Oral Metagenomic Analysis Techniques / 치위생과학회지

The modern era of microbial genome analysis began in earnest in the 2000s with the generalization of metagenomics and gene sequencing techniques. Studying complex microbial community such as oral cavity and colon by a pure culture is considerably ineffective in terms of cost and time. Therefore, various techniques for genomic analysis have been developed to overcome the limitation of the culture method and to explore microbial communities existing in the natural environment at the gene level. Among these, DNA fingerprinting analysis and microarray chip have been used extensively; however, the most recent method of analysis is metagenomics. The study summarily examined the overview of metagenomics analysis techniques, as well as domestic and foreign studies on disease genomics and cluster analysis related to oral metagenome. The composition of oral bacteria also varies across different individuals, and it would become possible to analyze what change occurs in the human body depending on the activity of bacteria living in the oral cavity and what causality it has with diseases. Identification, isolation, metabolism, and presence of functional genes of microorganisms are being identified for correlation analysis based on oral microbial genome sequencing. For precise diagnosis and treatment of diseases based on microbiome, greater effort is needed for finding not only the causative microorganisms, but also indicators at gene level. Up to now, oral microbial studies have mostly involved metagenomics, but if metatranscriptomic, metaproteomic, and metabolomic approaches can be taken together for assessment of microbial genes and proteins that are expressed under specific conditions, then doing so can be more helpful for gaining comprehensive understanding.

How Microbes Shape Their Communities? A Microbial Community Model Based on Functional Genes / 基因组蛋白质组与生物信息学报·英文版

Exploring the mechanisms of maintaining microbial community structure is important to understand biofilm development or microbiota dysbiosis. In this paper, we propose a functional gene-based composition prediction (FCP) model to predict the population structure composition within a microbial community. The model predicts the community composition well in both a low-complexity community as acid mine drainage (AMD) microbiota, and a complex community as human gut microbiota. Furthermore, we define community structure shaping (CSS) genes as functional genes crucial for shaping the microbial community. We have identified CSS genes in AMD and human gut microbiota samples with FCP model and find that CSS genes change with the conditions. Compared to essential genes for microbes, CSS genes are significantly enriched in the genes involved in mobile genetic elements, cell motility, and defense mechanisms, indicating that the functions of CSS genes are focused on communication and strategies in response to the environment factors. We further find that it is the minority, rather than the majority, which contributes to maintaining community structure. Compared to health control samples, we find that some functional genes associated with metabolism of amino acids, nucleotides, and lipopolysaccharide are more likely to be CSS genes in the disease group. CSS genes may help us to understand critical cellular processes and be useful in seeking addable gene circuitries to maintain artificial self-sustainable communities. Our study suggests that functional genes are important to the assembly of microbial communities.

Seroprevalence of microbial organisms during routine infertility evaluation at University of Benin Teaching Hospital, Benin-City, Nigeria

Background: The association of genital microorganism with infertility has been documented but no consensus exists. Understanding their prevalence amongst infertile clients may assist in facilitating better screening protocols. The objective of this study is to determine the prevalence of microorganisms routinely screened among women undergoing infertility evaluation at the University of Benin Teaching Hospital. Methods: A three year (January 2015 to December 2017) retrospective survey of all patients evaluated for infertility at the assisted reproduction unit of the hospital was undertaken. Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, cytomegalovirus (CMV), hepatitis B (HBV), hepatitis C (HCV) virus and the human immunodeficiency virus (HIV) were microorganisms serologically assayed at the unit. We analyzed data containing patients' demography and results of serological assay of these microorganisms. Results: There were 576 patients (288 couples) who completed their microbiological evaluation during the study period. The mean age (years) of female partners was 38.2±5.7, while the mean age of the male partners was 42.7±6.1. The frequency of CMV positive assay for infertile couples was 129 (22.4%); C. trachomatis 125 (21.7%); M. hominis 92 (15.9%) and U. urealyticum 76 (13.2%). Overall, more women (50.7%) were seropositive compared to men (26%). HIV was positive in 10 patients (1.73%) with 60% being women. HBV was seropositive in 8 (1.4%) (women 62.5% and men 37.5%) while HCV was positive in only 2 (0.3%) patient. Majority (over 80%) of couples were sero-discordant with 20% (2) concordance rate for HIV and 12.5% (1) for hepatitis B. Conclusion: Despite a relatively high seroprevalence rate of the studied microorganisms, the documented uncertainty on their association with infertility or its treatment limits justification for incorporation of routine screening of microbiological organisms into standard protocols for evaluation of infertile couples. A robust study on the impact of genital microorganism on specific infertility variables with comparison to fertile controls is recommended

