Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding / Isolamento e caracterização de bactérias associadas ao trato intestinal do bicho-da-seda sob alimentação larval tratada com antibióticos

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

Isolation and screening of chromium resistant bacteria from industrial waste for bioremediation purposes / Isolamento e triagem de bactérias resistentes ao cromo de resíduos industriais para fins de biorremediação

Abstract Chromium (VI) a highly toxic metal, a major constituent of industrial waste. It is continuously release in soil and water, causes environmental and health related issues, which is increasing public concern in developing countries like Pakistan. The basic aim of this study was isolation and screening of chromium resistant bacteria from industrial waste collected from Korangi and Lyari, Karachi (24˚52ʹ46.0ʺN 66˚59ʹ25.7ʺE and 24˚48ʹ37.5ʺN 67˚06ʹ52.6ʺE). Among total of 53 isolated strains, seven bacterial strains were selected through selective enrichment and identified on the basis of morphological and biochemical characteristics. These strains were designated as S11, S13, S17, S18, S30, S35 and S48, resistance was determined against varying concentrations of chromium (100-1500 mg/l). Two bacterial strains S35 and S48 showed maximum resistance to chromium (1600 mg/l). Bacterial strains S35 and S48 were identified through 16S rRNA sequence and showed 99% similarity to Bacillus paranthracis and Bacillus paramycoides. Furthermore, growth condition including temperature and pH were optimized for both bacterial strains, showed maximum growth at temperature 30ºC and at optimum pH 7.5 and 6.5 respectively. It is concluded that indigenous bacterial strains isolated from metal contaminated industrial effluent use their innate ability to transform toxic heavy metals to less or nontoxic form and can offer an effective tool for monitoring heavy metal contamination in the environment.

Resumo O cromo (VI), metal altamente tóxico, é um dos principais constituintes dos resíduos industriais. É liberado no solo e na água, causa problemas ambientais e de saúde de crescente preocupação pública em países em desenvolvimento como o Paquistão. O objetivo básico deste estudo foi o isolamento e a triagem de bactérias resistentes ao cromo de resíduos industriais coletados em Korangi e Lyari, Karachi (24˚52'46,0"N 66˚59'25,7"E e 24˚48'37,5"N 67˚06'52,6"E). Do total de 53 cepas isoladas, sete cepas bacterianas foram selecionadas por enriquecimento seletivo e identificadas com base em características morfológicas e bioquímicas. Essas cepas foram designadas como S11, S13, S17, S18, S30, S35 e S48, apresentaram alta resistência aos metais contra concentrações variáveis (100-1500 mg / l) de cromo. Já as cepas S35 e S48 foram identificadas por meio da sequência 16S rRNA e apresentaram 99% de similaridade com Bacillus paranthracis e Bacillus paramycoides. Além disso, as condições de crescimento incluindo temperatura e pH foram otimizadas e ambas as cepas bacterianas apresentaram crescimento máximo na temperatura de 30 ºC, enquanto seu pH ótimo foi observado em 7,5 e 6,5, respectivamente. Conclui-se que o potencial de resistência dessas bactérias resistentes ao cromo pode ser efetivamente utilizado na remoção de cromo de efluentes industriais contaminados. Técnicas de base biológica usando bactérias ajudarão a fornecer métodos mais baratos e ecológicos de remoção, recuperação e desintoxicação de cromo.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70'N - 90°45'W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Assessment of zinc solubilization potential of zinc-resistant Pseudomonas oleovorans strain ZSB13 isolated from contaminated soil / Avaliação do potencial de solubilização do zinco da cepa ZSB13 de Pseudomonas oleovorans resistente ao zinco isolada de solo contaminado

Abstract Zinc is an essential micronutrient that is required for optimum plant growth. It is present in soil in insoluble forms. Bacterial solubilization of soil unavailable form of Zn into available form, is an emerging approach to alleviate the Zn deficiency for plants and human beings. Zinc solubilizing bacteria (ZSB) could be a substitute for chemical Zn fertilizer. The present study aimed to isolate and characterize bacterial species from the contaminated soil and evaluate their Zn solubilizing potential. Zn resistant bacteria were isolated and evaluated for their MIC against Zn. Among the 13 isolated bacterial strains ZSB13 showed maximum MIC value upto 30mM/L. The bacterial strain with the highest resistance against Zn was selected for further analysis. Molecular characterization of ZSB13 was performed by 16S rRNA gene amplification which confirmed it as Pseudomonas oleovorans. Zn solubilization was determined through plate assay and broth medium. Four insoluble salts (zinc oxide (ZnO), zinc carbonate (ZnCO3), zinc sulphite (ZnS) and zinc phosphate (Zn3(PO4)2) were used for solubilization assay. Our results shows 11 mm clear halo zone on agar plates amended with ZnO. Likewise, ZSB13 showed significant release of Zn in broth amended with ZnCO3 (17 and 16.8 ppm) and ZnO (18.2 ppm). Furthermore, Zn resistance genes czcD was also enriched in ZSB13. In our study, bacterial strain comprising Zn solubilization potential has been isolated that could be further used for the growth enhancement of crops.

