Region Of 16S rDNA Gene Research Articles

First report of Candidatus Phytoplasma australasia (16SrII- subgroup D) associated with virescence of Chia (Salvia hispanica L.) from India.

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 - 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

Alterations of the Human Gut Microbiome in Patients With Hidradenitis Suppurativa: A Case-control Study and Review of the Literature.

Hidradenitis suppurativa (HS) is a chronic and systemic inflammatory disease that extends beyond the skin. The role of gut microbiome (GM) alterations in the pathogenesis of inflammatory and autoimmune disorders is remarkable. Based on the hypothesis that dysbiosis in the GM may trigger systemic inflammation in the pathogenesis of HS, this study aimed to investigate whether the GM is altered in HS patients compared with healthy subjects. In the present case-control study, fecal samples from 15 patients with HS and 15 age- and sex-matched healthy individuals were collected and analyzed using 16S rRNA-based metagenomic analysis, New Generation Sequencing (NGS). The V3 and V4-hypervariable regions of the bacterial 16S rDNA gene were amplified from all samples and sequenced by the Illumina MiSeq platform. Bioinformatics analyses were performed in QIIME2. Shannon alpha diversity index showed significantly reduced diversity in HS patients (P = 0.048). Bray-Curtis Dissimilarity and Jaccard Distance revealed that the gut microbial composition of HS patients was significantly distinctive from that of controls (P = 0.01 and P = 0.007, respectively). The relative abundance of unclassified Clostridiales, unclassified Firmicutes, and Fusicatenibacter in HS was significantly lower than that in controls (P = 0.005, P = 0.029, and P = 0.046, respectively). This study indicated that significant alterations in the GM of HS patients could play a critical role in the pathogenesis of HS and might be a trigger for systemic inflammation. Increased understanding of the pathogenesis of HS will shed light on the new potential therapeutic targets and novel treatment options.

Comparative and analytical characterization of the oral bacteriome of smokeless tobacco users with oral squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) is the most common type of head and neck cancer worldwide. Smokeless tobacco (SLT) has been well proven for its role in oral carcinogenesis due to the abundance of several carcinogens. However, the role of inhabitant microorganisms in the oral cavity of smokeless tobacco users has not yet been well explored in the context of OSCC. Therefore, the present investigation was conceived to analyze the oral bacteriome of smokeless tobacco users having OSCC (CP group). With the assistance of illumina-based sequencing of bacterial-specific V3 hypervariable region of 16S rDNA gene, 71,969 OTUs (operational taxonomic units) were categorized into 18 phyla and 166 genera. The overall analysis revealed that the oral bacteriome of the patients with OSCC, who were smokeless tobacco users, was significantly different compared to the healthy smokeless tobacco users (HTC group) and non-users (HI users). The appearance of 14 significantly abundant genera [FDR (false discovery rate) adjusted probability value of significance (p value) < 0.05] among the CP group showed the prevalence of tobacco-specific nitrosamines forming bacteria (Staphylococcus, Fusobacterium, and Campylobacter). The functional attributes of the oral bacteriome of the CP group can also be correlated with the genes involved in oncogenesis. This study is the first report on the oral bacteriome of Indian patients with OSCC who were chronic tobacco chewers. The results of the present study will pave the way to understand the influence of smokeless tobacco on the oral bacteriome of OSCC patients. KEY POINTS: • Oral bacteriome of OSCC patients differ from healthy smokeless tobacco (SLT) users and SLT non-users. • Smokeless tobacco influences the oral bacteriome of OSCC group. • Oral bacteriome specific diagnostics may be developed for pre-diagnosis of oral cancer.

Characteristics and Clinical Significance of Intestinal Microbiota in Patients with Chronic Hepatitis B Cirrhosis and Type 2 Diabetes Mellitus.

Background Chronic hepatitis B cirrhosis is often accompanied by glucose metabolism disorder, and intestinal microbiota was closely related to both cirrhosis and diabetes. There are few studies on the role of intestinal microbiota in hepatitis B liver cirrhosis and diabetes mellitus (LCDM). The purpose of this study was to investigate the characteristics of intestinal microbiota in patients with LCDM and to evaluate the relationship between the severity of intestinal microbiota imbalance and clinical significance. Methods A case-controlled study was conducted. People who met the inclusion and exclusion criteria of chronic HBV-related liver cirrhosis (LC), LCDM, and healthy controls (HC) were enrolled in, and their fecal and blood samples were collected. The V3-V4 region of 16s rDNA gene of fecal microbiota was sequenced; the bioinformatics analysis including α-diversity, β-diversity, and linear discriminant analysis (LDA) effect size (LEfSe) was performed; and the correlation between bacteria and clinical indexes was analyzed. Results A total of 70 participants completed fecal and blood tests, including 20 HC, 20 LCDM, and 30 LC. The α diversity of intestinal microbiota in the LCDM decreased than that in the HC. The abundance of Proteobacteria, Streptococcus, Escherichia-Shigella, and Lactobacillus increased, while the abundance of Bacteroidota, Bacteroides, Prevotella, Faecalibacterium, and Lachnospira decreased in the LCDM compared with the HC. The abundance of Lactobacillus, Roseburia, and Veillonella and the degree of hepatitis B cirrhosis dysbiosis indicator (HBCDI) increased in the LCDM than in the LC. The abundance of Escherichia-Shigella, Veillonella, and Lactobacillus positively correlated with liver injury and fasting blood glucose (FBG) level. The abundance of Escherichia-Shigella, Veillonella, Streptococcus, and Lactobacillus increased more significantly when FBG and glycosylated hemoglobin level increased. Conclusion Intestinal microbiota of patients with LCDM was significantly disordered, and the degree was more serious than that cirrhosis patients without diabetes.

First Report of Pectobacterium aroidearum Causing Soft Rot in Olecranon Honey Peach (Prunus persica) in China.

Olecranon honey peach (Prunus persica L.) is a popular fruit tree cultivated in Guangdong Province of China. Due to its excellent economic values and popularity, it has recently been widely adopted and planted in several other southern Provinces and Autonomous Region in China, including Yunnan, Hunan, Jiangxi, Guizhou, and Guangxi. In Lianping County of Guangdong Province alone, the annual peach fruit production was about 78,800 tonnes (Xie et al. 2017). In July 2021, peach fruits showing soft rot symptoms were collected from an olecranon honey peach plantation in Lechang, Guangdong, China. Symptoms included tissue disintegration with bacterial oozes and rotting smells. To isolate the causal agent of soft rot in the peach fruits, the bacterial oozes from various rotted fruits were streaked on the modified YEB agar plate (Huang et al. 2021), and 21 bacterial colonies were selected for PCR amplification using the primers targeting the conserved region of 16S rDNA gene (Wei et al. 2020). A blastN analysis of the DNA sequences of the obtained PCR fragments in NCBI website indicated that 17 isolates named as ZL strains were potential bacterial species of Pectobacterium with about 99% similarity (Genbank accession number of ZL1: OK189602) to Pectobacterium aroidearum SCRI 109T (Genbank accession number: NR_159926). Three of them (ZL1, ZL2 and ZL3) were selected for assay of pathogenicity. The bacterial suspensions (10 μl, 1×106 CFU/ml) of strains ZL1, ZL2 and ZL3 were injected into olecranon honey peach fruits by using a syringe. A portion of peach fruits were similarly injected with sterile distilled water as the negative control. After 18 h incubation at 25 °C, the typical symptom of soft rot, i.e., tissue decay, became visible on the peach fruits inoculated with the bacterial suspensions. After inoculation for 42 h, bacterial oozes were exuded from rotting tissues. Peach fruits without injuries were also sprayed with the bacterial suspensions under the same conditions, but decay symptoms were not observed, suggesting that the bacterial infection needs the wounding or injuries. To fulfill the Koch's postulates, bacterial colonies were re-isolated from bacterial oozes, and their conserved region of 16S rDNA fragments were amplified and sequenced. Bioinformatics analysis of the DNA sequence data confirmed that all the isolated colonies were Pectobacterium strains. Using the Biolog Gen III system, the representative strain ZL1 was identified as Pectobacterium (SIM 0.56). Transmission electron microscopy analysis showed that the bacterial cells of strain ZL1 were rod-shaped with peripheral flagella. To further determine the species of ZL strains, eight housekeeping genes (acnA, gapA, icd, mdh, mtlD, pgi, proA and rpoS) were analyzed by the methods described previously (Nabhan et al. 2013). The amplified DNA sequences analyzed by the blastN program in NCBI showed that the sequences of eight housekeeping genes from strains ZL1, ZL2 and ZL3 were identical to each other (Genbank accession number: OK274248 to OK274255), and most of the gene sequences shared over 99% similarity to their counterparts in P. aroidearum L6 (Genbank accession number: NZ_CP065044) (Xu et al. 2021), except that the acnA and proA genes showed about 98% and 96% similarity respectively to the corresponding genes of P. aroidearum L6. In addition, the multi-locus sequence analysis (MLSA) using DNA sequences of above eight housekeeping genes showed that ZL strains were grouped with other P. aroidearum strains. Taken together, the results of molecular and biochemical assays confirmed that ZL strains isolated from olecranon honey peach fruits were P. aroidearum. To our knowledge, this is the first report of P. aroidearum causing soft rot disease in olecranon honey peach in China. P. aroidearum is a relatively newly described soft rot pathogen (Nabhan et al. 2013). More recently, the pathogen was found causing soft rot infections in lettuce, Chinese cabbage, pepper (Capsicum annuum) fruits, konjac, carrot and Syngonium podophyllum (Barroso et al. 2019; Moraes et al. 2020; Sun et al. 2019; Tang et al. 2020; Xu et al. 2021). The results of this study add a new plant species to the host range of P. aroidearum.

