Prevalence of Bartonella species in shelter cats and their ectoparasites in southeastern Brazil / Prevalência de espécies de Bartonella em gatos de abrigos e seus ectoparasitas no Sudeste do Brasil

Abstract Feline Bartonella can be transmitted to humans through cat scratches or bites, and between cats, by the flea Ctenocephalides felis. The study was carried out in order to investigate the occurrence of Bartonella DNA in cats living in shelters and their ectoparasites and the relationship between the infection status of cats and ectoparasites they host. Bartonella DNA was detected in 47.8% of the cat blood samples, 18.3% of C. felis fleas, 13.3% of flea egg pools and 12.5% of lice pools. B. henselae and B. clarridgeiae DNA were detected in cat fleas, while B. henselae, B. clarridgeiae and B. koehlerae were found in blood samples from bacteremic cats. Cats infested by positive ectoparasites showed approximately twice the odds of being infected. Our results indicate that shelter cats have high prevalence of Bartonella species that are known to be human pathogens. This highlights the importance of controlling infestations by ectoparasites to avoid cat and human infection.

Resumo Algumas espécies de Bartonella têm os felinos como principais hospedeiros reservatórios. Tais patógenos são transmitidos ao homem por intermédio da arranhadura ou mordedura de gatos e entre os gatos, por meio da pulga Ctenocephalides felis. O objetivo deste estudo foi investigar a ocorrência de DNA de Bartonella spp. em gatos de abrigos e seus ectoparasitas e a relação entre o estado de infecção dos gatos e dos ectoparasitas albergados por estes. Material genético bacteriano foi detectado em 47,8% das amostras de sangue de gatos, 18,3% das pulgas C. felis, 13,3% dos "pools" de ovos de pulgas e 12,5% dos "pools" de piolhos. DNA de B. henselae e B. clarridgeiae foi detectado em pulgas, e B. henselae, B. clarridgeiae e B. koehlerae, em amostras de sangue de gatos. Gatos infestados por ectoparasitas que carreavam DNA de Bartonella spp. demonstraram aproximadamente o dobro de chance de estarem infectados. Esses resultados indicam que os gatos de abrigos têm alta prevalência de infecção por espécies de Bartonella, capazes de causar doenças no homem. E também destacam a importância do controle e prevenção da infestação por ectoparasitas, no intuito de prevenir a infecção em gatos e humanos.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates / Otimização de um método baseado em PCR em tempo real para rastreamento de bactérias em concentrados de plaquetas

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

The prevalence of novel periodontal pathogens and bacterial complexes in Stage II generalized periodontitis based on 16S rRNA next generation sequencing

Abstract Objective: To define the subgingival microbial profile associated with Stage II generalized periodontitis using next-generation sequencing and to determine the relative abundance of novel periodontal pathogens and bacterial complexes. Methodology: Subgingival biofilm samples were collected from 80 subjects diagnosed with Stage II generalized periodontitis. Bacterial DNA was extracted, and 16S rRNA-based bacterial profiling via next-generation sequencing was carried out. The bacterial composition and diversity of microbial communities based on the age and sex of the patients were analyzed. The bacterial species were organized into groups: bacterial complexes (red, orange, purple, yellow, and green), novel periodontal pathogens, periodontal health-related species, and unclassified periodontal species. The results were analyzed and statistically evaluated. Results: The highest number of bacteria belonged to the phylum Bacteroidetes and Firmicutes. In terms of relative abundance, the orange complex represented 18.99%, novel bacterial species (Fretibacterium spp. and Saccharibacteria spp.) comprised 17.34%, periodontal health-related species accounted for 16.75% and unclassified periodontal species represented (Leptotrichia spp. and Selenomonas spp.) 15.61%. Novel periodontal pathogens had outweighed the periodontal disease-related red complex (5.3%). The one-sample z-test performed was statistically significant at p<0.05. The Beta diversity based on the unweighted UniFrac distance at the species level demonstrated a total variance of 15.77% based on age and 39.19% on sex, which was not statistically significant. Conclusion: The bacterial species corresponding to the disease-related orange complex and novel periodontal pathogens are predominant in Stage II generalized periodontitis.

Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil / Diversidade de bactérias cultiváveis associadas a raízes de bromélias de afloramentos ferruginosos do Brasil central

Abstract Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the "cangas" in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the "cangas". This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.

Resumo Estudos sobre a diversidade bacteriana associada a plantas silvestres são raros, especialmente naqueles que crescem em associação com bromélias. No presente estudo, isolamos e identificamos bactérias epífitas e endofíticas das raízes das bromélias Dyckia excelsa, D. leptostachya e Deuterocohnia meziana ocorrentes nas "cangas" no Pantanal do Mato Grosso do Sul, Brasil. As bactérias epifíticas foram isoladas de raízes lavadas, enquanto as bactérias endofíticas foram isoladas de raízes desinfestadas na superfície. Representantes bacterianos correspondentes a cada perfil do BOX-PCR, bem como aqueles que não resultaram em amplificações, foram selecionados para o sequenciamento do gene 16S rDNA. Os dados da BOX-PCR mostraram diversidade intragênica e intraespecífica e puderam discriminar cepas e identificar suas características fenotípicas. A seqüência do gene 16S rDNA e a análise filogenética mostraram uma maior ocorrência de cepas pertencentes ao gênero Bacillus do que as bactérias Mycobacterium e Brevibacterium, encontradas em menor número. Espécies do gênero Bacillus são bem conhecidas por sua capacidade de esporulação e maior sobrevida em locais áridos, como as "cangas". Este estudo mostrou claramente que as espécies de bromélias representam um vasto reservatório de diversidade de comunidades bacterianas, e as linhagens cultiváveis podem representar uma nova fonte para a prospecção biotecnológica.

Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

Is there any association between Helicobacter pylori and otitis media with effusion? / Há associação entre Helicobacter pylori e otite média com efusão?

Abstract Introduction: It is proposed that Helicobacter pylori can be responsible for the development of otitis media with effusion. Objective: The aim of this study is to investigate the prevalence of H. pylori in the adenoid tissue and fluid of the middle ear in patients who suffer from adenoid hyperplasia and otitis media with effusion in comparison with those who suffer from adenoid hyperplasia without otitis media with effusion. Methods: This is a case-control study that was carried out in 50 children of age 2-7 years old who were admitted with adenoid hyperplasia. Patients were divided into case and control groups. The study group included 25 patients with adenoid hyperplasia and otitis media with effusion and the control group included 25 patients with adenoid hyperplasia without otitis media with effusion. The patients in both groups underwent surgical adenoidectomy. For the case group we carried out myringotomy and placement of tympanostomy tube, and fluid samples were collected under sterile conditions. The samples were sent to the laboratory for polymerase chain reactions. Results: In the case group H. pylori was found to be positive in 18 samples of the middle ear fluid (70%) and in 1 polymerase chain reaction adenoid tissue sample (4%). In the control group H. pylori was positive in 3 samples of adenoid tissues (12%). There was no gender difference. Conclusion: H. pylori is one of the important bacteria that plays a role in the pathogenesis of otitis media with effusion. Whether adenoid tissue may be a reservoir for H. Pylori is unclear.

Resumo Introdução: Propõe-se que o Helicobacter pylori possa ser responsável pelo desenvolvimento de otite média com efusão. Objetivo: Investigar a prevalência de H. pylori no tecido adenoideano e no fluido da orelha média em pacientes com hiperplasia de adenoide e otite média com efusão em comparação àqueles com hiperplasia de adenoide sem otite média com efusão. Método: Este é um estudo de caso-controle feito em 50 crianças de 2 a 7 anos, com sinais e sintomas de hiperplasia de adenoide. Os pacientes foram divididos em grupo de estudo e grupo controle. O grupo de estudo incluiu 25 pacientes com hiperplasia de adenoide e otite média com efusão e o grupo controle incluiu 25 pacientes com hiperplasia de adenoide sem otite média com efusão. Os pacientes dos dois grupos foram submetidos a adenoidectomia e, no grupo de estudo, realizou-se também miringotomia com colocação de tubo de ventilação e amostras de fluidos foram coletadas sob condições estéreis. As amostras foram enviadas para o laboratório, para investigação por reação de polimerase em cadeia. Resultados: No grupo de estudo, houve positividade para H. pylori em 18 amostras do fluido de orelha média (70%) e uma amostra de tecido adenoideano foi positiva na reação de polimerase em cadeia (4%). No grupo controle, houve positividade para H. pylori em 3 amostras de tecido adenoideano (12%). Não houve diferença entre os gêneros. Conclusão: H. pylori é uma das bactérias importantes que desempenham um papel na patogênese da otite médica com efusão. Se o tecido adenoideano pode ou não representar um reservatório para H. pylori ainda necessita ser esclarecido.