Resumo O zinco é um micronutriente essencial necessário para o crescimento ideal das plantas. Ele está presente no solo em formas insolúveis. A solubilização bacteriana da forma indisponível de Zn no solo para a forma disponível é uma abordagem emergente para aliviar a deficiência de Zn em plantas e seres humanos. Bactérias solubilizadoras de zinco (ZSB) podem ser um substituto para fertilizantes químicos de Zn. O presente estudo teve como objetivo isolar e caracterizar espécies bacterianas de solo contaminado e avaliar seu potencial de solubilização de Zn. Bactérias resistentes ao Zn foram isoladas e avaliadas quanto ao seu MIC contra o Zn. Entre as 13 cepas bacterianas isoladas, ZSB13 apresentou valor máximo de MIC de até 30 mM/L. A cepa bacteriana com maior resistência ao Zn foi selecionada para análise posterior. A caracterização molecular de ZSB13 foi realizada por amplificação do gene 16S rRNA que o confirmou como Pseudomonas oleovorans. A solubilização do Zn foi determinada através de ensaio em placa e meio caldo. Quatro sais insolúveis (óxido de zinco (ZnO), carbonato de zinco (ZnCO3), sulfito de zinco (ZnS) e fosfato de zinco (Zn3 (PO4) 2) foram usados ​​para o ensaio de solubilização. Nossos resultados mostram uma zona de halo clara de 11 mm em placas de ágar corrigidas com ZnO. Da mesma forma, ZSB13 mostrou liberação significativa de Zn em caldo alterado com ZnCO3 (17 e 16,8 ppm) e ZnO (18,2 ppm). Além disso, os genes de resistência ao Zn czcD também foram enriquecidos em ZSB13. Em nosso estudo, a cepa bacteriana compreendendo potencial de solubilização de Zn foi isolada e poderia ser usada posteriormente para o aumento do crescimento de safras.

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans / Disbiose intestinal, inflamação e diabetes tipo 2 em camundongos usando microbiota intestinal sintética de humanos diabéticos

Abstract The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice treated with GM+modified diet (HFD/CD) compared to strains Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629) which were isolated from mice receiving CD/HFD. In conclusion, these findings suggest that constitution of GM and diet plays significant role in inflammation leading to onset or/and possibly progression of T2D. .

Resumo O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados ​​como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P < 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-α, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) e Lactobacillus Gasseri (MT152635D), foram tratadas com dieta modificada / CD) em comparação com as linhagens Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629), que foram isoladas de camundongos recebendo CD / HFD. Em conclusão, esses resultados sugerem que a constituição de GM e dieta desempenham papel significativo na inflamação levando ao início ou/e possivelmente à progressão de T2D.

Biodegradation of petroleum by bacteria isolated from fishes of Indian Ocean / Biodegradação do petróleo por bactérias isoladas de peixes do oceano Índico

In this study, oil degrading bacteria discovered from fish living near the oil ports at Karachi in Pakistan were characterized. The bacteria isolated from skin, gills, and gut in fish could consume crude oil as a source of carbon and energy. Total 36 isolates were tested using Nutrient Agar (NA) and MSA media with different crude oil concentrations (0.2%, 0.5%, 0.7%, 1%, 2%, and 5%) and 4 out of 36 isolates (two Gram positive and two Gram negative bacteria) were selected for further identification. 16S rRNA gene sequencing revealed that the isolates are related to Bacillus velezensis, Bacillus flexus, Pseudomonas brenneri and Pseudomonas azotoforman. Oil degrading potential of these bacteria was characterized by GC-MS analysis of degradation of oil components in crude oil as well as engine oil. We found that one (2, 6, 10, 14-Tetramethylpentadecane) out of 42 components in the crude oil was fully eliminated and the other oil components were reduced. In addition, 26 out of 42 oil components in the engine oil, were fully eliminated and the rest were amended. Taken together, these studies identify that B. velezensis, B. flexus, P. brenneri and P. azotoforman have high oil degrading potential, which may be useful for degradation of oil pollutants and other commercial applications.

Neste estudo, bactérias degradadoras de óleo descobertas em peixes que vivem perto dos portos de petróleo em Karachi, no Paquistão, foram caracterizadas. As bactérias isoladas da pele, guelras e intestinos dos peixes podem consumir petróleo bruto como fonte de carbono e energia. No total, 36 isolados foram testados usando Agar Nutriente (NA) e meio MSA com diferentes concentrações de óleo bruto (0,2%, 0,5%, 0,7%, 1%, 2% e 5%) e 4 de 36 isolados (dois Gram positivos e duas bactérias Gram negativas) foram selecionadas para posterior identificação. O sequenciamento do gene 16S rRNA revelou que os isolados estão relacionados a Bacillus velezensis, Bacillus flexus, Pseudomonas brenneri e Pseudomonas azotoforman. O potencial de degradação do óleo dessas bactérias foi caracterizado pela análise de GC-MS da degradação dos componentes do óleo no óleo cru, bem como no óleo do motor. Descobrimos que um (2, 6, 10, 14-tetrametilpentadecano) de 42 componentes do óleo cru foi totalmente eliminado e os outros componentes do óleo foram reduzidos. Além disso, 26 dos 42 componentes do óleo do motor foram totalmente eliminados e o restante corrigido. Juntos, esses estudos identificam que B. velezensis, B. flexus, P. brenneri e P. azotoforman têm alto potencial de degradação de óleo, o que pode ser útil para a degradação de poluentes de óleo e outras aplicações comerciais.

