Clone Library Method Research Articles

Changes in Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria along a Glacier Retreating Chronosequence in the Tianshan Mountains, China.

Glaciers retreating due to global warming create important new habitats, particularly suitable for studying ecosystem development where nitrogen is a limiting factor. Nitrogen availability mainly results from microbial decomposition and transformation processes, including nitrification. AOA and AOB perform the first and rate-limiting step of nitrification. Investigating the abundance and diversity of AOA and AOB is essential for understanding early ecosystem development. The dynamics of AOA and AOB community structure along a soil chronosequence in Tianshan No. 1 Glacier foreland were analyzed using qPCR and clone library methods. The results consistently showed low quantities of both AOA and AOB throughout the chronosequence. Initially, the copy numbers of AOB were higher than those of AOA, but they decreased in later stages. The AOB community was dominated by "Nitrosospira cluster ME", while the AOA community was dominated by "the soil and sediment 1". Both communities were potentially connected to supra- and subglacial microbial communities during early stages. Correlation analysis revealed a significant positive correlation between the ratios of AOA and AOB with soil ammonium and total nitrogen levels. These results suggest that variations in abundance and diversity of AOA and AOB along the chronosequences were influenced by ammonium availability during glacier retreat.

Diversity and succession of chemolithoautotrophic microbial community along a recently deglaciation chronosequence on the Tibetan Plateau.

Glaciers in high-altitude mountain regions are retreating rapidly due to global warming, exposing deglaciated soils to extreme environmental conditions, and microbial colonization. However, knowledge about chemolithoautotrophic microbes, which play important roles in the development of oligotrophic deglaciated soils prior to plant colonization, remains elusive in deglaciated soils. Using real-time quantitative PCR and clone library methods, the diversity and succession of the chemolithoautotrophic microbial community harboring the cbbM gene across a 14-year deglaciation chronosequence on the Tibetan Plateau were determined. The abundance of the cbbM gene remained stable for the first 8 years after deglaciation and then increased significantly, ranging from 105 to 107 gene copies g-1 soil (P < 0.001). Soil total carbon increased gradually to 5-year deglaciation and then decreased. While total nitrogen and total sulfur levels were low throughout the chronosequence. Chemolithoautotrophs were related to Gammaproteobacteria and Betaproteobacteria, with the former dominating early deglaciated soils and the latter dominating older deglaciated soils. The diversity of chemolithoautotrophs was high in mid-age deglaciated soils (6-year-old) and was low in early (3-year-old) and older deglaciated soils (12-year-old). Our findings revealed that chemolithoautotrophic microbes colonize deglaciated soils quickly and follow a clear successional pattern across recently deglaciated chronosequences.

Comparative study of bacterial flora in bronchoalveolar lavage fluid of pneumonia patients based on their pneumonia subtypes and comorbidities using 16S ribosomal RNA gene analysis

IntroductionThe culture method is the gold standard for identifying pathogenic bacteria in patients with pneumonia but often does not reflect the exact bacterial flora in pulmonary lesions of pneumonia, partly owing to easiness or difficulties in culturing certain bacterial species. We aimed to evaluate bacterial flora in bronchoalveolar lavage fluid (BALF) samples directly obtained from pneumonia lesions using 16S ribosomal RNA (rRNA) gene analysis to compare the results of the BALF culture method in each category of pneumonia. MethodsBacterial florae were detected by a combination of the culture method, and the clone library method using the 16S rRNA gene sequencing in BALF directly obtained from pneumonia lesions in pneumonia patients from April 2010 to March 2020 at the University of Occupational and Environmental Health, Japan, and affiliated hospitals. Clinical information of these patients was also collected, and lung microbiome was evaluated for each pneumonia category. ResultsAmong 294 pneumonia patients (120 with community-acquired pneumonia (CAP), 101 with healthcare-associated pneumonia (HCAP), and 73 with hospital-acquired pneumonia (HAP)), significantly higher percentages of obligate anaerobes were detected in CAP than in HCAP and HAP patients by the clone library method. Corynebacterium species were significantly highly detected in HAP patients and patients with cerebrovascular diseases than in patients without, and Streptococcus pneumoniae was frequently detected in patients with diabetes mellitus. ConclusionObligate anaerobes may be underestimated in patients with CAP. Corynebacterium species should be regarded as the causative bacteria for pneumonia in patients with HAP and cerebrovascular diseases.

