Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds

Symbiotic bacteria play vital roles in the survival and health of marine sponges. Sponges harbor rich, diverse and species-specific microbial communities. Symbiotic marine bacteria have increasingly been reported as promising source of bioactive compounds. A culturomics-based study was undertaken to study the diversity of bacteria from marine sponges and their antimicrobial potential. We have collected three sponge samples i.e. Acanthaster carteri, Rhytisma fulvum (soft coral) and Haliclona caerulea from north region (Obhur) of Red Sea, Jeddah Saudi Arabia. Total of 144 bacterial strains were isolated from three marine sponges using culture dependent method. Screening of isolated strains showed only 37 (26%) isolates as antagonists against oomycetes pathogens (P. ultimum and P. capsici). Among 37 antagonistic bacteria, only 19 bacterial strains exhibited antibacterial activity against human pathogens (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 29212). Four major classes of bacteria i.e γ-Proteobacteria, α-Proteobacteria, Firmicutes and Actinobacteria were recorded from three marine sponges where γ-Proteobacteria was dominant class. One potential bacterial strain Halomonas sp. EA423 was selected for identification of bioactive metabolites using GC and LC-MS analyses. Bioactive compounds Sulfamerazine, Metronidazole-OH and Ibuprofen are detected from culture extract of strain Halomonas sp. EA423. Overall, this study gives insight into composition and diversity of antagonistic bacterial community of marine sponges and coral from Red Sea and presence of active metabolites from potential strain. Our results showed that these diverse and potential bacterial communities further need to be studied to exploit their biotechnological significance.     

A comparison of DNA- and RNA-based clone libraries from the same marine bacterioplankton community

Clones from the same marine bacterioplankton community were sequenced, 100 clones based on DNA (16S rRNA genes) and 100 clones based on RNA (16S rRNA). This bacterioplankton community was dominated by alpha-Proteobacteria in terms of repetitive DNA clones (52%), but gamma-Proteobacteria dominated in terms of repetitive RNA clones (44%). The combined analysis led to a characterization of phylotypes otherwise uncharacterized if only the DNA or RNA libraries would have been analyzed alone. Of the DNA clones, 25.5% were found only in this library and no close relatives were detected in the RNA library. For clones from the RNA library, 21.5% of RNA clones did not indicate close relatives in the DNA library. Based on the comparisons between DNA and RNA libraries, our data indicate that the characterization of the bacterial community based on RNA has the potential to characterize distinct phylotypes from the marine environment, which remain undetected on the DNA level.     

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype ({"type":"entrez-nucleotide","attrs":{"text":"AF479533","term_id":"28194589","term_text":"AF479533"}}AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.     

牛源多杀性巴氏杆菌的分离与初步鉴定

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。     

Physiological and phylogenetic diversity of thermophilic spore-forming hydrocarbon-oxidizing bacteria from oil fields

The distribution and population density of aerobic hydrocarbon-oxidizing bacteria in the high-temperature oil fields of Western Siberia, Kazakhstan, and China were studied. Seven strains of aerobic thermophilic spore-forming bacteria were isolated from the oil fields and studied by microbiological and molecular biological methods. Based on the 16S rRNA gene sequences, phenotypic characteristics, and the results of DNA-DNA hybridization, the taxonomic affiliation of the isolates was tentatively established. The strains were assigned to the first and fifth subgroups of the genusBacillus on the phylogenetic branch of the gram-positive bacteria. Strains B and 421 were classified asB. licheniformis. Strains X and U, located betweenB. stearothermophilus andB. thermocatenulatus on the phylogenetic tree, and strains K, Sam, and 34, related but not identical toB. thermodenitrificans andB. thermoleovorans, undoubtedly represent two new species. Phylogenetically and metabolically related representatives of thermophilic bacilli were found to occur in geographically distant oil fields     

Molecular diversity of the microsporidium Kneallhazia solenopsae reveals an expanded host range among fire ants in North America

Kneallhazia solenopsae is a pathogenic microsporidium that infects the fire ants Solenopsis invicta and Solenopsis richteri in South America and the USA. In this study, we analyzed the prevalence and molecular diversity of K. solenopsae in fire ants from North and South America. We report the first empirical evidence of K. solenopsae infections in the tropical fire ant, Solenopsis geminata, and S. geminata × Solenopsis xyloni hybrids, revealing an expanded host range for this microsporidium. We also analyzed the molecular diversity at the 16S ribosomal RNA gene in K. solenopsae from the ant hosts S.invicta, S. richteri, S. geminata and S. geminata × S. xyloni hybrids from North America, Argentina and Brazil. We found 22 16S haplotypes. One of these haplotypes (WD_1) appears to be widely distributed, and is found in S. invicta from the USA and S. geminata from southern Mexico. Phylogenetic analyses of 16S sequences revealed that K. solenopsae haplotypes fall into one of two major clades that are differentiated by 2-3%. In some cases, multiple K. solenopsae haplotypes per colony were found, suggesting either an incomplete homogenization among gene copies within the 16S gene cluster or multiple K. solenopsae variants simultaneously infecting host colonies.     