Rodents as potential reservoirs for Borrelia spp. in northern Chile / Roedores como potenciais reservatórios de Borrelia spp. no norte do Chile

Abstract Small mammals play an essential role in the transmission and maintenance cycles of Borrelia spirochetes. In Chile, recent studies have characterized novel Borrelia genotypes in ticks collected from small mammals, a fact that suggests these vertebrates are hosts for spirochetes from this genus. Considering this evidence, the goal of this study was to determine the presence of Borrelia DNA in small mammals inhabiting northern Chile. In winter of 2018, 58 small mammals were captured in five localities. Blood samples were collected from rodents and DNA was extracted to determine the presence of Borrelia DNA by PCR targeting the flaB gene and rrs-rrlA intergenic spacer (IGS). From three individuals (5%), belonging to two rodent species of Cricetidae family (Phyllotis xanthopygus and Oligoryzomys longicaudatus), we retrieved three flaB and two IGS Borrelia genotypes. Phylogenetic analyses performed with both Maximum Likelihood and Bayesian inferences showed that our sequences grouped with homologous genotypes from the relapsing fever and Lyme borreliosis groups. Our findings suggest that P. xanthopygus and O. longicaudatus rodents may play a role as reservoirs for borrelial spirochetes in Chile.

Resumo Pequenos mamíferos possuem um papel essencial na transmissão e manutenção de espiroquetas do gênero Borrelia. No Chile, estudos recentes têm descrito novos genótipos de Borrelia em carrapatos, parasitando pequenos mamíferos. Isso sugere que esses vertebrados podem atuar como possíveis reservatórios dessas espiroquetas. Considerando-se essa evidência, o objetivo deste estudo foi determinar a presença de DNA de Borrelia em pequenos mamíferos da região norte do Chile. Durante o inverno de 2018, 58 pequenos mamíferos foram capturados em cinco localidades. Amostras de sangue obtidas a partir dos indivíduos capturados foram submetidas à extração de DNA e ensaios de PCR, para a detecção de Borrelia spp. baseados no gene flaB e espaçador intergênico rrs-rrlA (IGS). A partir de três espécimes (5%) pertencentes a duas espécies de roedores da família Cricetidae (Phyllotis xanthopygus e Oligoryzomys longicaudatus) obtiveram-se três genótipos de Borrelia para o gene flaB e dois para IGS. Análises filogenéticas inferidas, usando-se os métodos Bayesiano e de Máxima Verossimilhança, indicaram que as sequências geradas neste estudo agrupam-se com borrelias do grupo da Febre Recorrente e Borreliose de Lyme. Os achados deste estudo sugerem que roedores P. xanthopygus e O. longicaudatus poderiam atuar como possíveis reservatórios para Borrelia spp. no Chile.

Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

Identification of plasmid IncQ1 and NTEKPC-IId harboring bla KPC-2 in isolates from Klebsiella pneumoniae infections in patients from Recife-PE, Brazil

Abstract INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.

Quantification of microbial DNA in laboratory environment during DNA extraction / 生物工程学报

Metagenomic sequencing provides a powerful tool for microbial research. However, traditional experimental DNA extraction process will inevitably mix with environmental microorganisms which float in the air. It is still unclear whether the mixed environmental microbial DNA will heavily affect the metagenomic results of samples with extremely low microbial content. In this study, we first collected environmental bacteria in the laboratory and quantified the mixed environmental microbial DNA content during DNA extraction based on a qPCR-based quantification assay. We then extracted DNA from pure water in order to determine the mixed microbial taxons during extraction under open environment. At last, we extracted total DNA from a skin sample in a Biosafety cabinet or under open laboratory environment, to assess the impact of the mixed environmental microorganisms on the metagenomic results. Our results showed that DNA extraction under open laboratory environment in Beijing region resulted in 28.9 pg contaminant, which may accout for 30% of total DNA amount from skin samples. Metagenomic analysis revealed that the main incorporated environmental taxons were Cutibacterium acnes and Escherichia coli. Tens of environmental bacteria were foisted in the skin DNA samples, which largely decreased the relative abundance of dominant species and thus deteriorated the result accuracy. Therefore, analyzing microbial composition of samples with extremely low DNA content should better performed under aseptic environment.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

New molecular target for the phylogenetic identification of Leptospira species directly from clinical samples: an alternative gene to 16S rRNA

Abstract INTRODUCTION: Phylogenetic analysis of the 16S ribosomal gene initial region is used to identify Leptospira isolates at the species level from clinical samples. Unfortunately, this method cannot differentiate between some intermediates and saprophytic species. METHODS: We used comparative genomic analysis between 35 Leptospira species to find new molecular targets for Leptospira species identification. RESULTS: We proposed the use of the rpoC gene, encoding the DNA-directed RNA polymerase β-subunit, for identifying 35 Leptospira species. CONCLUSIONS: The rpoC gene can be a molecular target to identify the main species of the Leptospira genus directly from clinical samples.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

Identification of Brucella abortus biovar 4 of bovine origin in Colombia / Identificación de Brucella abortus biovar 4 de origen bovino en Colombia

The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest -Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.

El objetivo de este estudio fue identificar 12 aislamientos de Brucella abortus de origen bovino procedentes del departamento de Narino, Colombia, hasta la descripción de biovar. Estos aislamientos conforman la colección del Banco de Germoplasma de Microorganismos de Interés en Salud Animal, Bacterias y Virus. La identificación se hizo mediante métodos convencionales, como la descripción morfológica macro y microscópica de actividad enzimática, de perfiles bioquímicos, de utilización de sustratos y de sensibilidad a colorantes. Se hizo una caracterización genotipica complementaria mediante PCR múltiple para Brucella abortus, Brucella melitensis, Brucella ovisy Brucella suis-eritritol (AMOS-ERY-PCR); RFLP-/S7II; hibridación Southern blot y análisis multi-locus de repeticiones en tándem de número variable (MLVA), empleando como marcadores moleculares el gen ery, la secuencia de inserción /S711 y el número variable de repeticiones en tándem (VNTR). Los resultados de la caracterización fenotípica y molecular permitieron identificar 12 aislamientos de campo como B. abortus biovar 4 y diferenciar cepas de campo de cepas vacunales. Este es el primer estudio de identificación fenotípica y molecular de aislamientos de B. abortus en Colombia. Por su importancia taxonómica y epidemiológica, la identificación de estos aislamientos hasta el nivel de biovar permitirá disponer de recursos genéticos que se pueden emplear como cepas de referencia en futuras investigaciones. Estos resultados pueden considerarse como una base para la identificación de biotipos no reportados en el país y podrán ser utilizados en programas de monitoreo y vigilancia de la brucelosis bovina en Colombia.

Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal / Filogeografia de genótipos msp4 de Anaplasma marginale em gado de corte no Pantanal brasileiro

Abstract The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1β gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.

Resumo O gene msp4 de A. marginale é unicodon, estável e pouco heterogêneo, sendo considerado como um marcador útil para caracterização filogeográfica desta bactéria. Este trabalho teve como objetivo analisar a filogeografia de A. marginale com base no gene msp4 em bovinos de corte do Pantanal Brasileiro, comparativamente a outra regiões do mundo. Alíquotas de sangue foram colhidas de 400 bovinos (200 vacas e 200 bezerros) em cinco propriedades de cria e recria extensiva. Como resultado, 56,75% (227/400) mostraram-se positivas para A. marginale pela qPCR para o gene msp1β e destas, 8,37% (19/227) amostras foram positivas na PCR convencional para o gene msp4. Na análise de distância Network, 14 sequências do Pantanal brasileiro foram agrupadas em um único grupo com as da Thailândia, Índia, Espanha, Colômbia, Paraná (Brasil), México, Portugal, Argentina, China, Venezuela, Austrália, Italia e Minas Gerais (Brasil). Dentre 68 sequências do Brasil e do mundo, constatou-se a presença de 15 genótipos, sendo o genótipo número um (#1) o mais distribuído. As sequências msp4 de A. marginale detectadas em bovinos de corte no Pantanal brasileiro apresentaram baixo polimorfismo com formação de dois genogrupos filogeneticamente relacionados àqueles encontrados em ruminantes de países das América do Sul e Central, Europa e Ásia.

A common Salmonella Enteritidis sequence type from poultry and human gastroenteritis in Ibagué, Colombia / Un tipo de secuencia común de Salmonella Enteritidis de origen aviar y de humano con gastroenteritis en Ibagué, Colombia

Abstract Introduction: Salmonella Enteritidis is a major cause of human salmonellosis in the world, with contaminated eggs and raw chicken meat as the main routes of infection. The main Salmonella spp. serovars circulating in laying hen farms, the surface of eggs, and in raw chicken carcasses have been identified in Ibagué, Colombia. However, it is unknown whether those serovars are responsible for human gastroenteritis. Objective: To evaluate the genetic relationship between gastroenteritis and Salmonella Enteritidis isolates from poultry and humans using multilocus sequence typing (MLST). Materials and methods: Salmonella spp. was isolated from clinical cases of gastroenteritis (n=110). Antibiotic susceptibility tests, followed by serotyping and MLST were conducted and S. Enteritidis was compared to those from laying hen farms and marketed eggs. Results: Ten isolates of Salmonella spp. were obtained from the stools of people with gastroenteritis. The prevalence of Salmonella spp. in human stools was 9.09%, and S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Uganda (n=1), S. Grupensis (n=1), and S. Braenderup (n=1) were the main serotypes. MLST indicated that a common S. Enteritidis sequence type (ST11) was present in all three sources and showed the same antibiotic resistance pattern. Conclusion: Salmonella Enteritidis ST11 constitutes a link between consumption and manipulation of contaminated eggs and human gastroenteritis in Ibagué. Additional studies would be required to establish if other Salmonella serovars isolated from raw chicken meat are also associated with human gastroenteritis.

Resumen Introducción. Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo; los huevos contaminados y la carne de pollo cruda son sus principales fuentes de infección. En Ibagué, Colombia, se han identificado los principales serovares que circulan en granjas, superficies de huevos y canales de pollo, pero se desconoce si esos serovares son responsables de la gastroenteritis. Objetivo. Evaluar la relación genética entre los aislamientos de Salmonella Enteritidis de aves de corral y de humanos con la gastroenteritis mediante tipificación de multiloci de secuencias (Multilocus Sequence Typing, MLST). Materiales y métodos. Se aisló Salmonella spp. de casos clínicos de gastroenteritis (n=110). Se hizo la prueba de sensibilidad antibiótica, así como la serotipificación y la tipificación mediante MLST, y se comparó S. Enteritidis de humanos con la hallada en granjas de gallinas ponedoras y en huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp. a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9,09%, y se identificaron los serotipos S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup presentes en pacientes con gastroenteritis. Mediante la MLST, se comprobó que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y presentó el mismo patrón de resistencia antibiótica. Conclusión. Salmonella Enteritidis ST11 constituye un vínculo entre el consumo y la manipulación de huevos contaminados, y la gastroenteritis en humanos en Ibagué. Se requieren estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis en humanos.

Genotipificación de aislamientos del complejo Mycobacterium tuberculosis mediante MIRU-VNTR, Cali, Colombia, 2013-2015 / MIRU-VNTR genotyping of Mycobacterium tuberculosis in a population of patients in Cali, Colombia, 2013-2015