Bacterial diversity in high Andean grassland soils disturbed with Lepidium meyenii crops evaluated by metagenomics / Diversidade bacteriana em solos de prados andinos elevados perturbados por culturas de Lepidium meyenii avaliadas por metagenômica

Soil quality is usually determined by its physical-chemical characteristics without taking into account the bacterial communities that play a fundamental role in the chemical decomposition of plant nutrients. In this context, the objective of the study was to evaluate bacterial diversity in high Andean grassland soils disturbed with Lepidium meyenii cultivation under different gradients of use (first, second and third use) and crop development (pre-sowing, hypocotyl development and post-harvest). The sampling was carried out in the Bombón plateau in the central Andes of Peru, during the rainy and low water seasons, by the systematic method based on a specific pattern assigned in a geometric rectangular shape at a depth of 0 - 20 cm. The characterization of the bacterial communities was carried out through the metagenomic sequencing of the 16S rRNA. 376 families of bacteria were reported, of which it was determined that there was a significant change in bacterial composition and distribution in relation to use pressure. There were no major changes due to the development of Lepidium meyenii. The families most sensitive to use pressure and soil poverty indicators were Verrucomicrobiaceae, Acidobacteraceae and Aakkermansiaceae.

A qualidade do solo é normalmente determinada pelas suas características físico-químicas sem ter em conta as comunidades bacterianas que desempenham um papel fundamental na decomposição química dos nutrientes das plantas. Neste contexto, o objetivo do estudo foi avaliar a diversidade bacteriana em solos de prados andinos elevados perturbados pelo cultivo de Lepidium meyenii sob diferentes gradientes de utilização (primeira, segunda e terceira utilizações) e desenvolvimento das culturas (pré-semeadura, desenvolvimento do hipocótilo e póscolheita). A amostragem foi realizada no planalto de Bombón, nos Andes centrais do Peru, durante as estações das chuvas e das águas baixas, pelo método sistemático baseado num padrão específico atribuído em forma geométrica retangular a uma profundidade de 0 - 20 cm. A caracterização das comunidades bacterianas foi realizada através da sequenciação metagenômica do rRNA 16S. Foram relatadas 376 famílias de bactérias, das quais se verificou uma alteração significativa na composição e distribuição bacteriana em relação à pressão de utilização. Não se registaram grandes alterações devido ao desenvolvimento do Lepidium meyenii. As famílias mais sensíveis à utilização de indicadores de pressão e pobreza do solo foram as Verrucomicrobiaceae, Acidobacteraceae e Aakkermansiaceae.

Clinical factors associated with composition of lung microbiota and important taxa predicting clinical prognosis in patients with severe community-acquired pneumonia / 医学前沿

Few studies have described the key features and prognostic roles of lung microbiota in patients with severe community-acquired pneumonia (SCAP). We prospectively enrolled consecutive SCAP patients admitted to ICU. Bronchoscopy was performed at bedside within 48 h of ICU admission, and 16S rRNA gene sequencing was applied to the collected bronchoalveolar lavage fluid. The primary outcome was clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy. Sixty-seven patients were included. Multivariable permutational multivariate analysis of variance found that positive bacteria lab test results had the strongest independent association with lung microbiota (R2 = 0.033; P = 0.018), followed by acute kidney injury (AKI; R2 = 0.032; P = 0.011) and plasma MIP-1β level (R2 = 0.027; P = 0.044). Random forest identified that the families Prevotellaceae, Moraxellaceae, and Staphylococcaceae were the biomarkers related to the positive bacteria lab test results. Multivariable Cox regression showed that the increase in α-diversity and the abundance of the families Prevotellaceae and Actinomycetaceae were associated with clinical improvements. The positive bacteria lab test results, AKI, and plasma MIP-1β level were associated with patients' lung microbiota composition on ICU admission. The families Prevotellaceae and Actinomycetaceae on admission predicted clinical improvements.

Investigating the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of severe scald rabbits based on 16S ribosomal RNA high-throughput sequencing / 中华烧伤杂志