Fecal microbiota profiling in irritable bowel syndrome and inflammatory bowel disease patients with irritable bowel syndrome-type symptoms

BackgroundThe intestinal microbiota is thought to be involved in the occurrence of inflammatory bowel disease in remission with irritable bowel syndrome (IBS)-type symptoms, but the specific distinct profile of these bacteria remains unclear. This cross-sectional study aims to investigate the fecal microbiota profiling in patients with these diseases.MethodsFecal samples from 97 subjects, including Crohn’s disease patients in remission with IBS-type symptoms (CDR-IBS+) or without IBS-type symptoms (CDR-IBS−), ulcerative colitis patients in remission with IBS-type symptoms (UCR-IBS+) or without IBS-type symptoms (UCR-IBS−), IBS patients and healthy controls, were collected and applied 16S ribosomal DNA (rDNA) gene sequencing. The V4 hypervariable regions of 16S rDNA gene were amplified and sequenced by the Illumina MiSeq platform. The differences in the sample diversity index in groups were analyzed with R software.ResultsThe richness of the intestinal microbiota in the CDR-IBS group was markedly lower than those in the control and IBS groups based on the analysis of observed species and the Chao index (P < 0.05). The observed species index in the CDR-IBS+ group was higher than that in the CDR-IBS− group (median index: 254.8 vs 203, P = 0.036). No difference was found in alpha diversity between UCR patients with IBS-type symptoms and those without related symptoms. At the genus level, the number of Faecalibacterium in CDR patients with IBS-type symptoms increased significantly, while Fusobacterium decreased versus those without such symptoms (mean relative abundance of Faecalibacterium: 20.35% vs 5.18%, P < 0.05; Fusobacterium: 1.51% vs 5.2%, P < 0.05). However, compared with the UCR-IBS− group, the number of Faecalibacterium in the UCR-IBS+ group decreased, while the number of Streptococcus increased, but there was no significant difference in the genus structure. The abundance and composition of the microbiota of IBS patients were not distinct from those of healthy controls.ConclusionsThe IBS-type symptoms in CD patients in remission may be related to an increase in Faecalibacterium and a decrease in Fusobacterium. The IBS-type symptoms in UC patients in remission cannot be explained by changes in the abundance and structure of the intestinal microbiota.

Environmental Influences on the Human Gut Microbiota: A Longitudinal Pilot Study

ObjectivesUrbanization has reduced environmental microorganism exposure, with most Americans spending over 90% of their time indoors. However, gardening remains a viable means of exposure to soil microorganisms and harvesting of edible produce. Accordingly, we aimed to determine relations between gardening, dietary habits, and gut microbiota. MethodsGardening families (N = 10) and non-gardening (control) families (N = 9) were enrolled in a longitudinal pilot trial. Families included two adults and a child (5–18 years) for a total sample size of 54 participants. Fecal samples were collected prior to and at the end of the gardening season. Garden soil samples (n = 9) were collected prior to and at the end of the season. Diet history questionnaires were collected at the beginning and end of the study to measure Healthy Eating Index (HEI) scores. Fecal and soil DNA were extracted, sequenced (V4 region of 16S rDNA gene), and analyzed using DADA2 and QIIME2. Alpha diversity measures were assessed, including Faith’s phylogenetic diversity (PD) and observed operational taxonomic units (OTUs). ResultsGardening families had significantly more fecal OTUs compared to control families (172.3 ± 44.2 vs. 157.0 ± 44.2, respectively; P = 0.03). Gardening families had greater (P = 0.02) Faith’s PD scores and tended (P = 0.08) to have more fecal OTUs than the control group at peak gardening season. In the gardening families, fecal OTUs and Faith’s PD were numerically but not statistically greater at the end of the season compared to baseline (all p’s > 0.05). Prior to the gardening season, gardening adults had greater HEI scores compared to control families (57 ± 9.1 vs. 49 ± 8.8, P = 0.03). HEI scores were not different between groups at the end of the study. ConclusionsThis study revealed that the fecal microbiota of families that garden differs from non-gardening families, and there are detectable changes in the fecal microbial community of gardeners and their family members over the course of the gardening season. Further research is needed to understand the role of diet in these changes and if microbes within the soil move between the soil and gastrointestinal environments. Funding SourcesThis research was funded by the Christopher Family Foundation Food and Family Grant Program.

HPV infection and bacterial microbiota in the semen from healthy men

BackgroundAberrant microbiota composition has been linked to disease development at numerous anatomical sites. Microbiota changes in reaction to viral infections, such as human papillomavirus (HPV), have been investigated almost exclusively in the female reproductive tract. However, HPV infection may also affect male health by reducing semen quality and fertility. The aim of this study was to investigate whether present HPV DNA is associated with detectable changes in semen bacterial microbiota composition and diversity.MethodsThis study relied on stored semen samples from 31 fertile healthy men who participated in the Finnish family HPV Study during the years 1998–2001. DNA was extracted from semen with PCR template preparation kit. HPV was genotyped using Luminex-based Multimetrix® assay. Microbiota was analyzed from the V3-V4 region of 16S rDNA gene following sequencing on an Illumina MiSeq platform. All statistical analyses were performed with Calypso software version 8.84.ResultsHPV DNA was detected in 19.4% (6/31) of the semen samples. HPV status in the semen did not impact the α-diversity estimations, as measured by Chao1 and Shannon indices, nor ß-diversity. Nevertheless, HPV-positive semen samples exhibited differences in the taxonomic composition of the bacterial microbiota including higher abundances of Moraxellaceae (p = 0.028), Streptococcus (p = 0.0058) and Peptostreptococcus (p = 0.012) compared to HPV-negative semen samples.ConclusionHPV infection is associated with altered bacterial microbiota composition in semen, and this might have in impact to male health in general. As of present, it is unclear whether these changes result from HPV infection or whether altered bacterial microbiota increases susceptibility to HPV infection. More research is needed on viral-bacterial interactions in the male reproductive system.

Gender-Specific Differences in Gut Microbiota Composition Associated with Microbial Metabolites for Patients with Acne Vulgaris

BackgroundThe gut microbial dysbiosis and gender differences in the pathogenesis of acne vulgaris have long been postulated respectively. However, there was no data about a gender-related discrepancy in gut microbiota and microbial metabolism in acne.ObjectiveThis study aimed at identifying the underlying gender-related difference in gut microbiota and metabolism in acne vulgaris.MethodsFecal samples were collected from 43 acne patients and 43 age and gender-matched controls. Gut microbiota was analyzed by sequencing the V3-V4 region of 16SrDNA gene and microbial metabolites were quantitatively detected using gas chromatography time-of-flight mass spectrometry.ResultsCompared with healthy controls, the men had a lower abundance of 18 microbes such as Butyricicoccus, Clostridium sensu stricto, Faecalibaculum, Bacillus, Lactococcus, Blautia, Clostridiales, Lachnospiracea incertae sedis, Ruminococcus at genus level. However, the female patients only showed increased Clostridium sensu stricto and declined Oscillibacter and Odoribacterin. Additionally, the disordered metabolism of fatty acids was identified in male patients, while the dysbiosis of amino acids metabolism in female ones.ConclusionThe disorder of gut microbiota and metabolism in acne vulgaris was gender-specific, which supported the potential role of gender difference in the pathogenesis of this disease.

Characteristics of intestinal flora in patients with primary Sjögren syndrome

To investigate changes in intestinal flora in patients with primary Sj?gren syndrome (pSS) and explore the relationship between pSS disease activity and intestinal flora structure. Fecal samples were collected from 18 female pSS patients, including 9 patients with active disease (group A) and 9 with disease inactivity or low activity (group B), with 10 healthy subjects as the control group. The total bacterial DNA was extracted from the fecal samples for PCR amplification, and Illumina Hiseq 2500 high-throughput sequencing was performed for the v3-v4 region of 16Sr DNA gene to obtain the biological information of the intestinal flora. The intergroup OTU analysis, structural diversity analysis, significant difference analysis and LEFSE analysis were performed with information mining of the literature think tanks. The dilution curves generated based on the OTUshannon index for analysis of sample complexity showed that the measured data were relatively complete and could reflect the diversity of the microorganisms in the subjects. Analysis of the Alpha diversity index showed that the Shannon index differed significantly between group A and group B, and the Simpson index differed significantly between group A and group B and between group A and the control group (P &lt; 0.05). Sequence analysis the 3 groups all consisted mainly of 4 phylum (Firmicutes, Bacteroidetes, Actinobacteria, showed that the intestinal flora in and proteobacteria) and 4 genera (finegoldia, Prevotella, Streptococcus, and Corynebacterium_1), all showing no significant differences among the 3 groups (P &gt; 0.05) with the exception of Streptococcus genus, which differed significantly among the 3 groups (P &lt; 0.05). The 16S v3-v4 region in the genus Alloscardovia, Bacteroides, Barnesiella, Butyricicoccus, Facklamia, Faecalibacterium, Lachnospiraceae_FCS020_group, Lachnospiraceae_ND3007_group, Lachnospiraceae_UCG-001, Lachnospirace, Lachnospirace, Ruminococcaceae_UCG-002, Streptococcus and Coprococcus_1 differed significantly among the 3 groups (P &lt; 0.05). The high-dimensional biometrics and genomic characteristics of the intestinal microorganisms differed significantly among the 3 groups (P &lt; 0.05). According to the size of LDA SCORE (effect size), the core flora in group A included the genera Barnesiellaccae, Aerococcaceae, Family-XIII, Bacteroidaceae, Lachnospiraceae_UCG-001, Barnesiella, Facklamia, Alloscardovia, Faecalibacterium and Bacteroides, as compared with the genera Streptococcaceae, Streptococcus, Coprococcus_1, Ruminococcaceae_ucg-002, Lachnospiraceae_FCS020_group, Lachnospiraceae_ucg-004, Lachnospiraceae_ND3007_group, Lachnospiraceae_ucg-008 and Butyricicoccus in the control group. Patients with pSS have significant changes in the diversity of intestinal flora, especially in some specific bacteria in Streptococcu genus and in 16S v3-v4 region of the bacteria. The differences in the core bacteria in the intestinal flora of pSS patients suggest the role of flora structure changes in the pathogenesis of pSS.