Resumen Introducción. La tuberculosis continúa siendo uno de los problemas de salud más importantes a nivel mundial y, con la infección por el virus de la inmunodeficiencia humana (HIV), constituye la principal causa de muerte por infecciones. En el 2016, se notificaron 6,3 millones de casos nuevos de la enfermedad. Objetivo. Describir los patrones genéticos determinados mediante la genotipificación del número variable de repeticiones en tándem de unidades repetitivas interespaciadas de micobacterias (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats, MIRU-VNTR) en la población de estudio y compararlos con los hallados en otros estudios locales e internacionales. Materiales y métodos. Mediante MIRU-VNTR, entre el 2013 y el 2015 se hizo la genotipificación de 105 muestras de ADN extraídas del esputo o de aislamientos en cultivo de M. tuberculosis provenientes de pacientes residentes en Cali con diagnóstico de tuberculosis pulmonar. La amplificación de 24 loci MIRU-VNTR se hizo por medio de la reacción en cadena de la polimerasa (PCR). Los amplicones resultantes se visualizaron por electroforesis en geles de agarosa (2 %) teñidos con SYBR Safe™. La asignación de los alelos se hizo con un análisis gráfico con el programa GelAnalyzer 2010. Los resultados obtenidos se analizaron con el algoritmo UPGMA y se compararon con las bases de datos internacionales MIRU-VNTRplus y SITVITWEB. Resultados. Se genotipificaron por completo 62 de las muestras y se obtuvieron 58 perfiles diferentes de MIRU-VNTR. Al comparar con las bases de datos internacionales, su distribución por linajes fue la siguiente: 54,8 % para el LAM, 25,8 % para el Haarlem, 14,5 % para el S, 3,2 % para el Beijing y 1,6 % para el Cameroon. Los patrones MIRU-VNTR correspondieron a 20 tipos internacionales de MIRU (MIRU International Types, MIT) diferentes, y los más frecuentes fueron el MIT 190 y el MIT 110, con 22,6 y 6,5 %, respectivamente. Conclusión. Estos resultados confirmaron hallazgos previos sobre el predominio de los linajes LAM y Haarlem en la ciudad y la presencia de los MIT encontrados en otra ciudad de Colombia.

Abstract Introduction: Tuberculosis continues to be one of the main public health problems in the world. Together with the HIV infection, it is one of the main causes of death due to infections worldwide. In 2016, 6.3 million new cases of the disease were reported. Objective: To describe the genetic patterns determined by genotyping using variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) in the study population and compare them with other studies carried out in Cali, Colombia, and the world. Materials and methods: We genotyped a total of 105 DNA samples extracted from sputum or culture isolates of the Mycobacterium tuberculosis complex, which were obtained from pulmonary tuberculosis diagnosed patients over the period 2013-2015, in Cali. We performed PCR amplification of 24 loci by MIRU-VNTR on the DNA extracted from the samples. The amplicons were visualized in agarose gel electrophoresis (2%) with SYBR Safe™ staining. Then, the alleles were designated by graphical analysis using the GelAnalyzer 2010 software. These results were analyzed using the UPGMA logarithm and compared with the registers from the MIRU-VNTR plus and SITVITWEB databases. Results: We genotyped 62 of the samples completely and we obtained 58 different MIRU-VNTR profiles. By comparing with the international databases, we determined the following distributions per lineage: LAM, 54.8%; Haarlem,25.8%; S, 14.5%; Beijing, 3.2%, and Cameroon, 1.6%. The MIRU-VNTR patterns corresponded to 17 different MITs; the most frequent were MIT 190 and MIT 110, with 22.6% and 6.5%, respectively. Conclusions: These results demonstrated previous observations about the predominance of the LAM and Haarlem lineages in the city, and the presence of the MITs found in another city of Colombia.

Helicobacter pylori: resistencia múltiple en pacientes de Bogotá, Colombia / Helicobacter pylori: Multiple resistance in patients from Bogotá, Colombia