Global distribution and diversity of marine Verrucomicrobia

Verrucomicrobia is a bacterial phylum that is commonly detected in soil, but little is known about the distribution and diversity of this phylum in the marine environment. To address this, we analyzed the marine microbial community composition in 506 samples from the International Census of Marine Microbes as well as 11 coastal samples taken from the California Current. These samples from both the water column and sediments covered a wide range of environmental conditions. Verrucomicrobia were present in 98% of the analyzed samples, and thus appeared nearly ubiquitous in the ocean. Based on the occurrence of amplified 16S ribosomal RNA sequences, Verrucomicrobia constituted on average 2% of the water column and 1.4% of the sediment bacterial communities. The diversity of Verrucomicrobia displayed a biogeography at multiple taxonomic levels and thus, specific lineages appeared to have clear habitat preference. We found that subdivision 1 and 4 generally dominated marine bacterial communities, whereas subdivision 2 was more frequent in low salinity waters. Within the subdivisions, Verrucomicrobia community composition were significantly different in the water column compared with sediment as well as within the water column along gradients of salinity, temperature, nitrate, depth and overall water column depth. Although we still know little about the ecophysiology of Verrucomicrobia lineages, the ubiquity of this phylum suggests that it may be important for the biogeochemical cycle of carbon in the ocean.     

Photobacterium marinum sp. nov., a marine bacterium isolated from a sediment sample from Palk Bay,India

The novel, cream colored, Gram-staining-negative, rod-shaped, motile bacteria, designated strains AK15^T and AK18, were isolated from sediment samples collected from Palk Bay, India. Both strains were positive for arginine dihydrolase, lysine decarboxylase, oxidase, nitrate reduction and methyl red test. The major fatty acids were C_16:0, C_{18:1 ω7c}, C_{16:1 ω7c} and/or C_{16:1 ω6c} and/or iso-C_15:0 2-OH (summed feature 3). Polar lipids content of strains AK15^T and AK18 were found to bephosphatidylethanolamine (PE), two unidentified phospholipids (PL1 and PL2) and three unidentified lipids (L1-L3). The 16S rRNA gene sequence analysis indicated strains AK15^T and AK18 as the members of the genus Photobacterium and closely related to the type strain Photobacterium jeanii with pair-wise sequence similarity of 96.7%. DNA–DNA hybridization between strain AK15^T and AK18 showed a relatedness of 87%. Based on data from the current polyphasic study, strains AK15^T and AK18 are proposed as novel species of the genus Photobacterium, for which the name Photobacterium marinum sp. nov. is proposed. The type strain of Photobacterium marinum is AK15^T (=MTCC 11066^T = DSM 25368^T).     

人体肠道微生物多样性和功能研究进展

人体肠道中庞大而复杂的微生物群落对人体自身代谢表型有深远的影响.肠道微生物群落在亚种或菌株水平上表现出极大的多样性.利用微生物分子生态学、元基因组学和代谢组学研究方法,发现肠道微生物与宿主表现出共进化的特点,肠道微生物群落及其基因组为宿主提供了互补的遗传和代谢功能,表现出互惠共生关系.但是,肠道微生物群落中影响宿主代谢表型的关键功能菌鉴定及其作用模式问题仍然悬而未决,综合运用多种高通量研究方法和多维数据分析方法可能成为解决这个问题的突破口.     

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.     

Characterization and Phylogenetic Diversity of Carboxymethyl Cellulase Producing <Emphasis Type="Italic">Bacillus</Emphasis> Species from a Landfill Ecosystem

Total population of cellulose degrading bacteria was studied in a landfill ecosystem as a part of microbial diversity study. Samples were obtained from 3 and 5 feet depth of a local landfill being operated for past 10 years. Among many isolates, 22 bacterial strains were selected based on their capability to decompose carboxymethyl cellulose (CMC). These isolates were cultivated on agar medium with CMC as the carbon source. All isolates were Gram positive, endospore forming and alkalophilic bacteria with optimum growth pH 9–10. They were grouped based on the phenotypic and chemotaxonomic characters and representative strains of different groups along with high carboxymethyl cellulase (CMCase) producing strains were included for further characterization. Analysis of 16S rRNA gene indicated that these strains belong to different species of the genus Bacillus. Maximum CMCase activity of 4.8 U/ml at 50°C was obtained by strain LFC15. Results in the present study indicated the potential of waste land ecosystems such as landfill are potential source for isolation of industrially important microorganisms.     