Objective: To investigate the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of in severe scald rabbits based on 16S ribosomal RNA (16S rRNA) high-throughput sequencing. Methods: The experimental research method was adopted. Ninety Japanese big-ear rabbits regardless gender, aged 6 to 8 months, were randomly divided into normal control group, scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group, with 18 rabbits in each group. The rabbits in normal control group were free to eat and drink, and the rabbits in scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group were intragastrically administered normal saline, 0.2 g/mL Modified Sijunzi Decoction, 1.0 g/mL Modified Sijunzi Decoction, and 5.0 g/mL Modified Sijunzi Decoction, respectively for 7 days after sustaining full-thickness scalding of 30% total body surface area. On the 1st, 3rd, and 7th day after grouping, the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-10 in ileal mucosa tissue of rabbits in each group were determined by enzyme-linked immunosorbent assay, and the number of samples in each group at each time point was 6. According to the above experimental results, another 9 rabbits were selected and divided into normal control group, scald alone group and scald+medium-dose group, with 3 rabbits in each group. The grouping and treatment methods of rabbits in each group were the same as before. On the 7th day after grouping, the V3, V4 region of 16S rRNA of ileum mucosa of rabbits in three groups were sequenced by high-throughput sequencing technology. The number of quality bacteria was counted by QIME software. The classifications of phylum, class, order, family and genus of microflora were analyzed by RDP Classifier software. The α diversity (Ace, Chao1, Simpson, and Shannon indexes) and β diversity were analyzed by Illumina MiSeq sequencing technology, and the number of experiment samples in each group was 3. Data were statistically analyzed with analysis for variance of factorial design, SNK test, and Bonferroni correction. Results: Compared with that in normal control group, the levels of TNF-α of ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, and scald+high-dose group on the 1st, 3rd, and 7th day after grouping and scald+medium-dose group on the 1st and 3rd day after grouping were all significantly increased (P<0.01), the levels of IL-1β in ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, scald+medium-dose group and scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly increased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly decreased (P<0.01). Compared with that in scald alone group, the levels of TNF-α in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 3rd and 7th day after grouping, and scald+medium-dose group on the 1st day after grouping were all significantly decreased (P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 3rd and 7th day after grouping and scald+medium-dose group on the 1st day after grouping were all significantly decreased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+low-dose group on the 7th day after grouping and scald+medium-dose group on the 1st, 3rd, and 7th day after grouping and scald+high-dose group on the 3rd and 7th day after grouping were all significantly increased (P<0.05 or P<0.01). Compared with that in scald+low-dose group, the levels of TNF-α in ileal mucosa tissue of rabbits in medium-dose scald alone group on the 1st, 3rd, and 7th day after grouping and in high-dose scald alone group on the 3rd and 7th day after grouping were significantly decreased (P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in medium-dose scald alone group on the 1st, 3rd, and 7th day after grouping and in high-dose scald alone group on the 3rd and 7th day after grouping were all significantly decreased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+medium-dose group on the 1st, 3rd, and 7th day after grouping and in scald+high-dose group on the 7th day after grouping were all significantly increased (P<0.05 or P<0.01). Compared with that in scald medium-dose group, the levels of TNF-α in ileal mucosa tissue of rabbits in scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly increased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly decreased (P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in scald+high-dose group on the 7th day after grouping was significantly decreased (P<0.01). Compared with that on the 1st day after grouping, the levels of TNF-α in ileal mucosa tissue of rabbits in scald alone group on the 3rd and 7th day after grouping and in normal control group on the 3rd day after grouping were all significantly increased (P<0.05 or P<0.01), and the levels of IL-1β in ileal mucosa tissue of rabbits in scald alone group both on the 3rd and 7th day after grouping were significantly increased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in both scald+low-dose group and scald+high-dose group on the 7th day after grouping and scald+medium-dose group both on the 3rd and 7th day after grouping were significantly increased (P<0.05 or P<0.01), and the levels of TNF-α in ileal mucosa tissue of rabbits in scald+high-dose group on the 3rd and 7th day after grouping and in scald+medium-dose group on the 7th day after grouping were all significantly decreased (P<0.05 or P<0.01), and the level of IL-1β in ileal mucosa tissue of rabbits in scald+medium-dose group on the 7th day after grouping was significantly decreased (P<0.01), and the level of IL-10 in ileal mucosa tissue of rabbits in scald alone group on the 7th day after grouping was significantly decreased (P<0.01). Compared with that on the 3rd day after grouping, the levels of TNF-α and IL-1β in ileal mucosa tissue of rabbits in scald alone group and the levels of IL-10 in ileal mucosa tissue of rabbits in normal control group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 7th day after grouping were all significantly increased (P<0.05 or P<0.01); and the levels of TNF-α in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 7th day after grouping were all significantly decreased (P<0.05), and the levels of IL-1β in ileal mucosa tissue of rabbits both in scald+medium-dose group and scald+high-dose group on the 7th day after grouping were significantly decreased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald alone group on the 7th day after grouping was significantly decreased (P<0.01). On the 7th day after grouping, the high-quality sequences obtained from the microflora in ileum mucosa of rabbits in normal control group, scald alone group, and scald+medium-dose group were 96 023, 107 365, and 95 921, respectively. At the classification level of phylum, class, order, family, and genus of the microflora in ileum mucosa of rabbits in three groups were all Bacteroidetes and Firmicutes, Clostridium and Bacteroidetes, Clostridium and Bacteroidetes, Rumenobacteriaceae and Clostridium and Bacteroideaceae, Clostridium and Bacteroidetes and rumen bacteria mainly, while the percentage of microflora in each group was different. There were no significant differences in Ace, Chao1, Simpson, Shannon indices (P>0.05), and no obvious difference in β diversity of microflora in ileal mucosa tissue of rabbits among three groups. Conclusions: After severe scalding, the inflammatory response of rabbit ileal mucosa tissue is obvious and increased in a time-dependent manner. Modified Sijunzi Decoction can reduce inflammation with optimal therapeutic concentration of 1.0 g/mL. The technology of high-throughput sequencing can reflect the structural composition of the intestinal microflora accurately. The ileal microflora of the severe scald rabbit can be regulated by the administration of Modified Sijunzi Decoction.