Resumen Introducción. La resistencia a los antibióticos es la principal causa del fracaso del tratamiento contra Helicobacter pylori; la claritromicina y el metronidazol son los antibióticos que generan mayor resistencia. En Colombia, la resistencia primaria a estos dos antibióticos y el uso excesivo de levofloxacina han alcanzado los límites aceptados (13,6, 83 y 16 %, respectivamente). A pesar de ello, se usa el tratamiento empírico combinando estos antibióticos en pacientes en los que ha fallado anteriormente. Objetivo. Determinar la resistencia a los antibióticos en pacientes previamente tratados para H. pylori en Bogotá, Colombia. Materiales y métodos. Se llevó a cabo un estudio descriptivo en el que se evaluó mediante dilución en agar la resistencia a la amoxicilina, la claritromicina, la levofloxacina y el metronidazol en 10 aislamientos provenientes de 5 pacientes con tres o cuatro tratamientos fallidos para H. pylori. La resistencia a los antibióticos se confirmó mediante secuenciación de ADN (Magrogen, Korea). Resultados. Ocho de los aislamientos presentaron resistencia a dos o más antibióticos y todos fueron resistentes a la levofloxacina. Los patrones de sensibilidad de los aislamientos provenientes del antro pilórico y del cuerpo del estómago, fueron diferentes en tres de los pacientes. Conclusión. Hasta donde se sabe, esta es la primera evidencia de resistencia múltiple de H. pylori en Colombia en pacientes previamente tratados. Los resultados evidenciaron las consecuencias del uso de un esquema ineficaz de tratamiento antibiótico y la necesidad de evaluar la sensibilidad a los antibióticos en diferentes sitios anatómicos del estómago. La resistencia múltiple limita el número de antibióticos útiles para erradicar H. pylori.

Abstract Introduction: The main cause for Helicobacter pylori infection treatment failure is antibiotic resistance, where clarithromycin and metronidazole play the main role. In Colombia, primary resistance as a consequence of the use of these two antibiotics and excessive levofloxacin use is above the accepted limit (13.6%, 83%, and 16%, respectively). Despite this fact, empirical therapies that include the combination of these antibiotics are used in patients with previous therapeutic failure. Objective: To determine antibiotic resistance in patients previously treated for H. pylori in Bogotá, Colombia. Materials and methods: We conducted a descriptive study that included ten isolates obtained from five patients with three or four previous failed treatments for H. pylori. Antibiotic resistance to amoxicillin, clarithromycin, levofloxacin, and metronidazole was investigated by agar dilution and confirmed by DNA sequencing (Magrogen, Korea). Results: Eight isolates were resistant to two or more antibiotics. All isolates were resistant to levofloxacin. Susceptibility patterns in isolates from the gastric antrum and the body of the stomach were different in three patients. Conclusion: As far as we know, this is the first evidence of multiple H. pylori resistance in Colombia in previously treated patients. Results demonstrated the consequences of using an ineffective antibiotic scheme and the need to assess antibiotic susceptibility in different anatomical sites of the stomach. The consequences of multiple resistance decrease possible antibiotic effectiveness to eradicate H. pylori in the future.

Detection of multidrug-resistant Enterobacteriaceae isolated from river waters flowing to the Guanabara Bay and from clinical samples of hospitals in Rio de Janeiro, Brazil / Detección de enterobacterias multirresistentes aisladas en aguas de los ríos que desembocan en la bahía de Guanabara y en muestras de hospitales de Río de Janeiro, Brasil

Abstract Introduction: The use of antibiotics in humans, animal husbandry and veterinary activities induces selective pressure leading to the colonization and infection by resistant strains. Objective: We evaluated water samples collected from rivers of the Guanabara Bay, which have suffered minor and major environmental degradation, and clinical samples of hospital origin to detect evidence of the presence of resistance genes to aminoglycosides, beta-lactam antibiotics and fluoroquinolones in strains of Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and Escherichia coli. Materials and methods: For isolation of the water strains we employed culture media containing 32 μg/ml cephalotin and 8 μg/ml gentamicin. The strains from clinical materials were selected using culture media containing 8 μg/ml gentamicin. The strains were identified and subjected to antimicrobial susceptibility testing (AST), plasmid DNA extraction and polymerase chain reaction (PCR) to detect genes encoding enzymes modifying aminoglycosides (EMA), extended-spectrum beta-lactamases (ESBL) and plasmid mechanisms of quinolone resistance (PMQR). Results: The AST of the isolates recovered from water samples showed multidrug-resistance profiles similar to those found in isolates recovered from clinical materials. All isolates from water samples and 90% of the isolates from clinical samples showed at least one plasmid band. In the PCR assays, 7.4% of the isolates recovered from water samples and 20% of those from clinical materials showed amplification products for the three antimicrobial classes. Conclusion: We believe that the detection of microorganisms presenting genetic elements in environments such as water is necessary for the prevention and control of their dissemination with potential to infect humans and other animals in eventual contact with these environments.

