropneumoniae reference strains generated a very distinctive pattern for each one of thetested strains. Moreover, all samples presented a large enough number of amplicons (bands) as to enable an unequivocaldifferentiation of each sample. Reproducibility of the developed ERIC-PCR method was assessed by means of duplicate...(AU)

Development and application of an ERIC-PCR method for genotyping and differentiating Actinobacillus pleuropneumoniae isolates

Background: The pleuropneumonia caused by Actinobacillus pleuropneumoniae is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for the diagnosis and characterization of this bacterium. However, in some isolates, confl icting results are found. There are at least 15 serotypes with signifi cant differencesin virulence that have been identifi ed until now. Moreover, cross reactions between serotypes are not uncommon. Theserotype determination from isolates occurring in outbreaks is an important procedure in prophylaxis and control of thedisease. The present work focuses on the application of an ERIC-PCR technique for genotyping and differentiation ofA. pleuropneumoniae isolates.Materials, Methods & Results: Fifteen reference strains for the recognized A. pleuropneumoniae serotypes were analyzedin this work, alongside with 27 fi eld isolates that had been previously characterized regarding biochemical, serological andmolecular features. Total DNA from each sample was purifi ed and subjected to PCR amplifi cation using ERIC-specifi cprimers (ERIC1R and ERIC2). The resulting amplicons were analyzed by agarose gel electrophoresis and their sizes wereestimated from the gel images. Bands with similar sizes were identifi ed and used to construct a binary matrix that tookinto account the presence or absence of individual bands in all lanes. Pair-wise similarity coeffi cients were computed fromthe binary matrix and the similarity matrices obtained were utilized to construct an UPGMA-based dendrogram. The amplicons obtained from the A. pleuropneumoniae reference strains generated a very distinctive pattern for each one of thetested strains. Moreover, all samples presented a large enough number of amplicons (bands) as to enable an unequivocaldifferentiation of each sample. Reproducibility of the developed ERIC-PCR method was assessed by means of duplicate...

Molecular epidemiology of Listeria monocytogenes isolated from different sources in Brazil / Epidemiologia molecular de Listeria monocytogenes isoladas de diferentes fontes no Brasil

Listeria monocytogenes is an important foodborne pathogen that primarily affects pregnant women, neonates, the elderly and immune-compromised individuals, and it may cause abortion, septicemia, and meningitis. From the 13 capsular groups described, serotypes 4b, 1/2b and 1/2a are most closely related to human infection. For this reason, serotyping has limited value as an epidemiological tool; thus, improved discriminatory typing methods are required to enhance knowledge of L. monocytogenes contamination and infection. The aim of this study was to characterize the genetic diversity of L. monocytogenes isolates in the pork processing industry in Sao Paulo, Brazil and human infection isolates by ERICPCR and single enzyme AFLP. Serotypes 1/2c and 4b were frequent among isolates from pork and slaughterhouse/market environments, whereas serotypes 4b and 1/2a were observed among human isolates. ERIC-PCR and AFLP revealed 34 and 31 distinct profiles, respectively, which had tendencies of separation according to serogroup and isolate origin. The genetic profiles from slaughterhouse and market environments suggest the possibility of different sources of Listeria contamination in the environment, although in certain cases, continuous contamination caused by the persistence of clonal strains is also a possibility.(AU)

Listeria monocytogenes é um importante patógeno de origem alimentar que afeta principalmente grávidas, neonatos, idosos e indivíduos imunocomprometidos, e pode causar abortamento, septicemia e meningite. Dos 13 grupos capsulares descritos, os sorotipos 4b, 1/2b e 1/2a são os mais relacionados à infecção humana. Por esta razão, a sorotipagem possui valor limitado como ferramenta epidemiológica e, dessa forma, métodos mais discriminatórios são necessários para melhorar o conhecimento sobre a contaminação e a infecção por L. monocytogenes. O objetivo deste estudo foi caracterizar a diversidade genética de isolados de L. monocytogenes da indústria de processamento de carne suína noEstado de São Paulo, Brasil, e compará-los a isolados de casos de infecção humana através do ERIC-PCR e AFLP com uma única enzima. Os sorotipos 1/2c e 4b foram frequentes em carne suína e ambientes de abatedouros e mercados, enquanto os sorotipos 4b e 1/2a foram observados nos isolados de humanos. ERIC-PCR e AFLP resultaram em 34 e 31 perfis distintos, respectivamente, com uma tendência a separar de acordo com o sorogrupo e a origem do isolado. Os perfis genéticos de ambiente dos abatedouros e mercados sugerem a possibilidade de diferentes origens de contaminação por Listeria nos ambientes estudados, porém, em alguns casos, é possível que ocorra a persistência de cepas clonais causando contaminação contínua.(AU)