Relationships between lead-induced learning and memory impairments and gut microbiota disturbance in mice / 中华劳动卫生职业病杂志

Objective: To explore the effect of lead exposure on the neurobehavior and gut microbiota community structure in mice. Methods: In August 2019, 64 C57BL/6 mice were randomly divided into 4 groups: control group (0 ppm) , low lead exposure group (20 mg/l) , medium lead exposure group (100 mg/l) and high lead exposure group (500 mg/l) . During the experiment, they were free to eat and drink. The drinking water of the lead exposure group was mixed with lead acetate, and sodium acetate was added in the control group. After 10 weeks of exposure, the Morris water maze was used to test the learning and memory ability of each group of mice, and then they were sacrificed for sampling. ICP-MS was used to detect lead content in whole blood and brain tissue. ELISA was used to determine the level of IL-1β in mouse serum. 16S rRNA sequencing was used to detect the structural diversity of the intestinal flora in feces, and then the correlation between the flora and behavior indicators was analyzed. Results: In the Morris water maze experiment, compared with the control group, there was no significant difference in the body weight and swimming speed of the mice in the lead exposure groups. The escape latency of the mice in the 100 mg/l and 500 mg/l dose groups was prolonged, and the number of platform crossings decreased (P<0.05) ; meanwhile, the staying time of the mice in the 500 mg/l Pb-treated group in the target quadrant was lower than that of the control group, and the difference was statistically significant (P<0.05) . Compared with the control group, the blood lead content of the mice in each lead exposure group was significantly increased, and the brain lead content of mice in the 500 mg/l dose group was significantly elevated (P<0.05) . The serum IL-1β levels of mice in each lead exposure group were higher than those of the control group (P<0.05) . At the phylum level, the relative abundance of the Proteobacteria phylum in all of Pb-treated groups was significantly increased (P<0.05) ; at the genus level, Allobaculum, Desulfovibrio, Lachnospiraceae_NK4A136_group, Turicibacter and Ureaplasma were significantly increased (P<0.05) . Among them. The relative abundance of Desuffaoibrio, Turici bacter, and Ureaplasma was negatively correlated with the residence time of mice in the quadrant of the platform (r=-0.32, -0.29, -0.44, P<0.05) . Conclusion: Lead exposure induced learning and memory impairments in mice, which may be related to the disturbance of the gut microbiota.

Analysis of factors associated with the structure of the gut microbial community in HIV/AIDS patients in some areas of Henan province / 中华流行病学杂志

Objective: To investigate the related factors associated with the structure of the gut microbial community in HIV infection/AIDS cases (HIV/AIDS) in Henan province. Methods: The convenience sampling method was used to select 122 cases who were receiving Antiviral Treatment (ART) or ART-naive in Henan. Whole blood and stool specimens were collected. Genomic DNA of stool samples was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using Illumina NovaSeq 6000 high-throughput sequencing system. The analysis was performed mainly at the genus level, and the 30 genera with the highest abundance were selected as a measure of the gut microbial community structure. The correlation between community structure and related factors was analyzed using redundancy analysis and Envfit function. Results: 122 cases were finally completed sequencing and analysis, the average BMI was (23.62±2.78) kg/m2 and the average age was (47±13) years. Among them, male accounted for 66.39% (81/122), and heterosexual transmission route constituted the largest ratio, accounting for 51.64% (63/122). 36 cases were treatment naive (29.51%, 36/122). The top five dominant genera of the total population (122 cases) were Prevotella, Roseburia, Megamonas, Bacteroides and Faecalibacterium and the top five dominant genera of the ART population (86 cases) were Prevotella, Megamonas, Bacteroides, Roseburia and Faecalibacterium. The top five dominant genera of the ART-naive population (36 cases) appeared as Prevotella, Faecalibacterium, Roseburia, Bacteroides and Megamonas. In the total population, ART (P<0.001) was the most significant factors of community structure. Other significant factors were: duration of diagnosis (P=0.009), viral load (P=0.022) and anti-HCV (P=0.018). ART was positively correlated with Megamonas and negatively correlated with Prevotella, Roseburia and Faecalibacterium, while the other three factors of duration of diagnosis, viral load and anti-HCV were positively correlated with Prevotella, Roseburia and Faecalibacterium and negatively correlated with Megamonas. In the ART-naive population, duration of diagnosis (P=0.003) were the factors significantly associated with community structure. Duration of diagnosis was positively correlated with Roseburia, Faecalibacterium, Megamonas and Prevotella and negatively correlated with Bacteroides. Conclusion: ART and duration of diagnosis were factors significantly associated with gut microbial community structure and had a significant impact on multiple high-abundance genera.

Determining the protective effects of Ma-Mu-Ran Antidiarrheal Capsules against acute DSS-induced enteritis using 16S rRNA gene sequencing and fecal metabolomics / 中国天然药物

Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) is traditional Chinese medicine that has been used to treat diarrhea caused by acute enteritis (AE) and bacillary dysentery in Xinjiang (China) for many years. However, the potential therapeutic mechanism of MMRAC for AE and its regulatory mechanism on host metabolism is unclear. This study used fecal metabolomics profiling with GC/MS and 16S rRNA gene sequencing analysis to explore the potential regulatory mechanisms of MMRAC on a dextran sulfate sodium salt (DSS)-induced mouse model of AE. Fecal metabolomics-based analyses were performed to detect the differentially expressed metabolites and metabolic pathways. The 16S rRNA gene sequencing analysis was used to assess the altered gut microbes at the genus level and for functional prediction. Moreover, Pearson correlation analysis was used to integrate differentially expressed metabolites and altered bacterial genera. The results revealed that six intestinal bacteria and seven metabolites mediated metabolic disorders (i.e., metabolism of amino acid, carbohydrate, cofactors and vitamins, and lipid) in AE mice. Besides, ten altered microbes mediated the differential expression of eight metabolites and regulated these metabolisms after MMRAC administration. Overall, these findings demonstrate that AE is associated with metabolic disorders and microbial dysbiosis. Further, we present that MMRAC exerts protective effects against AE by improving host metabolism through the intestinal flora.