Composition and Diversity of Bacterial Community on the Ocular Surface of Patients With Meibomian Gland Dysfunction.

To investigate the composition and diversity of bacterial community on the ocular surface of patients with meibomian gland dysfunction (MGD) via 16S rDNA sequencing. Forty-seven patients with MGD, who were divided into groups of mild, moderate, and severe MGD, and 42 sex- and age-matched participants without MGD (control group) were enrolled. Samples were collected from the upper and lower conjunctival sac of one randomly chosen eye of each participant. Through sequencing the hypervariable region of 16S rDNA gene obtained from samples, differences in the taxonomy and diversity between groups were compared. Principle coordinate analysis showed significantly distinct clustering of the conjunctival sac bacterial community between the severe MGD group and the other groups. At the phylum level, the relative abundances of Firmicutes (31.70% vs. 19.67%) and Proteobacteria (27.46% vs. 14.66%) were significantly higher (P < 0.05, Mann-Whitney U), and the abundance of Actinobacteria (34.17% vs. 56.98%) was lower in MGD than controls (P < 0.05, Mann-Whitney U). At the genus level, the abundances of Staphylococcus (20.71% vs. 7.88%) and Sphingomonas (5.73% vs. 0.79%) in patients with MGD were significantly higher than the controls (P < 0.05, Mann-Whitney U), while the abundance of Corynebacterium (20.22% vs. 46.43%) was significantly lower (P < 0.05, Mann-Whitney U). The abundance of Staphylococcus was positively correlated with the meiboscores in patients with MGD (r = 0.650, P < 0.001, Spearman). Patients with MGD can have various degrees of bacterial microbiota imbalance in the conjunctival sac. Staphylococcus, Corynebacterium, and Sphingomonas may play roles in the pathophysiology of MGD.

2665. Intestinal Microbiome of Patients Submitted to Hematopoietic Stem Cell Transplantation Using Lactobacillus plantarum to Decolonized Multidrug-Resistant Bacteria

BackgroundPatients colonized by multidrug-resistant bacteria (MDR) have high risk for infection after hematopoietic stem cell transplantation (HSCT). Probiotic is a strategy that can be used to decolonize patients. Our aim was to describe the impact of use of Lactobacillus plantarum (LP) on decolonization, MDR infections and intestinal microbiome (IM) of autologous HSCT patients colonized by vancomycin-resistant Enterococcus (VRE) and carbapenem-resistant Gram-negative (CRGN).MethodsA prospective study was conducted at Hospital das Clinicas of University of Sao Paulo Brazil from 2017 to 2018. Candidates for auto-HSCT colonized by MDR received LP in capsules, 5 × 109 CFU twice daily until neutropenia (NP). Rectal swabs were performed and cultured using selective media as well as PCR for carbapenemases, vanA and vanB. Stool samples for IM analysis were collected weekly as baseline (before LB use) until the NP. The V4 region of 16S rDNA gene were sequenced by Ion Torrent PGM and analyzed using alpha and β diversities by Qiime. Demographic and clinical data including previous antibiotics use were evaluated; CDC criteria was applied for colonization and infection.ResultsAll of seven patients were colonized by VRE and CRGN (Table 1). Only one patient remained colonized by CRGN after 61 days of LP. Four patients developed seven bloodstream infections (BSI) during the NP, two of them by CRGN. There was no infection caused by VRE neither by LP. One patient, with low adherence to LP use (66%), died due to MDR K. pneumoniae BSI. We observed a decrease of Clostridia, Verrucomicrobiae, Blautia and an increase of Enterobacteriaceae. Baseline samples from patients who used TMP/SMX had higher concentrations of Bacteroidetes when compared with those who had not use it. The Shannon index in controls ranged 1.98–5.55 and during NP 1.15–5.99. The β diversity analysis showed no clear patterns between patients.ConclusionWe observed a heterogeneity among IM of auto-HSCT patients prior and after LP. It was not possible to establish an IM pattern, probably, because of small number of patients. Although, clinical infections by CRGN occurred despite of LP use, no cases of colonization and infection by VRE were identified. Thus, it seems that LP is a good and safe strategy to decolonized HSCT. Disclosures All authors: No reported disclosures.

Correlation analysis of pediatric sepsis and intestinal flora imbalance

Objective To investigate the interaction between the gut microbiota and the sepsis in children by comparing the difference of gut microbiota between septic children and healthy children. Methods Genome was extracted from excrements of 18 cases of sepsis and 6 cases of healthy children.After genomic extraction, the hypervariable region of 16S rDNA gene were amplified and a small fragment library was constructed, and high-throughput sequencing was carried out, then the data of the lower machine was effectively sequenced by biological information processing.We could seek for the species that had changed significantly due to sepsis by comparing the diversity and the differences in the composition of intestinal flora between the two groups. Results The gut microbiome of the sepsis group was distinct from that of the health group.The operational taxonomic units in the sepsis group were significantly reduced compared with healthy group(P=0.001). The gut microbiome of children with sepsis had significantly lower diversity and richness compared with healthy group(P<0.05). A total of 7 species were shown to be differentially abundant between septic patients and healthy controls.The genus Pseudomonadales, Carnobacteriaceae and Granulicatella_elegans were significantly more abundant in the sepsis group; meanwhile the genus Pasteurellaceae, Ruminococcus, Lactobacillus_rogosae and Anaerostipes_butyratucus were less abundant in the sepsis group.In addition, the Granulicatella_elegans was characteristically present in the intestine of children with sepsis(P<0.001). Conclusion The microbial diversity and structure of the gut microbiome in children with sepsis are significantly different from those of healthy children.Our data suggest biomarkers identified in this study might participate in the pathogenesis or development process of sepsis. Key words: Sepsis; Gut microbiota; 16S rDNA; Children

Canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems.

The objective of this study was to characterize the normal microbiome of healthy canine vagina and endometrium and to determine the effect of the stage of estrous, on the resident microbiome. Cranial vaginal swabs and uterine biopsy samples were collected from twenty-five bitches in five different stages of estrous at elective ovariohysterectomy (OVH). Over 4 million reads of the V4 region of 16S rDNA gene were obtained and used for further analyses. A total of 317 genera belonging to 24 known phyla were identified. The endometrium was higher in bacterial diversity while the vagina was higher in richness. Proteobacteria, Bacteroidetes and Firmicutes were the most abundant phyla observed across all samples. Hydrotalea, Ralstonia, and Fusobacterium accounted for nearly 60% of the OTUs identified in the vagina while organisms identified in the endometrium were more evenly distributed. Pseudomonas, Staphylococcus, and Corynebacterium were the prominent genera in the endometrium. The microbiome of the endometrium was distinctly different from that of the vagina. There was large animal-to-animal variation. Other than the vaginal microbiome of bitches in estrus (i.e. in heat), there were no distinct clustering of the organisms based on the stage of estrous. These findings establish the presence of a resident microbiome of the endometrium throughout all stages of estrous cycle.

Disease-Specific Enteric Microbiome Dysbiosis in Inflammatory Bowel Disease.

Inflammatory Bowel disease (IBD) is traditionally divided into Crohn's disease (CD) and ulcerative colitis (UC). UC is a relapsing non-transmural inflammatory disease that is restricted to the colon and is characterized by flare-ups of bloody diarrhea. CD is a chronic, segmental localized granulomatous disease that can affect any part of the entire gastrointestinal tract. Ileo-anal pouch is a procedure restoring functionality of the rectum after a colectomy. IBD is a multifactorial disease and flares of IBD are probably triggered by changes in the intestinal microbiota followed by an abnormal immune response. In this study, we aim to analyze the intestinal bacterial diversity in IBD patients during various stages of disease compared with healthy controls. Permission for human experiments and recruitment of participants was obtained from the Ethic Committee for Copenhagen County hospitals (Permission no. KA-03019, Permission no. KA-20060159). Stools from 26 healthy controls, 42 CD, 38 UC and 18 pouch patients were collected. Stool DNA extraction was performed using Qiagen, DNA mini stool kit Denmark. DGGE-PCR amplifying the V2-V3 region of 16S-rDNA gene of the bacteria was amplified by universal primers HDA1 and HDA2. Analysis of DGGE was performed blinded using BioNumerics version 7.5. After normalization, a DGGE gel band matching was performed. The similarities between profiles were calculated with a ranked Pearson correlation coefficient based on the band matching results using band intensities. Simpson's index of diversity and Pielou's species evenness were calculated. Based on the Shannon Diversity Index, UC patients had lower species diversity and bacterial evenness in comparison to healthy persons, p < 0.05. However, only CD and disease pouch patients had lower species diversity compared to those with inactive disease and healthy controls. Well-functioning pouch patients had decreased species evenness in comparison to diseased pouch patients and control group. During the active disease stage in CD and pouch, the patients have a low bacterial diversity in their gut when compared to the inactive stage. In UC patients, a generally low diversity was observed at all stages of the disease compared to healthy controls.

Mycoplasma pneumoniae in Community-Acquired Lower Respiratory Tract Infections.

To determine the role of Mycoplasma pneumoniae (M. pneumoniae) in pediatric lower respiratory tract infections (LRTIs) employing serological tests and polymerase chain reaction (PCR) analysis. In this prospective study, 200 children aged 6 mo to 12 y hospitalized with acute LRTIs were investigated for M. pneumoniae. Serum samples were collected for serological analysis of M. pneumoniae. Throat swab samples were obtained on admission to amplify 277-base pair region of 16S rDNA gene of M. pneumoniae by PCR. In the present study, 40(26.1%) children <5 y and 28(59.5%) children ≥5 y age group were positive for M. pneumoniae infection and this difference was statistically significant (P < 0.001). M. pneumoniae was positive in 32(41%) female and 36(29.5%) male children though this difference was statistically insignificant (P = 0.12). The clinical profile across M. pneumoniae positive and negative cases were comparable except for presence of chest pain which was statistically significant (P = 0.023). None of the radiological findings was statistically associated with incidence of M. pneumoniae infection. Serological evidence of acute M. pneumoniae infection was observed in 64(32%) patients with sensitivity 66.6% and specificity 70.1% while PCR positivity in 12(6%) patients with sensitivity 12.5% and specificity 97%. Together, serology and PCR detected M.pneumoniae infection in 68(34%) patients. The present study underlines the role of M. pneumoniae in children with community- acquired LRTIs and more particularly in ≥5 y of age.

Analysis of Bacterial Diversity in Different Heavy Oil Wells of a Reservoir in South Oman with Alkaline pH.

The identification of potential hydrocarbon utilizing bacteria is an essential requirement in microbial enhanced oil recovery (MEOR). Molecular approaches like proteomic and genomic characterization of the isolates are replacing the traditional method of identification with systemic classification. Genotypic profiling of the isolates includes fingerprint or pattern-based technique and sequence-based technique. Understanding community structure and dynamics is essential for studying diversity profiles and is challenging in the case of microbial analysis. The present study aims to understand the bacterial community composition from different heavy oil contaminated soil samples collected from geographically related oil well areas in Oman and to identify spore-forming hydrocarbon utilizing cultivable bacteria. V4 region of 16S rDNA gene was the target for Ion PGM™. A total of 825081 raw sequences were obtained from Ion torrent from all the 10 soil samples. The species richness and evenness were found to be moderate in all the samples with four main phyla, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, the most abundant being Firmicutes. Bacillus sp. ubiquitously dominated in all samples followed by Paenibacillus, which was followed by Brevibacillus, Planococcus, and Flavobacterium. Principal Coordinate Analysis (PCoA) and UPGMA dendrogram clustered the 10 soil samples into four main groups. Weighted UniFrac significance test determined that there was significant difference in the communities present in soil samples examined. It can be concluded that the microbial community was different in all the 10 soil samples with Bacillus and Paenibacillus sp. as predominating genus. The 16S rDNA sequencing of cultivable spore-forming bacteria identified the hydrocarbon utilizing bacteria as Bacillus and Paenibacillus sp. and the nucleotide sequences were submitted to NCBI GenBank under accession numbers KP119097–KP119115. Bacillus and Paenibacillus sp., which were relatively abundant in the oil fields, can be recommended to be chosen as candidates for hydrocarbon utilization study.

First Report on the Occurrence of Virescence of Chrysanthemum Associated with 16SrII-A Group Phytoplasma in India