Perspective on the clone library method for infectious diseases.

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.

Association between obligatory anaerobes and empyema caused by Streptococcus anginosus group bacteria.

Co-infections of Streptococcus anginosus group (SAG) bacteria and obligate anaerobes are observed in patients with empyema; however, their epidemiology and pathology remain unknown. A retrospective study was performed with 44 patients who underwent pleural effusion microbiota evaluation between January 2006 and March 2018, using the clone library method for detecting empyema caused by SAG bacteria. Based on culture analysis of pleural effusion, 12 patients were diagnosed with empyema caused by SAG bacteria. Obligate anaerobe phylotypes were detected in eight patients (66.7%) using the clone library method, whereas anaerobic culture analysis detected anaerobes in only two patients (16.7%). No significant difference was observed between the clinical features of SAG-mediated empyema with and without anaerobes using the clone library method, except for chest computed tomographic data. Co-infection of SAG bacteria and obligate anaerobes may be underestimated if conventional culture methods are used. SAG-mediated empyema with and without anaerobes may present different radiological features; therefore, further studies are required.

Coinfection With Multiple Nontuberculous Mycobacteria as a Possible Exacerbating Factor in Pulmonary Nontuberculous Mycobacteriosis: Clone Library Analysis Using the 16S Ribosomal RNA Gene

Mycobacterial culture is the gold standard for the diagnosis of nontuberculous Mycobacterium (NTM) infections. However, this method is not suitable for detection of coinfection with different NTMs. The goal of this study was to determine if clone library analysis of BAL fluid (BALF) was useful for detection of NTM phylotypes, including multiple NTM phylotypes, in pulmonary NTM infections. BALF samples obtained from 120 patients with suspected pulmonary NTM infections were retrospectively evaluated by using the mycobacterial culture and clone library methods between July 2010 and August2016. In total, 55 (45.8%) patients were diagnosed as NTM positive according to results of mycobacterial culture, and 52 patients were NTM positive as determined by using the clone library method. Furthermore, 45 (86.5%) and seven (13.5%) patients exhibited a single phylotype (mono-phylotype group) and multiple phylotypes of NTM (multi-phylotype group), respectively. Compared with the mono-phylotype group, the multi-phylotype group had a significantly higher incidence of adverse chest CT findings (P= .048). In addition, 11 patients who were NTM negative according to results of BALF mycobacterial culture were determined to be NTM positive according to the clone library method. Six of these 11 patients were eventually diagnosed as NTM positive by using mycobacterial culture results within 6.2 ± 2.1months following the initial sample collection. Coinfection multiple phylotypes could be associated with adverse clinical findings. In addition, patients who test positive for NTM genes but negative for mycobacterial culture may be diagnosed with NTM lung infection within 1 year of the initial sample collection. Further follow-up of these patients may facilitate early detection of NTM species.

Effect of transient exposure to carbaryl wettable powder on the gut microbial community of honey bees

Bees are important pollinators in agriculture. The bee population has recently begun to decline possibly due to pesticides. The bee gut microbiota strongly influences the health of bees. The gut microbiota of bees is composed of distinct members belonging to selective taxa. Chemicals like pesticides can alter the gut microbiota. The present study investigated the effect of carbaryl pesticides on gut microbiota of honey bees, which had come in contact with rapeseed plants (Brassica napus) sprayed with carbaryl wettable powder during the honey bee brood test under semi-field condition. Molecular techniques (conventional and quantitative polymerase chain reaction (PCR), clone library method, and DNA sequencing) were employed to analyze changes in the microbial communities between the pesticide-exposed and unexposed bees. Phylogenetic analysis of 16S rRNA genes of the clones from both groups, showed differences in their respective compositions of core and non-core bacteria. Both groups contained carbohydrate-degrading bacteria such as Gilliamella apicola and Lactobacillus. However, the unexposed bees harbored Alphaproteobacteria, which were absent in the exposed bees. Microorganisms found in honey bee guts such as Snodgrassella alvi and L. kullabergensis, however, were observed only in the exposed bees, but not in the unexposed bees. The difference between the two groups was distinctly recognized when copy numbers of 16S rRNA genes were compared by quantitative PCR. Results showed that the average gene copy number for the unexposed bees was higher than that for the exposed bees. This may indicate the toxic effect of pesticides on bees and gut microbiota.