Sequence-based identification of microbial contaminants in non-parenteral products

ABSTRACT Phenotypic profiles for microbial identification are unusual for rare, slow-growing and fastidious microorganisms. In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rRNA sequencing has played a pivotal role in the accurate identification of microorganisms and the discovery of novel isolates in microbiology laboratories. The 16S rRNA region is universally distributed among microorganisms and is species-specific. Accordingly, the aim of our study was the genotypic identification of microorganisms isolated from non-parenteral pharmaceutical formulations. DNA was separated from five isolates obtained from the formulations. The target regions of the rRNA genes were amplified by PCR and sequenced using suitable primers. The sequence data were analyzed and aligned in the order of increasing genetic distance to relevant sequences against a library database to achieve an identity match. The DNA sequences of the phylogenetic tree results confirmed the identity of the isolates as Bacillus tequilensis, B. subtilis, Staphylococcus haemolyticus and B. amyloliqueficians. It can be concluded that 16S rRNA sequence-based identification reduces the time by circumventing biochemical tests and also increases specificity and accuracy.

RESUMO Os perfis fenotípicos para identificação microbiana são incomuns para micro-organismos raros, de crescimento lento e exigentes. Na última década, em resultado do uso generalizado de PCR e sequenciação de DNA, a sequenciação do rRNA 16S tem desempenhado papel crucial na identificação precisa do micro-organismo e a descoberta de novos isolados em laboratórios de microbiologia. A região de rRNA 16S é universalmente distribuída entre micro-organismos e é espécie-específica. A genotipagem foi realizada sobre os organismos isolados a partir de formulações farmacêuticas não parenterais. O DNA foi separado dos cinco isolados obtidos a partir das formulações. As regiões alvo dos genes de rRNA foram amplificados por PCR e sequenciados utilizando os iniciadores adequados. Os dados dos sequência foram analisados e alinhados na ordem crescente de distância genética de sequências relevantes contra biblioteca de dados para obter a identidade. A sequência de DNA de árvores filogenéticas confirma a identidade dos isolados como Bacillus-tequilensis, B. subtilis, Staphylococcus haemolyticus e B. amyloliqueficians. Pode-se concluir identificação baseada na sequência do rRNA 16S reduz o tempo por evitar testes bioquímicos e também aumenta a especificidade e a precisão.

Participação da resposta inflamatória induzida por Chlamydophila pneumoniae e Mycoplasma pneumoniae no infarto agudo do miocárdio / Participation of the inflammatory response induced by Chlamydophila pneumoniae and Mycoplasma pneumoniae in acute myocardial infarction

Os agentes infecciosos têm sido considerados iniciadores da desestabilização da placa de ateroma. Este mecanismo pode estar relacionado a uma intensificação do processo inflamatório através da interação dos receptores de membrana CD14 e TLR com os microorganismos. Para avaliar esta hipótese, estudou-se a participação da resposta inflamatória induzida por Chlamydophila pneumoniae (Cp) e Mycoplasma pneumoniae (Mp) em indivíduos com infarto agudo do miocárdio (IAM). Avaliou-se também, a possível associação entre polimorfismos dos genes CD14, TLR2, TLR4 e TNFA e a expressão dos genes IL6, TLR2, TLR4 e TNFA em leucócitos do sangue periférico, assim como a sua associação com o IAM. Para isso, foi realizado um estudo caso-controle constituído por pacientes com IAM e por indivíduos sem evidência de doença cardiovascular (grupo controle). As imunoglobulinas IgM e IgG séricas anti-Cp foram detectadas por imunofluorescência indireta. O DNA dos agentes infecciosos foi detectado no sangue periférico pela PCR em tempo real. A genotipagem dos polimorfismos TNFA -308G>A, IL6 -174G>C, CD14 -260C>T, TLR4 (Asp299Gli e Thr39911e) e TLR2 Arg753Gln e a quantificação relativa da expressão gênica nas células sanguíneas foram analisados pela PCR em tempo real. A porcentagem de positividade para DNA de Cp foi de 18,0% e 8,1% nos grupos IAM e controle (p=0,071), respectivamente, (p=0,071). Foram positivos para DNA de Mp, 5,0% e 11,2% dos indivíduos nos grupos IAM e controle, respectivamente (p=0,318). Sete indivíduos (7,1%) do grupo IAM tiveram títulos anti-Cp IgG positivos (1:512) e 3,9% dos indivíduos do grupo controle (p=0,718). A expressão do TLR4 foi significantemente menor no grupo IAM (0,00113±0,00102) comparado ao grupo controle (0,00144±0,000806; p=0,003). As frequências genotípicas e alelicas dos polimorfismos TNFA -308G>A, CD14 -260C>T, TLR4 (Asp299GIi e Thr39911e) e TLR2 Arg753Gln foram similares entre os grupos estudados (p>0,05) sugerindo que esses polimorfismos não estão associados com IAM nesta amostra populacional. No grupo IAM, houve associação entre o genótipo -260CT+TI CD14 com títulos IgG anti-Cp detectados na diluição 1:16 (p=0,042). Da mesma forma, o alelo A do polimorfismo -308G>A TNF-α foi associado com títulos positivos de IgG anti-Cp na diluição 1:512 (p=0,0058). No grupo IAM, pacientes positivos para DNA de Cp tiveram maiores concentrações de fibrinogênio do que pacientes negativos para este agente infeccioso (541,8±161,5mg/dL e 450,5±196,8mg/dL, respectivamente; p=0.043). Os agentes infecciosos Chlamydophila pneumoniae e Mycoplasma pneumoniae não foram significantemente mais frequentes em indivíduos que tiveram infarto agudo do miocárdio em relação ao grupo controle, porém houve uma associação, no grupo IAM, entre positividade para DNA de C. pneumoniae e concentrações mais elevadas de fibrinogênio

Atheroma plaque instability has been attributed to the presence of some infectious agents. This mechanism may be related with increased stimulus of inflammatory process through interactions of CD14 and TLR with infectious agents. In this present study, it was evaluated the association of the presence of Chlamydophila pneumoniae and Mycoplasma pneumonia with acute myocardial infarction (AMI). A case-control study was conducted with AMI patients and non-AMI individuais as controls. Immunoglobulin G (lgG) and IgM antibodies anti-Chlamydophila pneumoniae were detected by indirect immunifluorescent assay and the Cp DNA and Mp DNA were detected by real time PCR (RT-PCR) in peripheral blood cells. Using the same method, the individuals were genotyped and the gene expressions of TLR2, TLR4, IL-6 e TNF-α were evaluated by RT-qPCR. In AMI patients, Cp DNA and Mp DNA were positive in 18,0% and 5,0% samples, respectively. In controls, 8,1% and 11,2% were positive for Cp DNA and Mp DNA, respectively. TLR4 expression was significantly decreased in AMI patients (0.00113±0.001 02) compared with controls (0.00144±0.000S06; p=0.003). The frequencies of -308G>A TNF-α., -260C>T CD14, Asp299Gli TLR4, Thr39911e TLR4e Arg753Gln TLR2 SNPs in AMI group were similar to those found in controls. On the other hand, In AMI group, the -260CT+TT CD14 genotype was associated with anti-CP IgG antibody titer of 1/16. Likewise, the rare allele of -308G>A TNF-α was associated with anti-CP IgG antibody titer of 1/16. Cp DNA positive patients had high concentration of fibrinogen when compared with negative patients. In conclusion, Cp DNA and Mp DNA positivity were not associated with AMI