Patógenos Respiratórios dos Suínos: Diversidade genética de 15 Mycoplasma hyopneumoniae em Minas Gerais e dinâmica de 16 colonização e resposta imune por Haemophilus parasuis

A doença de Glasser, causada pela bactéria Haemophilus parasuis, e a pneumonia enzoótica, causada pelo Mycoplasma hyopneumoniae, são duas das principais enfermidades sistêmicas e respiratórias de suínos na creche, recria e terminação. Os fatores que desencadeiam a doença não foram completamente esclarecidos. Várias estratégias já foram utilizadas para controlar estas doenças, mas nenhuma delas é completamente eficaz. Em relação ao M. hyopneumoniae, o primeiro capítulo teve como objetivo obter um panorama sobre a diversidade genética desse agente relacionado a lesões pneumônicas em suínos de rebanhos das diferentes mesorregiões produtoras do estado de Minas Gerais. Isso foi possível através da genotipagem pela análise de MLVA, uma técnica altamente discriminatória para a espécie. Os resultados mostraram que há alta prevalência de granjas positivas para o agente e um alto polimorfismo das cepas circulantes no estado de Minas Gerais, sem um padrão genotípico específico de determinada região geográfica. Em relação ao H. parasuis, o segundo capítulo objetivou esclarecer, através de um estudo longitudinal em uma granja comercial de ciclo completo, o efeito da associação de diferentes antimicrobianos aplicados no manejo em diferentes fases de criação dos suínos sobre a dinâmica de colonização por H. parasuis e resposta imune humoral contra essa bactéria. Os resultados mostraram que o uso intenso de antimicrobianos na creche pode reduzir a pressão de infecção na granja, mas interfe na colonização pela bactéria e desloca o início da resposta imune ativa para semanas posteriores. Nas condições de campo encontradas nesse estudo, não existiram diferenças significativas no tratamento com enrofloxacina sobre a dinâmica de colonização em leitões clinicamente saudáveis. Em relação à cinética de colonização global dos leitões, nem todos os leitões são expostos ao agente e recebem imunidade materna em uma idade precoce a níveis detectáveis, e a presença de anticorpos maternos parece reduzir a colonização até mesmo por cepas possivelmente comensais

Glassers disease, caused by the bacterium Haemophilus parasuis, e enzootic pneumonia, caused by Mycoplasma hyopneumoniae, are two of the main causes of systemic and respiratory diseases in swine after weaning, respectively. The factors involved in the occurrence of disease are not completely clarified. Several strategies used to control these diseases are not completely efficient. In relation to Mycoplasma hyopneumoniae, the first chapter focused on obtaining an overview about the distribution and genetic diversity of this agent related to pneumonic lesions of swine reared in herds from different production areas of Minas Gerais state. This was accomplished by the use of MLVA genotyping analysis, which has proved worldwide to be a highy discriminatory technique for this bacterium. The results showed that there is a high prevalence of positive farms for the agent and a high polymorphism among circulating strains of the state of Minas Gerais, without a specific genotypic pattern of a geographic region. Regarding H. parasuis, the second chapter aimed to clarify, though a longitudinal study in a commercial farm, the effect of the association with different antimicrobials used in different stages of pig breeding on colonization dynamics by H. parasuis and on humoral response against this bacterium. Results showed that the intensive use of antibiotics in nursery can reduce the infection pressure in the farm, but interfers with the colonization by bacteria and shifting the onset of active immune response to subsequent weeks. In the field conditions of this study, there was no significant difference on the treatment with enrofloxacin on the colonization dynamics in clinically healthy piglets. Regarding the global colonization kinetics, not all piglets are exposed to the agent and receive maternal immunity in an early age, and the presence of maternal antibodies seems to reduce colonization even by putative comensal strains.

Caracterização fenotípica e genotípica de animais da raça Hereford criados na região sul do estado do RS durante surtos de ceratoconjuntivite infecciosa bovina

Esta dissertação foi dividida em quatro capítulos, tendo como principais objetivos: i) realizar a identificação bacteriológica dos agentes envolvidos na Ceratoconjuntivite Infecciosa Bovina (CIB), por meio de diferentes técnicas: análise morfo-tintorial e bioquímica, Espectrometria de massas com fonte de ionização e dessorção a laser assistida por matriz e analisador de massas do tipo tempo-de-voo (MALDI-TOF), Reação em cadeia da polimerase (PCR) e sequenciamento. ii) avaliar a diversidade genética de bactérias das espécies Moraxella bovis e Moraxella bovoculi isoladas de surtos de CIB entre 2015 e 2017, utilizando as técnicas de ERIC-PCR (enterobacterial repetitive intergenic consensus), RAPD-PCR (random amplification of polymorphic DNA) e uma variação do RAPD-PCR com os primers JWP1 e JWOPA07. iii) estimar os parâmetros e valores genéticos por meio da avaliação tradicional pelo modelo animal para a incidência de CIB. iv) realizar estudo de associação ampla do genoma (GWAS) para identificação de regiões genômicas e SNPs mais representativos para susceptibilidade à CIB. Logo no início do surto de CIB amostras de secreções oculares e nasais foram coletadas de bovinos da raça Hereford para possibilitar a identificação do agente bacteriológico causador da enfermidade, com isso confirmar o surto de CIB. Os animais eram provenientes de cinco fazendas, criados no sul do estado do Rio Grande do Sul. Foram realizadas quatro avaliações, com intervalo médio de 30 dias. O monitoramento teve como finalidade a realização da fenotipagem com relação à susceptibilidade à doença, por meio do acompanhamento da evolução dos surtos de CIB nas propriedades, a partir da atribuição de escores de lesão ocular. Foram atribuídos escores para as lesões de CIB, para cada um dos olhos, em uma escala de zero a quatro, na qual zero indicava o olho normal e quatro o estágio mais avançado da doença, implicando em perda ocular. As melhores concordâncias obtidas entre os métodos de identificação bacteriológica estudada foram à identificação por MALDI-TOF e a identificação molecular (PCR), confirmadas por sequenciamento, a partir do DNA extraído das colônias de Moraxella spp. Ademais, foi confirmada a existência de variabilidade genética entre as cepas de Moraxella bovis e Moraxella bovoculi isoladas de bovinos da raça Hereford, acometidos por CIB, pertencentes a cinco diferentes municípios situados no estado do Rio Grande do Sul. Os coeficientes de herdabilidade preditos foram de 0,14, 0,096 e 0,099 para as categorias (c_2, c_3 e c_9) para incidência a CIB. Os estudos de GWAS apontaram janelas genômicas significativas nos cromossomos 3, 11, 14, 18, 26 e 27 associados à CIB. As análises de enriquecimento funcional revelaram oito termos significativos (p<0,05) associados com a susceptibilidade a CIB: Babesia, Plasmodium vivax, Babesiosis, Bood bactericidal activity, Retinal cone photorecptor cells, Animal nutritional physiological phenomena, COS cells e 5 Flanking region. Os termos MeSH, apontam para importantes processos biológicos que estão relacionados com a expressão fenotípica das lesões ocorridas pela CIB. Concluindo, as análises fenotípicas e genômicas realizada ao longo deste estudo podem auxiliar na concepção de estratégias de prevenção e tratamento contra o CIB, podendo colaborar com estratégias de seleção por animais resistentes a CIB.

This dissertation was divided into four chapters, with the following main objectives: i) to carry out the bacteriological identification of the agents involved in Infectious Bovine Keratoconjunctivitis (IBK) by means of different techniques: morpho-tintorial and biochemical analysis, mass spectrometry with ionization source and matrixassisted laser desorption and time-of-flight mass analyzer (MALDI-TOF), polymerase chain reaction (PCR) and sequencing. ii) to evaluate the genetic diversity of Moraxella bovis and Moraxella bovoculi bacteria isolated from IBK outbreaks between 2015 and 2017, using ERIC-PCR (enterobacterial repetitive intergenic consensus), RAPD-PCR (random amplification of polymorphic DNA) and a variation of the RAPD-PCR with primers JWP1 and JWOPA07. iii) to estimate the genetic parameters and values through the traditional evaluation by the animal model for the incidence of IBK. iv) carry out genome association study (GWAS) to identify genomic regions and most representative SNPs for susceptibility to IBK. At the beginning of the outbreak of IBK samples of ocular and nasal secretions were collected from Hereford cattle to enable the identification of the bacteriological agent causing the disease, thus confirming the IBK outbreak. The animals came from five farms, located in the southern of Rio Grande do Sul state. Four evaluations were performed, with an average interval of 30 days. The purpose of the monitoring was to perform the phenotyping with regard to susceptibility to the disease, by monitoring the evolution of IBK outbreaks in the properties, from the attribution of ocular lesion scores. Scores were assigned to IBK lesions for each eye on a scale of zero to four, in which zero indicated the normal eye and four the most advanced stage of the disease, implying ocular loss. The best concordances obtained between the methods of bacteriological identification studied were the identification by MALDI-TOF and the molecular identification (PCR), confirmed by sequencing, from the DNA extracted from the colonies of Moraxella spp. In addition, the existence of genetic variability between Moraxella bovis and Moraxella bovoculi strains isolated from Hereford cattle, affected by IBK, belonging to five different municipalities located in the state of Rio Grande do Sul was confirmed. The predicted heritability coefficients were 0.14, 0.096 and 0.099 for the categories (c2, c3 and c9) for incidence at IBK. GWAS studies showed significant genomic windows on chromosomes 3, 11, 14, 18, 26 and 27 associated with IBK. Functional enrichment analyzes revealed eight significant (p <0.05) terms associated with IBK susceptibility: Babesia, Plasmodium vivax, Babesiosis, Bood bactericidal activity, Retinal cone photorecptor cells, Animal nutritional physiological phenomena, COS cells e 5 Flanking region. The terms MeSH, point to important biological processes that are related to the phenotypic expression of the lesions occurred by IBC. In conclusion, the phenotypic and genomic analyzes carried out throughout this study may help in the design of strategies for prevention and treatment against CIB, and may collaborate with selection strategies for CIB resistant animals.

Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections

Pathogenic strains of Escherichia coli are the most common bacteria associated with urinary tract infections in both humans and companion animals. Standard biochemical tests may be useful in demonstrating detailed phenotypical characteristics of these strains. Thirteen strains of E. coli isolated from dogs with UTIs were submitted to biochemical tests, serotyping for O and H antigens and antimicrobial resistance testing. Furthermore, the presence of papC, sfa, and afa genes was evaluated by PCR, and genetic relationships were established using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The antimicrobial that showed the highest resistance rate among the isolates was nalidixic acid (76.9%), followed by cephalotin (69.2%), sulfamethoxazole + trimethoprim (61.5%), tetracycline (61.5%), streptomycin (53.8%), ciprofloxacin (53.8%), ampicillin (46.2%), gentamicin (30.8%) and chloramphenicol (23.1%). No isolate was resistant either to meropenem or nitrofurantoin. Among the five clusters that were identified using ERIC-PCR, one cluster (A) had only one strain, which belonged to a serotype with zoonotic potential (O6:H31) and showed the genes papC+, sfa+, afa-. Strains with the genes papC-, sfa+, afa- were found in two other clusters (C and D), whereas all strains in clusters B and E possessed papC-, sfa-, afa- genes. Sucrose and raffinose phenotypic tests showed some ability in discriminating clusters A, B and C from clusters D and E.

Resistência bacteriana a antimicrobianos em uma comunidade remota da Floresta Amazônica

O aparecimento e a disseminação de bactérias multi-resistentes estão estreitamente relacionados ao uso excessivo e inadequado de antimicrobianos em medicina humana/veterinária e na produção animal. Entretanto, alguns estudos têm relatado a presença dessas bactérias em comunidades remotas com baixa ou mínima exposição a antimicrobianos. Assim, o objetivo deste trabalho foi investigar a presença de bactérias produtoras de -lactamases adquiridas, mediadas por plasmídeos, na microbiota Gram-negativa comensal de humanos e animais domésticos de diferentes etnias indígenas na região da Floresta Amazônica, e elucidar o seu contexto genético. No mês de março de 2013 foram coletadas amostras de fezes de indivíduos atendidos (n = 36) e funcionários (n = 9) de um centro assistencial em saúde restrito a comunidades indígenas. Adicionalmente, no mês de julho, foram coletadas amostras de swab retal de animais de companhia (n = 20) da comunidade. A identificação das espécies bacterianas foi feita por MALDI-TOF e a avaliação da susceptibilidade aos antimicrobianos foi realizada pela técnica de Kirby-Bauer. A detecção de genes de resistência e a determinação de filogrupos de E. coli foi feita por PCR. A genotipagem dos isolados foi feita por ERIC-PCR e MLST. As avaliações de transferibilidade, tamanho e grupos de incompatibilidade plasmidial (Inc) foram realizadas por conjugação e transformação, S1-PFGE e PBRT, respectivamente. Isolados representativos de cada grupo clonal tiveram o genoma completo sequenciado. Surpreendentemente, foram identificados 30 isolados de origem humana e cinco de origem animal apresentando resistência a cefalosporinas e fluoroquinolonas. A presença dos genes blaTEM-1 (78,6%) > blaCTX-M-15 (71,4%) > blaCTX-M-14 (21,4%) > blaCTX-M-8 e blaGES-5 (7,1% cada) foi confirmada em 14 isolados (provenientes de 10 indivíduos), sendo a maioria identificada como E. coli (70%). Adicionalmente, a presença dos genes blaCTX-M-14 (60%) > blaTEM-1 (40%) > blaCTX-M-2 e blaCTX-M-8 (20% cada) foi confirmada em cinco cepas de E. coli isoladas de cães. O filogrupo D de E. coli foi predominantemente encontrado, tanto em isolados de humanos (n = 6) quanto de animais (n = 4). Foi observada relação clonal entre cinco isolados de E. coli de humanos e três de animais (ST38 e ST6993), e todos os isolados de K. pneumoniae foram representados por um único clone (ST198). A transferibilidade plasmidial foi observada em nove isolados carregando os genes blaTEM, blaCTX-M-15, blaCTX-M-14 e blaCTX-M-8. O Inc mais encontrado foi IncF (n = 11), classificado em seis replicons diferentes; seguido por IncI1 (n = 5), classificado no ST80 e ST131; e IncL/M (n = 1), em plasmídeos variando de 97-291 kb. O sequenciamento de genoma completo permitiu a detecção de outros genes de resistência a -lactâmicos e a outras classes de antimicrobianos, bem como diversos genes de virulência. A presença de genes codificadores de ESBL e carbapenemase na microbiota comensal de indivíduos de uma comunidade remota no Brasil é um resultado inédito que demonstra que a disseminação de um problema endêmico em áreas urbanas para uma comunidade em teoria com baixa exposição a antibacterianos está acontecendo, sendo que as fontes e/ou veículos de aquisição são dignas de investigação, para um controle epidemiológico.