Chrysanthemum (Chrysanthemum indicum L.) is an important floriculture crop grown in different parts of Karnataka, India for its beautiful cut flowers. Recently, there has been a decline in flower quality due to major pests and diseases. A field survey conducted during 2012-2013 in chrysanthemum-growing areas revealed the occurrence of virescence with an incidence of 5 to 10% in nearly 6 to 8 ha of chrysanthemum fields in Mandya and Mysore districts. Symptoms including malformed flowers with greenish florets and reduced flower size and floral virescence with sepaloidy were observed. Diseased leaves and flower parts were collected and used for identification of the causative agent. Ten samples (6 symptomatic, 2 healthy, and 2 phytoplasma-associated with phyllody of Catheranthus roseus as positive control) were used for total genomic DNA isolation using GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) and the DNA was used as template to amplify the phytoplasma-specific 16SrDNA gene using P1/P7 primers followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected only with DNA from symptomatic and positive control samples. Nested PCR products of all six phytoplasma-associated samples were purified and sequenced from both the directions using a ABIx370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The 16SrDNA sequences were aligned using CodonCode Aligner (Centerville, MA) and analyzed by BLASTn. The analysis revealed that all six sequences shared 100% identity with Cassava witches broom (KM280680.1) and Peanut witches broom (JX403944.1) phytoplasma. The in-silico RFLP pattern of F2n/R2 primed region of 16SrDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 99.8% nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup A (Peanut witches broom phytoplasma; Accession No. L33765.1). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chrysanthemum virescence was identified as a member of 16SrII-A group. The representative sequence was deposited in GenBank with Accession Number KP027534.1. Phytoplasma associated with little leaf and witches broom of chrysanthemum have been reported earlier from India and were assigned to groups 16SrI-A and 16SrII, respectively (Kumar et al. 2012; Raj et al. 2007). Chrysanthemum virescence phytoplasma belonging to Candidatus phytoplasma aurantifolia has been reported from Japan and has not been assigned to any group (Naito et al. 2007). The occurrence of virescence is of great importance as it affects the overall productivity and quality of flowers, which reduces the market value of chrysanthemum. To our knowledge, this is the first report of a group 16SrII-A phytoplasma associated with chrysanthemum virescence in India.

High-throughput sequencing of 16S rDNA amplicons characterizes bacterial composition in cerebrospinal fluid samples from patients with purulent meningitis.

Purulent meningitis (PM) is a severe infectious disease that is associated with high rates of morbidity and mortality. It has been recognized that bacterial infection is a major contributing factor to the pathogenesis of PM. However, there is a lack of information on the bacterial composition in PM, due to the low positive rate of cerebrospinal fluid bacterial culture. Herein, we aimed to discriminate and identify the main pathogens and bacterial composition in cerebrospinal fluid sample from PM patients using high-throughput sequencing approach. The cerebrospinal fluid samples were collected from 26 PM patients, and were determined as culture-negative samples. The polymerase chain reaction products of the hypervariable regions of 16S rDNA gene in these 26 samples of PM were sequenced using the 454 GS FLX system. The results showed that there were 71,440 pyrosequencing reads, of which, the predominant phyla were Proteobacteria and Firmicutes; and the predominant genera were Streptococcus, Acinetobacter, Pseudomonas, and Neisseria. The bacterial species in the cerebrospinal fluid were complex, with 61.5% of the samples presenting with mixed pathogens. A significant number of bacteria belonging to a known pathogenic potential was observed. The number of operational taxonomic units for individual samples ranged from six to 75 and there was a comparable difference in the species diversity that was calculated through alpha and beta diversity analysis. Collectively, the data show that high-throughput sequencing approach facilitates the characterization of the pathogens in cerebrospinal fluid and determine the abundance and the composition of bacteria in the cerebrospinal fluid samples of the PM patients, which may provide a better understanding of pathogens in PM and assist clinicians to make rational and effective therapeutic decisions.

High-throughput sequencing of 16S rDNA amplicons characterizes bacterial composition in bronchoalveolar lavage fluid in patients with ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) is a life-threatening disease that is associated with high rates of morbidity and likely mortality, placing a heavy burden on an individual and society. Currently available diagnostic and therapeutic approaches for VAP treatment are limited, and the prognosis of VAP is poor. The present study aimed to reveal and discriminate the identification of the full spectrum of the pathogens in patients with VAP using high-throughput sequencing approach and analyze the species richness and complexity via alpha and beta diversity analysis. The bronchoalveolar lavage fluid samples were collected from 27 patients with VAP in intensive care unit. The polymerase chain reaction products of the hypervariable regions of 16S rDNA gene in these 27 samples of VAP were sequenced using the 454 GS FLX system. A total of 103,856 pyrosequencing reads and 638 operational taxonomic units were obtained from these 27 samples. There were four dominant phyla, including Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. There were 90 different genera, of which 12 genera occurred in over ten different samples. The top five dominant genera were Streptococcus, Acinetobacter, Limnohabitans, Neisseria, and Corynebacterium, and the most widely distributed genera were Streptococcus, Limnohabitans, and Acinetobacter in these 27 samples. Of note, the mixed profile of causative pathogens was observed. Taken together, the results show that the high-throughput sequencing approach facilitates the characterization of the pathogens in bronchoalveolar lavage fluid samples and the determination of the profile for bacteria in the bronchoalveolar lavage fluid samples of the patients with VAP. This study can provide useful information of pathogens in VAP and assist clinicians to make rational and effective therapeutic decisions.