Features of intestinal flora in children with food protein-induced proctocolitis based on high-throughput sequencing / 中国当代儿科杂志

OBJECTIVES@#To study the features of intestinal flora in children with food protein-induced proctocolitis (FPIP) by high-throughput sequencing.@*METHODS@#A total of 31 children, aged <6 months, who experienced FPIP after exclusive breastfeeding and attended the outpatient service of the Third Affiliated Hospital of Zunyi Medical University from October 2018 to February 2021 were enrolled as the FPIP group. Thirty-one healthy infants were enrolled as the control group. Fecal samples were collected to extract DNA for PCR amplification. High-throughput sequencing was used to perform a bioinformatics analysis of 16S rDNA V3-V4 fragments in fecal samples.@*RESULTS@#The diversity analysis of intestinal flora showed that compared with the control group, the FPIP group had a lower Shannon index for diversity (P>0.05) and a significantly higher Chao index for abundance (P<0.01). At the phylum level, the intestinal flora in both groups were composed of Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Compared with the control group, the FPIP group had a significant reduction in the composition ratio of Actinobacteria (P<0.001) and a significant increase in the composition ratio of Proteobacteria (P<0.05). At the genus level, the intestinal flora in the FPIP group were mainly composed of Escherichia, Clostridium, Enterococcus, Klebsiella, and Bifidobacterium, and the intestinal flora in the control group were mainly composed of Bifidobacterium and Streptococcus. Compared with the control group, the FPIP group had a significant reduction in the composition ratio of Bifidobacterium and Ruminococcus (P<0.05) and significant increases in the composition ratios of Clostridium and Shigella (P<0.05).@*CONCLUSIONS@#Compared with the control group, the FPIP group has a reduction in the diversity of intestinal flora and an increase in their abundance, and there are certain differences in several bacterial genera. These results suggest that changes in the composition of intestinal flora at genus level may play an important role in the development and progression of FPIP.

Change in intestinal flora after treatment in children with focal epilepsy / 中国当代儿科杂志

OBJECTIVES@#To study the difference in intestinal flora between children with focal epilepsy and healthy children and the change in intestinal flora after treatment in children with epilepsy.@*METHODS@#A total of 10 children with newly diagnosed focal epilepsy were recruited as the case group and were all treated with oxcarbazepine alone. Their clinical data were recorded. Fecal specimens before treatment and after 3 months of treatment were collected. Fourteen aged-matched healthy children were recruited as the control group. Total bacterial DNA was extracted from the fecal specimens for 16S rDNA sequencing and bioinformatics analysis.@*RESULTS@#After 3 months of carbamazepine treatment, the seizure frequency was reduced by >50% in the case group. At the phylum level, the abundance of Actinobacteria in the case group before treatment was significantly higher than that in the control group (P<0.05), and it was reduced after treatment (P<0.05). At the genus level, the abundances of Escherichia/Shigella, Streptococcus, Collinsella, and Megamonas in the case group before treatment were significantly higher than those in the control group (P<0.05), and the abundances of these bacteria decreased significantly after treatment (P<0.05).@*CONCLUSIONS@#There is a significant difference in intestinal flora between children with focal epilepsy and healthy children. Oxcarbazepine can significantly improve the symptoms and intestinal flora in children with epilepsy.

Evaluation of the Gastric Microbiome in Patients with Chronic Superficial Gastritis and Intestinal Metaplasia / 中国医学科学杂志(英文版)

Objective To evaluate the gastric microbiome in patients with chronic superficial gastritis (CSG) and intestinal metaplasia (IM) and investigate the influence of Helicobacter pylori (H. pylori) on the gastric microbiome. Methods Gastric mucosa tissue samples were collected from 54 patients with CSG and IM, and the patients were classified into the following four groups based on the state of H. pylori infection and histology: H. pylori-negative CSG (n=24), H. pylori-positive CSG (n=14), H. pylori-negative IM (n=11), and H. pylori-positive IM (n=5). The gastric microbiome was analyzed by 16S rRNA gene sequencing. Results H. pylori strongly influenced the bacterial abundance and diversity regardless of CSG and IM. In H. pylori-positive subjects, the bacterial abundance and diversity were significantly lower than in H. pylori-negative subjects. The H. pylori-negative groups had similar bacterial composition and bacterial abundance. The H. pylori-positive groups also had similar bacterial composition but different bacterial relative abundance. The relative abundance of Neisseria, Streptococcus, Rothia, and Veillonella were richer in the I-HP group than in G-HP group, especially Neisseria (t=175.1, P<0.001). Conclusions The gastric microbial abundance and diversity are lower in H. pylori- infected patients regardless of CSG and IM. Compared to H. pylori-positive CSG group and H. pylori-positive IM, the relative abundance of Neisseria, Streptococcus, Rothia, and Veillonella is higher in H. pylori-positive patients with IM than in H. pylori-positive patients with CSG, especially Neisseria.

Fecal transplantation can alleviate tic severity in a Tourette syndrome mouse model by modulating intestinal flora and promoting serotonin secretion / 中华医学杂志(英文版)

BACKGROUND@#: Tourette syndrome (TS) is a neuropsychiatric disorder with onset in childhood that warrants effective therapies. Gut microbiota can affect central physiology and function via the microbiota-gut-brain axis. Therefore, the gut microbiota plays an important role in some mental illnesses. A small clinical trial showed that fecal microbiota transplantation (FMT) may alleviate TS symptoms in children. Herein, FMT effects and mechanisms were explored in a TS mouse model.@*METHODS@#: TS mice model (TSMO) (n = 80) were established with 3,3'-iminodipropionitrile, and 80 mice were used as controls. Mice were grouped into eight groups and were subjected to FMT with feces from children or mice with or without TS, or were given probiotics. Fecal specimens were collected 3 weeks after FMT. 16S rRNA sequencing, behavioral observation, and serum serotonin (5-HT) assay were performed. Differences between groups were analyzed using Mann-Whitney U test and Kolmogorov-Smirnov (KS) tests.@*RESULTS@#: A total of 18 discriminative microbial signatures (linear discriminant analysis score > 3) that varied significantly between TS and healthy mice (CONH) were identified. A significant increase in Turicibacteraceae and Ruminococcaceae in TSMO after FMT was observed (P  < 0.05). Compared with non-transplanted TSMO, the symptoms of those transplanted with feces from CONH were alleviated (W = 336, P = 0.046). In the probiotic and FMT experiments, the serum 5-HT levels significantly increased in TSMO that received probiotics (KS = 1.423, P = 0.035) and in those transplanted with feces from CONH (W = 336.5, P = 0.046) compared with TSMO without transplantation.@*CONCLUSIONS@#: This study suggests that FMT may ameliorate TS by promoting 5-HT secretion, and it provides new insights into the underlying mechanisms of FMT as a treatment for TS.

Avaliação da microbiota intestinal em adultos com esofagite erosiva / Assessment of the intestinal microbiota in adults with erosive esophagitis

ABSTRACT BACKGROUND: The intestinal microbiota influences the appropriate function of the gastrointestinal tract. Intestinal dysbiosis may be associated with a higher risk of esophageal lesions, mainly due to changes in gastroesophageal motility patterns, elevation of intra-abdominal pressure, and increased frequency of transient relaxation of the lower esophageal sphincter. OBJECTIVE: The aim of this study was to evaluate the intestinal microbiota in individuals with erosive esophagitis and in healthy individuals using metagenomics. METHODS: A total of 22 fecal samples from adults aged between 18 and 60 years were included. Eleven individuals had esophagitis (eight men and three women) and 11 were healthy controls (10 men and one woman). The individuals were instructed to collect and store fecal material into a tube containing guanidine solution. The DNA of the microbiota was extracted from each fecal samples and PCR amplification was performed using primers for the V4 region of the 16S rRNA gene. The amplicons were sequenced using the Ion Torrent PGM platform and the data were analyzed using the QIIME™ software version 1.8. Statistical analyses were performed using the Mann-Whitney non-parametric test and the ANOSIM non-parametric method based on distance matrix. RESULTS: The alpha-diversity and beta-diversity indices were similar between the two groups, without statistically significant differences. There was no statistically significant difference in the phylum level. However, a statistically significant difference was observed in the abundance of the family Clostridiaceae (0.3% vs 2.0%, P=0.032) and in the genus Faecaliumbacterium (10.5% vs 4.5%, P=0.045) between healthy controls and esophagitis patients. CONCLUSION: The findings suggest that reduced abundance of the genus Faecaliumbacterium and greater abundance of the family Clostridiaceae may be risk factors for the development of erosive esophagitis. Intervention in the composition of the intestinal microbiota should be considered as an adjunct to current therapeutic strategies for this clinical condition.

RESUMO CONTEXTO: A doença do refluxo gastroesofágico (DRGE) é uma das enfermidades mais comuns na prática clínica e possui fisiopatologia multifatorial. Disbiose da microbiota intestinal pode ter influência em mecanismos envolvidos nesta doença, como mudanças nos padrões motores gastrointestinais, elevação da pressão intra-abdominal e aumento da frequência de relaxamentos transitórios do esfíncter esofágico inferior. Contudo, a avaliação da microbiota intestinal, neste contexto, ainda é pouco documentada. OBJETIVO: Este estudo avaliou a microbiota bacteriana intestinal, em indivíduos com doença do refluxo gastroesofágico erosivo e em indivíduos saudáveis, utilizando técnicas de metagenômica. MÉTODOS: Estudo incluiu amostras fecais de 22 adultos, com idades entre 18 e 60 anos: 11 com esofagite erosiva (oito homens e três mulheres) e 11 controles saudáveis (dez homens e uma mulher). Os pacientes foram orientados a coletar e armazenar o material fecal em tubo contendo solução de guanidina. O DNA da microbiota foi extraído das amostras de fezes e amplificação por PCR foi realizada usando iniciadores para a região V4 do gene 16S rRNA. Os amplicons foram seqüenciados usando a plataforma Ion PGM Torrent e os dados foram analisados usando o software QIIME™ versão 1.8 (Quantitative Insights Into Microbial Ecology). Análise de estatística foi realizada utilizando-se o teste não paramétrico de Mann-Whitney e o teste ANOSIM, método não paramétrico baseado em matriz de distância. RESULTADOS: Os índices de alfa-diversidade e beta-diversidade foram semelhantes entre os dois grupos, sem diferença estatisticamente significante. Não houve diferença estatisticamente significante no nível de filo, classe e ordem. Entretanto, observou-se diferença estatisticamente significante na abundância da família Clostridiaceae (0,3% vs 2,0%, P=0,032) e no gênero Faecaliumbacterium (10,5% vs 4,5%, P=0,045) entre controles saudáveis e pacientes com DRGE erosiva, respectivamente. CONCLUSÃO: Os achados sugerem que menor abundância do gênero Faecaliumbacterium e maior abundância da família Clostridiaceae, nos pacientes com DRGE, podem influenciar na fisiopatologia desta doença.