纳木错湖水体固碳微生物数量、群落结构及其驱动因子

PDF HTML阅读 XML下载 导出引用 引用提醒 纳木错湖水体固碳微生物数量、群落结构及其驱动因子 DOI: 10.5846/stxb201805081021 作者: 作者单位: 西南医科大学附属医院，肝胆外科,中国科学院青藏高原研究所,中国科学院青藏高原研究所,中国科学院青藏高原研究所,中国科学院青藏高原研究所,中国科学院青藏高原研究所 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金面上项目（41471054）；中国科学院百人计划项目（KZZD-EW-TZ-14） Abundance, community structure, and the driving factors of Carbon fixing microorganisms in the Nam Co Lake Author: Affiliation: Department of Hepatobiliary Surgery, The Affiliated Hospital of Southwest Medical University,Institute of Tibetan Plateau Research, Chinese Academy of Sciences,,,, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:湖泊是微生物固碳的主要生态系统之一，但青藏高原湖泊水体固碳微生物群落的研究还罕见报道。以纳木错为例，采用定量PCR和克隆文库方法，研究湖水中cbbL ID基因丰度和固碳微生物群落组成，并分析其与环境参数的关系。结果显示：纳木错湖水中存在较高丰度的cbbL ID类型固碳微生物，从表层到底层呈增加趋势，T2点底层达到最高值（6.37×108 拷贝L-1湖水）。cbbL ID类型固碳微生物共分四个类群，即不等鞭毛类（Stramenopiles），定鞭藻纲（Haptophyceae），蓝藻（Cyanobacteria）和隐藻门（Cryptophyta）。其中占主要的是Stramenopiles和Haptophyceae。Stramenopiles类群的多样性较高（含7个纲，13个科），其他类群只有1个科。相关性分析表明Stramenopiles和Haptophyceae出现频率存在显著的负相关关系（P<0.01）。湖水深度和pH与湖水cbbL ID基因丰度显著相关（P<0.05，P<0.01）。叶绿素含量与Stramenopiles和Haptophyceae出现频率显著相关（P<0.01）。 Abstract:Lakes are one of the main ecosystems for carbon fixation; however, the microbial community for carbon fixation in the lakes of the Tibetan Plateau have rarely been reported. In this study, the Nam Co Lake was chosen to study the abundance of cbbL ID genes and their composition using quantitative PCR and a clone library method. We also discuss their relationship with environmental parameters. The results showed that there was a high abundance of the cbbL ID gene in the Nam Co Lake, and there was an increasing trend from the surface to the bottom, and the highest amount was in the bottom sample of T2 (6.37×108 copies/L water). The community was mainly composed of Stramenopiles and Haptophyceae, and a few Cyanobacteria and Cryptophyta appeared on individual layers. The Stramenopiles group had a higher diversity (including 7 classes and 13 families) than that of the other groups, and there was only 1 family of another taxa. A correlation analysis showed there was a significant negative correlation (P<0.01) of the occurrence frequency of Stramenopiles and Haptophyceae. Water depth and pH had a significant correlation with cbbL ID gene abundance (P<0.05, P<0.01, respectively). Chlorophyll content had a significant correlation with the occurrence frequency of Stramenopiles and Haptophyceae. 参考文献 相似文献 引证文献

Changes in soil microbial community response to precipitation events in a semi-arid steppe of the Xilin River Basin, China

In the context of climate change, precipitation is predicted to become more intense at the global scale. Such change may alter soil microbial communities and the microbially mediated carbon and nitrogen dynamics. In this study, we experimentally repackaged precipitation patterns during the growing season (from June to September) of 2012 in a semi-arid temperate steppe of the Xilin River Basin in Inner Mongolia of China, based on the 60-year growing season precipitation data. Specifically, a total amount of 240 mm simulated precipitation was assigned to experimental plots by taking the following treatments: (1) P6 (6 extreme precipitation events, near the 1st percentile); (2) P10 (10 extreme precipitation events, near the 5th percentile); (3) P16 (16 moderate precipitation events, near the 50th percentile); and (4) P24 (24 events, 60-year average precipitation, near the 50th percentile). At the end of the growing season, we analyzed soil microbial community structure and biomass, bacterial abundance, fungal abundance and bacterial composition, by using phospholipid fatty acid (PLFA), real-time quantitative polymerase chain reaction (RT-qPCR) and 16S rRNA gene clone library methods. The extreme precipitation events did not change soil microbial community structure (represented by the ratio of PLFA concentration in fungi to PLFA concentration in bacteria, and the ratio of PLFA concentration in gram-positive bacterial biomass to PLFA concentration in gram-negative bacterial biomass). However, the extreme precipitation events significantly increased soil microbial activity (represented by soil microbial biomass nitrogen and soil bacterial 16S rRNA gene copy numbers). Soil fungal community showed no significant response to precipitation events. According to the redundancy analysis, both soil microbial biomass nitrogen and soil ammonium nitrogen (NH4-N) were found to be significant in shaping soil microbial community. Acidobacteria, Actinobacteria and Proteobacteria were the dominant phyla in soil bacterial composition, and responded differently to the extreme precipitation events. Based on the results, we concluded that the extreme precipitation events altered the overall soil microbial activity, but did not impact how the processes would occur, since soil microbial community structure remained unchanged.

Bacterial Diversity of a Microbial Mat from Hot Spring at Wartawan Beach, Lampung and Its Potential as a Source of Hydrogenases

Biohydrogen produced from thermophilic hydrogenases is an ideal and clean energy sources. As the biggest tectonic area in the world, Indonesia is potential for thermophile isolation. The aims of this study were to analyze the bacterial diversity of a microbial mat from hot spring at Wartawan beach, Lampung and to analyze the potency of microbial mat for hydrogenases, using clone library method. The diversity of 16S rRNA showed that the microbial mat sample contained 9 phyla of bacteria, and dominated by Cyanobacteria and Proteobacteria. These phyla indicate that the bacterial community of the microbial mat consisted of phototrophic and heterotrophic groups. In addition, a microbial mat of Wartawan beach environment might be influenced by marine environment and hydrothermal vent which was indicated by detection of both associated bacteria. The diversity of hydrogenase genes using NiFe hydrogenase (NiFe) and FeFe hydrogenase (FeFe) genes showed that Cyanobacteria was specifically related to NiFe, while Firmicutes was associated with FeFe. Proteobacteria and Bacteroidetes, however, were detected for both genes. The detected hydrogenase genes indicate that the microbial mat from hot spring at Wartawan beach is a promising source for hydrogenases isolation and further applications for biohydrogen production as a renewable energy.

Comparative diversity analysis of ruminal methanogens in Murrah buffaloes (Bubalus bubalis) in four states of North India

We compared the community structure of methanogens in Murrah breed of buffaloes of four states of north India using 16S rRNA gene clone library method. The results revealed the dominance of methanogens related to Methanobrevibacter in three states, while Methanomicrobium-related methanogens were abundant in one state.