Resumen Introducción. El uso de antibióticos en seres humanos, en la industria pecuaria y en las actividades veterinarias induce una presión selectiva que resulta en la colonización e infección con cepas resistentes. Objetivo. Determinar la presencia de genes de resistencia a aminoglucósidos, betalactámicos y fluoroquinolonas en cepas de Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae y Escherichia coli, obtenidas de muestras de agua de los ríos que desembocan en la bahía de Guanabara y de muestras clínicas de hospitales de Río de Janeiro. Materiales y métodos. En la selección de las cepas resistentes obtenidas de las muestras de agua de los ríos, se emplearon medios de cultivo que contenían 32 μg/ml de cefalotina y 8 μg/ ml de gentamicina. En el caso de las muestras de especímenes clínicos, se usaron medios de cultivo que contenían 8 μg/ml de gentamicina. Las cepas se identificaron y se sometieron a pruebas de sensibilidad antimicrobiana, extracción de ADN plasmídico y pruebas de reacción en cadena de la polimerasa (PCR) para detectar los genes que codifican aquellas enzimas que modifican los aminoglucósidos, las betalactamasas de espectro extendido (BLEE) y los mecanismos de resistencia a las quinolonas mediados por plásmidos. Resultados. Se encontraron perfiles de resistencia a los antimicrobianos similares en los dos grupos. En todas las bacterias obtenidas de las muestras de agua y en 90 % de las muestras clínicas, se evidenciaron bandas de plásmidos asociados con la transferencia de genes de resistencia. En las pruebas de PCR, se obtuvieron productos de amplificación de los genes de resistencia para las tres clases de antimicrobianos analizados, en el 7,4 % de las bacterias recuperadas de las muestras de agua y en el 20 % de aquellas recuperadas de las muestras clínicas. Conclusión. La detección de microorganismos con elementos genéticos que confieren resistencia a los antibióticos en ambientes como el agua, es una estrategia necesaria para prevenir y controlar la diseminación de estos agentes patógenos con potencial para infectar a humanos y a otros animales en dichos ambientes.

Molecular detection of Mycoplasma suis in extensive pig production systems in the State of Maranhão, northeast Brazil / Detecção molecular de Mycoplasma suis em suínos de criações extensivas no estado do Maranhão, nordeste do Brasil

Abstract Mycoplasma suis is a bacterium that causes hemoplasmosis in pigs. This agent is capable of adhering to the surface of porcine erythrocytes, inducing structural changes on these cells. In Brazil, there are few reports about the disease, its causal agent, and the economic impact of this pathogen on pig production systems and farm sanitation. The present study aimed to investigate the occurrence of M. suis in extensive swine farms located in the counties of Itapecuru Mirim, Santa Rita and Rosario, State of Maranhão, northeast Brazil. For such purpose, 64 blood samples of pigs from these facilities were tested for M. suis using a 16S rRNA gene-based quantitative real-time PCR (qPCR); 82.3%, 65.2% and 25% of blood samples of swine from farms in the cities of Itapecuru Mirim, Santa Rita and Rosario were positive for M. suis by qPCR, respectively. This study shows, for the first time, that M. suis circulates in pig populations from the state of Maranhão, Northeast Brazil.

Resumo Mycoplasma suis é uma bactéria que causa a hemoplasmose em suínos. Este agente é capaz de se aderir à superfície dos eritrócitos de suínos, ocasionando deformações estruturais nestas células. No Brasil, poucos são os relatos acerca do parasita, da infecção e de seus impactos econômicos nas esferas produtiva e sanitária. O objetivo deste estudo foi investigar, por meio da PCR em tempo real quantitativa (qPCR) baseada no gene 16S rRNA, a ocorrência de M. suis em 64 amostras de sangue de suínos de criações extensivas dos municípios de Itapecuru Mirim, Santa Rita e Rosário, localizados no estado do Maranhão. Foram obtidos um percentual de 82,3%, 65,2% e 25% de amostras positivas na qPCR para M. suis nos municípios de Itapecuru Mirim, Santa Rita e Rosário, respectivamente. Este estudo mostra que M. suis circula entre os suínos de criações extensivas no estado do Maranhão.