Using PacBio sequencing to investigate the effects of treatment with lactic acid bacteria or antibiotics on cow endometritis

BACKGROUND: Endometritis is the most common disease of dairy cows and traditionally treated with antibiotics. Lactic acid bacteria can inhibit the growth of pathogens and also have potential for treatment of endometritis. Using PacBio single-molecule real-time sequencing technology, we sequenced the fulllength l6S rRNA of the microbiota in uterine mucus samples from 31 cows with endometritis, treated with lactic acid bacteria (experimental [E] group) and antibiotics (control [C] group) separately. Microbiota profiles taken before and after treatment were compared. RESULTS: After both treatments, bacterial species richness was significantly higher than before, but there was no significant difference in bacterial diversity. Abundance of some bacteria increased after both lactic acid bacteria and antibiotic treatment: Lactobacillus helveticus, Lactococcus lactis, Lactococcus raffinolactis, Pseudomonas alcaligenes and Pseudomonas veronii. The bacterial species that significantly decreased in abundance varied depending on whether the cows had been treated with lactic acid bacteria or antibiotics. Abundance of Staphylococcus equorum and Treponema brennaborense increased after lactic acid bacteria treatment but decreased after antibiotic treatment. According to COG-based functional metagenomic predictions, 384 functional proteins were significantly differently expressed after treatment. E and C group protein expression pathways were significantly higher than before treatment (p < 0.05). CONCLUSIONS: In this study, we found that lactic acid bacteria could cure endometritis and restore a normal physiological state, while avoiding the disadvantages of antibiotic treatment, such as the reductions in abundance of beneficial microbiota. This suggests that lactic acid bacteria treatment has potential as an alternative to antibiotics in the treatment of endometritis in cattle.

'Candidatus Mycoplasma haemoalbiventris' and tick-borne pathogens screening in white-eared opossums (Didelphis albiventris) from Curitiba and Foz do Iguaçu Cities, Paraná State, southern Brazil / 'Candidatus Mycoplasma haemoalbiventris' e triagem para patógenos transmitidos por carrapatos em gambás de orelha branca (Didelphis albiventris) das cidades de Curitiba e Foz do Iguaçu, Estado do Paraná, sul do Brasil

Abstract Hemoplasmas are epierythrocytic bacteria that infect mammals. 'Candidatus Mycoplasma haemoalbiventris' was detected in white-eared opossums (Didelphis albiventris) from southern and central-western Brazil. The present study aimed at: i) screening opossums for tick-borne (TBP) pathogens (Piroplasmida and Anaplasmataceae) and ii) detecting and characterizing hemoplasma species infecting opossums from Curitiba and Foz do Iguaçu cities in the Paraná State, southern Brazil. Thirty blood samples from white-eared opossums were evaluated by PCR assays. Animals were not infested by ectoparasites. The mammalian endogenous gapdh gene was consistently amplified in all samples. All opossums tested negative for Theileria/Babesia spp. and Ehrlichia/Anaplasma spp. by PCR based on 18S rRNA and 16S rRNA genes, respectively. A genus-specific PCR assay based on the 16S rRNA gene of hemoplasmas showed that three/13 (23.08%; CI 95%: 8.18-50.26%) opossums from Foz do Iguaçu were positive for hemotropic Mycoplasma sp. All opossums from Curitiba tested negative for hemoplasmas. Sequencing of both the 16S and 23S rRNA genes revealed that the animals were infected by 'Ca. M. haemoalbiventris'. Although 'Ca. M. haemoalbiventris' is prevalent in opossums in Brazil, clinical signs associated with its infection and its putative vectors remain unknown.

Resumo Hemoplasmas são bactérias epieritrocíticas que infectam mamíferos. 'Candidatus Mycoplasma haemoalbiventris' foi detectado previamente em gambás-de-orelha-branca (Didelphis albiventris) das regiões sul e centro-oeste do Brasil. O presente estudo objetivou: i) triar os gambás para as doenças transmitidas por carrapatos (Piroplasmida e Anaplasmataceae); e ii) detectar e caracterizar as espécies de hemoplasma que infectam gambás nas cidades de Curitiba e Foz do Iguaçu, no Estado do Paraná, sul do Brasil. Trinta amostras de sangue de gambás-de-orelha-branca foram analisadas por PCR. Os animais não estavam infestados por ectoparasitos. O gene endógeno de mamífero gapdh foi amplificado em todas as amostras. Todos os gambás testaram negativos para Theileria/Babesia spp. e Ehrlichia/Anaplasma spp. por PCR, respectivamente, para os genes 18S rRNA e 16S rRNA. Uma PCR gene-específica, baseada no gene 16S rRNA de hemoplasmas, mostrou que três/13 (23,08%; CI 95%: 8,18-50,26%) gambás de Foz do Iguaçu foram positivos para Mycoplasma sp. hemotrópico. Todos os gambás de Curitiba testaram negativos para hemoplasmas. O sequenciamento de fragmentos dos genes 16S e 23S rRNA revelou que os animais estavam infectados pelo 'Ca. M. haemoalbiventris'. Embora 'Ca. M. haemoalbiventris' seja prevalente em gambás no Brasil, os sinais clínicos associados à infecção e os prováveis vetores permanecem desconhecidos.