Acclimatization and Community Structure Analysis of the Microbial Consortium in Nitrate-Dependent Anaerobic Methane Oxidation

Methane oxidation coupled with denitrification is an effective way to reduce the discharge of nitrate and methane. The anaerobic sludge from a laboratory wastewater treatment facility, anaerobic sludge from a wastewater treatment plant, and soil from a landfill were selected as inoculum to enrich the consortium for anaerobic methane oxidation in cooperation with nitrate reduction. The investigation of methane oxidation was carried out in these systems. The results showed that the maximum methane consumption rate of 0.05 mg·d-1 was obtained when the anaerobic sludge from a wastewater treatment plant served as inoculum. The population of bacteria and archaea were assayed by the clone library method. The Methanosarcinales and Methanomicrobiales were present in methane oxidation as methane oxidizing archea. The Pseudomonas, Clostridia, and Thermomonas were identified as nitrate reducing bacteria in the process of nitrate reduction. Both the methane conversion rate and microbial population varied with the amount of nitrate. The nitrate reduction bacteria were Pseudomonas and Clostridia when the nitrate concentration was 200 mg·L-1. The Pseudomonas and Thermomonas emerged when the nitrate concentration increased to 500 mg·L-1, and the rate of methane conversion was increased by 34.7%. The results provided science evidence for the co-treatment of methane and nitrate.

Analysis of Sulfate-Reducing and Sulfur-Oxidizing Prokaryote Community Structures in Marine Sediments with Different Sequencing Technologies

Sulfate-reducing prokaryotes (SRP) and sulfur-oxidizing prokaryotes (SOP) play vital roles in the sulfur cycle. The SRP community was used to represent a microbial community with high richness and diversity. The 454 pyrosequencing, Illumina high-throughput sequencing, and traditional clone library methods that target the dissimilatory sulfite reductase β subunit gene (dsrB), which encodes a key enzyme in the sulfate reduction pathway, were used to compare the differences in SRP community characteristics. Comparative analyses suggested that Illumina high-throughput sequencing was a more appropriate method for SRP (high richness and diversity) community studies. The SOP soxB gene (~750 bp) was used as a representative of the long-sequence segment. The 454 pyrosequencing and Illumina high-throughput sequencing methods were used to compare the differences in SOP community characteristics. The results revealed that 454 pyrosequencing did not reflect its advantage of a longer read length; whereas, the Illumina high-throughput sequencing with more numerous and shorter sequence reads was more suitable when the soxB gene was used to investigate the community composition and diversity of SOP.

Polyhydroxyalkanoate accumulation ability and associated microbial community in activated sludge-derived acetate-fed microbial cultures enriched under different temperature and pH conditions

The influence of temperature and pH during enrichment on the polyhydroxyalkanoate (PHA) accumulation ability and composition of PHA-accumulating microorganisms (PHAAMOs) in enrichment cultures was investigated. Enrichment of PHAAMOs from activated sludge was conducted in acetate-fed sequencing batch reactors using a feast-famine regime under different temperature (20°C, 28°C, and 36°C) and pH (controlled at 7.2 or not) conditions. PHA accumulation ability, which was evaluated in nitrogen- and phosphorus-deficient 24-h single-batch cultures, was greatly enhanced by enrichment, irrespective of the temperature and pH. Enrichment at 20°C or 28°C and without pH control seemed most appropriate for strong PHA accumulation. Analyses of the PHAAMO composition by the clone library method targeting phaC genes, which encode the class I and II PHA synthases, revealed that Burkholderiales were the dominant PHAAMOs in the seed sludge, while Rhodocyclales, specifically Azoarcus spp. and Thauera spp., were dominant after enrichment without pH control, showing a strong ability to accumulate PHA. The results indicated that Azoarcus spp. and Thauera spp. are key PHAAMOs in an enrichment culture based on the feast-famine method, with high PHA accumulation ability.

Monitoring of microbial communities in anaerobic digestion sludge for biogas optimisation

This study characterised and compared the microbial communities of anaerobic digestion (AD) sludge using three different methods – (1) Clone library; (2) Pyrosequencing; and (3) Terminal restriction fragment length polymorphism (T-RFLP). Although high-throughput sequencing techniques are becoming increasingly popular and affordable, the reliance of such techniques for frequent monitoring of microbial communities may be a financial burden for some. Furthermore, the depth of microbial analysis revealed by high-throughput sequencing may not be required for monitoring purposes. This study aims to develop a rapid, reliable and economical approach for the monitoring of microbial communities in AD sludge. A combined approach where genetic information of sequences from clone library was used to assign phylogeny to T-RFs determined experimentally was developed in this study. In order to assess the effectiveness of the combined approach, microbial communities determined by the combined approach was compared to that characterised by pyrosequencing. Results showed that both pyrosequencing and clone library methods determined the dominant bacteria phyla to be Proteobacteria, Firmicutes, Bacteroidetes, and Thermotogae. Both methods also found that sludge A and B were predominantly dominated by acetogenic methanogens followed by hydrogenotrophic methanogens. The number of OTUs detected by T-RFLP was significantly lesser than that detected by the clone library. In this study, T-RFLP analysis identified majority of the dominant species of the archaeal consortia. However, many of the more highly diverse bacteria consortia were missed. Nevertheless, the combined approach developed in this study where clone sequences from the clone library were used to assign phylogeny to T-RFs determined experimentally managed to accurately predict the same dominant microbial groups for both sludge A and sludge B, as compared to the pyrosequencing results. Results showed that the combined approach of clone library and T-RFLP accurately predicted the dominant microbial groups and thus is a reliable and more economical way to monitor the evolution of microbial systems in AD sludge.

Microbial population structure in near-ground aerosols during fog-haze days in northern China

Studies on microbial populations in near-ground aerosols during fog–haze days could help enhance our knowledge concerning the relationship between fog–haze and human health. In this study, microbial populations in aerosols near-ground during a fog–haze event in Beijing were analyzed using clone library methods. Results showed that the bacterial diversity in aerosols during fog–haze days was lower than that after fog–haze days. Proteobacteria alone and Proteobacteria with Firmicutes were respectively detected in 1.5 and 20 m aerosols during fog–haze days. In addition to Proteobacteria and Firmicutes, Bacteroidetes alone and Acidobacteria with Verrucomicrobia were respectively found in aerosols at 20 and 1.5 m after fog–haze days. The fungal species observed during fog–haze days were completely different from those detected after fog–haze days. Ascomycota and Basidiomycota were respectively detected during and after fog–haze days. The distribution of microbial diversity in aerosols exhibited meteorological and site-associated variations. The same potential pathogenic microorganisms were detected at different heights during fog–haze days. This study on the characteristics of microbial population in aerosols could provide a comprehensive understanding of the factors causing the harmful effects of particles on humans during fog–haze days.

Identification of microbial populations contributing to nitrification-associated nitrous oxide emission during cattle manure composting process with forced aeration

This study was conducted to clarify the fates of whole nitrogen cycle-associated microorganisms during laboratory-scale composting of cattle manure with continuous forced aeration, with the primary goal of identifying microbial populations that contributed greatly to high nitrous oxide (N2O) emissions triggered by nitrification. DNA microarray analysis targeting functional genes associated with the entire microbial nitrogen cycle was applied for the first time to investigate the fates of entire nitrogen cycle-associated microbial community during composting, and revealed increased diversity of nitrogen cycle-associated microorganisms, including ammonia-oxidizing populations, during active nitrification and N2O emission periods. Further analyses of the compositions of ammonia-oxidizing populations by DNA microarray, PCR-denaturing gradient gel electrophoresis and clone library methods revealed that Nitrosomonas europaea, Nitrosomonas eutropha, and their relatives were the dominant ammonia-oxidizing populations. Overall, the analyses of entire nitrogen cycle-associated microbial community and specific functional groups by a combination of different molecular biological methods in this study could confirm that N. europaea/eutropha and their relatives contributed greatly to high N2O emissions via active nitrification during cattle manure composting with forced aeration.

Determining the Possible Etiology of Hospital-Acquired Pneumonia Using a Clone Library Analysis in Japan

Obtaining precise etiological information regarding causative bacteria is important for the proper use of antimicrobials in hospital-acquired pneumonia (HAP), which is associated with a high rate of mortality. The aim of this study was to comparatively investigate the bacterial diversity in bronchoalveolar lavage fluid (BALF) in Japanese patients with HAP by the clone library method using the 16S rRNA gene. This study included Japanese patients with HAP who were treated at our hospital and referring hospitals. BALF specimens were obtained from pneumonia lesions identified on chest radiographs and/or computed tomography. Sputum specimens were also evaluated in patients with sputum production. Sixty-eight patients were ultimately enrolled. BALF cultivation revealed bacterial positivity in 53 of 68 (77.9%) patients, and Staphylococcus aureus (30.9%) was the most frequently isolated, followed by Pseudomonas aeruginosa (16.2%), and Escherichia coli (10.3%). In contrast, the clone library analysis identified the presence of some bacterial phenotype in 65 of 68 (95.6%) patients, and streptococci (16.2%), Corynebacterium species (11.8%), anaerobes (10.3%) were frequently detected as the predominant phylotypes. Both methods tended to detect S. aureus, Klebsiella pneumoniae, and E. coli in patients with late-onset pneumonia. In addition, the cases that phylotypes of S. aureus and P. aeruginosa were found to account for > 5% of the bacterial flora of each case were 42.9% and 72.7%, respectively. These results indicate that attention should be paid to the roles of gram-positive bacilli such as streptococci, Corynebacterium species and anaerobes, in addition to Gram-negative bacilli, in the pathogenesis of HAP.

Diversity and abundance of ammonia-oxidizing bacteria (AOB) revealed by PCR amplification of amoA gene in a polyacrylamide transportation system of an oilfield

Microorganisms were detected in the hydrolyzed high-molecular-weight polyacrylamide (HPAM) transportation system. The diversity of both general bacteria and ammonia-oxidizing bacteria (AOB) in the online distribution system was further analyzed using 16S rRNA and the functional amoA genes through PCR amplification and clone library methods together with the physicochemical properties and environmental factors of samples. In total, twenty-four families of bacteria were identified in the system; biofilm sample showed higher diversity and production water showed lower diversity than the other samples. The AOB-like phylotypes in sample S6 mainly showed high alignment to Nitrosospira briensis while others in samples showed low alignment to the known uncultrued AOB, indicative of new AOB phylotypes possibly. The abundance of AOB amoA gene abundance ranged from 3.54 × 102 to 2.04 × 103 copies·ml−1, where were significantly lower than those of bacterial 16S rRNA gene copies from 2.94 × 106 to 6.33 × 108 copies·ml−1 in samples S4–S6. The RDA analysis showed that the AOB amoA gene was positively related to the concentration of NH4+, NO2−, NO3−, SO42− and S2−. The results showed the distribution of bacterial amoA gene for nitrification in this polyacrylamide transporting system and the information enriches our knowledge of microbial community diversity in the nitrogen cycle of the polyacrylamide transportation system.

Dominance of Desulfotignum in sulfate-reducing community in high sulfate production-water of high temperature and corrosive petroleum reservoirs

Petroleum reservoirs souring and pipelines corrosion are severe in Jiangsu Oilfield of China, and microorganisms have a major role in the related problems. This study revealed the microbial diversity and composition of both bacteria and archaea by using 16S rRNA gene clone library method, and those of sulfate-reducing prokaryotes (SRP) by dissimilatory sulfate reductase α-subunit encoding gene (dsr) clone library in two different samples of high temperature petroleum reservoirs containing high concentration of sulfate and with known corrosion. In addition, the abundance of bacteria and SRP in the samples was quantified by real-time PCR assays based on 16S rRNA and dsr genes. The results showed that both production water samples from Wei 2-53 and Wei 11-7 wells exhibited very rich bacterial diversity, and both Gammaproteobacteria and Alphaproteobacteria were the main abundant ones. Thioalkalivibrio-related species belonging to Gammaproteobacteria were also observed in both of them. Methanomicrobia, Archaeoglobi and Thermococci were common archaea in these samples, with Archaeoglobus as the dominant genus in both of them. The dsrA libraries analysis showed that a low diversity of sulfidogenic communities with Desulfotignum spp. as the most dominant SRP in the samples. The data reported here enhance our knowledge on microbial community and abundance including SRP in high temperature and corrosive reservoirs, providing a better basis for further research on and management control of reservoir souring and biocorrosion in oil production systems.