Emergence and spread of multiresistant bacteria are closely related to antimicrobial misuse in human and veterinary medicine and livestock. However, some studies have reported the presence of these bacteria in remote communities with low or minimal exposure to antimicrobials. Thus, the aim of this study was to investigate the presence of plasmid-mediated -lactamase in the commensal Gram-negative microbiota of humans and domestic animals of different indigenous ethnic groups in the Amazon Forest region, and to elucidate their genetic context. In March 2013, stool samples were collected from individuals attended in a health care center restricted to indigenous communities (n = 36) and from their local staff (n = 9). Additionally, in July 2013, rectal swab samples were collected from companion animals (n = 20) in the community. Bacterial species identification was performed by MALDI-TOF and antimicrobial susceptibility was assessed by the Kirby-Bauer technique. Antimicrobial resistance genes and E. coli phylo-groups were detected by PCR. Clonal relation among the isolates was investigated by ERIC-PCR and MLST. Plasmid mobility, size, and incompatibility groups (Inc) were evaluated by conjugation and transformation, S1-PFGE, and PBRT, respectively. Representative isolates of each clonal group were submitted to whole genome sequencing. Surprisingly, 30 isolates from humans and five isolates from animals have presented resistance to cephalosporins and fluoroquinolones. The presence of blaTEM-1 (78.6%) > blaCTX-M-15 (71.4%) > blaCTX-M-14 (21.4%) > blaCTX-M-8, and blaGES-5 (1% each) genes was confirmed in 14 isolates (from 10 individuals), most of them identified as E. coli (70%). In addition, the presence of blaCTX-M-14 (60%) > blaTEM-1 (40%) > blaCTX-M-2, and blaCTX-M-8 (20% each) genes was confirmed in five E. coli strains isolated from dogs. E. coli phylo-group D was predominantly found in both human (n = 6) and animal (n = 4) isolates. A clonal relationship was observed among five E. coli isolates from humans and three from animals (ST38 and ST6993), and all K. pneumoniae isolates were represented by a single clone (ST198). Plasmid mobility was observed in nine isolates harboring blaTEM-1, blaCTX-M-15, blaCTX-M-14, and blaCTX-M-8 genes. IncF was the most prevalent Inc group (n = 11), classified in six different replicons; followed by IncI1 (n = 5), assigned to ST80 and ST131; and IncL/M (n = 1), in plasmids ranging from 97-291 kb. The whole genome sequencing allows the identification of other -lactam resistance genes and genes conferring resistance to other antimicrobial classes, as well as several virulence genes. The presence of genes encoding ESBL and carbapenemase in the commensal microbiota of individuals from a remote community in Brazil is an unprecedented result. It demonstrates that the dissemination of an urban endemic problem to a community in theory with low antimicrobial exposure is occurring. Therefore, the sources and/or vehicles of acquisition are worthy of investigation in order to establish epidemiologic control measures.