Biodegradation of microcystin-RR by Bacillus flexus isolated from a Saudi freshwater lake

A bacterium capable of degrading microcystin-RR (MC-RR) was isolated from a Saudi eutrophic lake which was previously reported to have microcystin-producing cyanobacteria. Based on the analysis of the 16S rRNA gene sequence, the isolated strain SSZ01, most likely belong to the genus Bacillus with a highest sequence similarity (99%) with Bacillus flexus strain EMGA5. It was found that B. flexus strain SSZ01, possesses an mlrA gene encoding the most important enzyme for MC degradation. This strain was capable of degrading MC-RR, at a concentration of 10 mg l⁻¹, in batch experiments under environmentally relevant conditions. The degradation of MC-RR was completely removed within 4 d. The degradation of MC-RR by this strain occurred in a rich medium nutrient broth (NB), indicating that this could likely occur along with other organic compounds found in the environment. Therefore, the coexistence of such bacteria with MCs in the same environment can contribute to the self-purification of the ecosystem from such potent toxins. This is the first study to report that B. flexus can degrade MCs.     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied. A. Sghir and E. Ammar have equally contributed to this work.     

Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin

High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5–8 years old orchards of kinnow mandarin {Citrus reticulate Balanco (‘King’ × ‘Willow mandarin’)} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus (‘Ca L. asiaticus’) showing maximum identity of 95.9% with ‘Ca L. asiaticus’ from USA and Brazil in 16S rRNA. The study indicates definite association of ‘Ca L. asiaticus’ with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.     

Characterization of <Emphasis Type="Italic">Vibrio</Emphasis> species isolated from freshwater fishes by ribotyping

Three Vibrio species from the resident microflora of gastrointestinal tract of freshwater carps and prawns were isolated and confirmed biochemically as V. fluvialis from Cyprinus carpio/Labeo rohita; V. parahaemolyticus from Macrobrachium rosenbergii and V. harveyi from Macrobrachium malcomsoni. The genetic relationship among these Vibrio species was carried out by polymerase chain reaction (PCR) amplification of 16S rRNA gene followed by restriction digestion with Hae III, Bam HI and Pst I. Dendogram based on ribotyping showed the isolated Vibrios were differentiated into three clusters. V. harveyi was closely related to V. vulnificus (reference Microbial type Culture Collection (MTCC) strain) and distantly related to V. parahaemolyticus as well as V. fluvialis.     

Shivajiella indica gen. nov., sp. nov., a marine bacterium of the family "Cyclobacteriaceae" with nitrate reducing activity

Novel orange pigmented, Gram-negative-staining, rod-shaped, non-motile, strictly aerobic strains designated NIO-S1(T) and NIO-S2 were isolated from the water sample of a pond adjacent to the coast and an algal mat from a fish pond, respectively, at Kakinada, India. Both strains were positive for oxidase, catalase and β-galactosidase activities. The predominant fatty acids in NIO-S1(T) were iso-C(15:0) (39.6%), anteiso-C(15:0) (9.9%), iso-C(17:0) 3OH (10.9%) and C(16:1)ω7c/C(16:1)ω6c (summed feature 3) (5.7%). The strains contained MK-7 as the major respiratory quinine, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids as the polar lipids. Phylogenetic analysis indicated that strain NIO-S1(T) was a member of the family "Cyclobacteriaceae" of the class "Sphingobacteriia" and it clustered with the genera Fontibacter, Cecembia and Aquiflexum with phylogenetic distances of 6.8, 9.0 and 12.2% (93.2, 91.0 and 87.8% similarity), respectively. DNA-DNA hybridization between strains NIO-S1(T) and NIO-S2 showed a relatedness of 93% and rep-PCR banding patterns were similar. Based on data from the current polyphasic study, it is proposed that the new isolates be placed in a new genus and species with the name Shivajiella indica gen. nov., sp. nov. The type strain of Shivajiella indica is NIO-S1(T) (= KCTC 19812(T)=MTCC 11065(T)).     

Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir.