Targeting methanotrophs and isolation of a novel psychrophilic Methylobacter species from a terrestrial Arctic alkaline methane seep in Lagoon Pingo, Central Spitsbergen (78° N).

The microbial diversity associated with terrestrial groundwater seepage through permafrost soils is tightly coupled to the geochemistry of these fluids. Terrestrial alkaline methane seeps from Lagoon Pingo, Central Spitsbergen (78°N) in Norway, with methane-saturated and oxygen-limited groundwater discharge providing a potential habitat for methanotrophy. Here, we report on the microbial community's comparative analyses and distribution patterns at two sites close to Lagoon Pingo's methane emission source. To target methane-oxidizing bacteria from this system, we analysed the microbial community pattern of replicate samples from two sections near the main methane seepage source. DNA extraction, metabarcoding and subsequent sequencing of 16S rRNA genes revealed microbial communities where the major prokaryotic phyla were Pseudomonadota (42-47%), Gemmatimonadota (4-14%) and Actinobacteriota (7-11%). Among the Pseudomonadota, members of the genus Methylobacter were present at relative abundances between 1.6 and 4.7%. Enrichment targeting the methane oxidising bacteria was set up using methane seep sediments as inoculum and methane as the sole carbon and energy source, and this resulted in the isolation of a novel psychrophilic methane oxidizer, LS7-T4AT. The optimum growth temperature for the isolate was 13 °C and the pH optimum was 8.0. The morphology of cells was short rods, and TEM analysis revealed intracytoplasmic membranes arranged in stacks, a distinctive feature for Type I methanotrophs in the family Methylomonadaceae of the class Gammaproteobacteria. The strain belongs to the genus Methylobacter based on high 16S rRNA gene similarity to the psychrophilic species of Methylobacter psychrophilus Z-0021T (98.95%), the psychrophilic strain Methylobacter sp. strain S3L5C (99.00%), and the Arctic mesophilic species of Methylobacter tundripaludum SV96T (99.06%). The genome size of LS7-T4AT was 4,338,157 bp with a G + C content of 47.93%. The average nucleotide identities (ANIb) of strain LS7-T4AT to 10 isolated strains of genus Methylobacter were between 75.54 and 85.51%, lower than the species threshold of 95%. The strain LS7-T4AT represents a novel Arctic species, distinct from other members of the genus Methylobacter, for which the name Methylobacter svalbardensis sp. nov. is proposed. The type of strain is LS7-T4AT (DSMZ:114308, JCM:39463).

A community-based cluster randomized controlled trial (cRCT) to evaluate the impact and operational assessment of "safe motherhood and newborn health promotion package": study protocol.

BACKGROUND: Despite considerable progress in reduction of both under-five and maternal mortality in recent decades, Bangladesh is still one of the low and middle income countries with high burden of maternal and neonatal mortality. The primary objective of the current study is to measure the impact of a comprehensive package of interventions on maternal and neonatal mortality. In addition, changes in coverage, quality and utilization of maternal and newborn health (MNH) services, social capital, and cost effectiveness of the interventions will be measured. METHODS: A community-based, cluster randomized controlled trial design will be adopted and implemented in 30 unions of three sub-districts of Chandpur district of Bangladesh. Every union, the lowest administrative unit of the local government with population of around 20,000-30,000, will be considered a cluster. Based on the baseline estimates, 15 clusters will be paired for random assignment as intervention and comparison clusters. The primary outcome measure is neonatal mortality, and secondary outcomes are coverage of key interventions like ANC, PNC, facility and skilled provider delivery. Baseline, midterm and endline household survey will be conducted to assess the key coverage of interventions. Health facility assessment surveys will be conducted periodically to assess facility readiness and utilization of MNH services in the participating health facilities. DISCUSSION: The current study is expected to provide essential strong evidences on the impact of a comprehensive package of interventions to the Bangladesh government, and other developmental partners. The study results may help in prioritizing, planning, and scaling-up of Safe Motherhood Promotional interventions in other geographical areas of Bangladesh as well as to inform other developing countries of similar settings. TRIAL REGISTRATION: NCT03032276 .

Methane oxidation at 55 degrees C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum.

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at approximately 55 degrees C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kam1 will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum.

A Novel Moderately Thermophilic Facultative Methylotroph within the Class Alphaproteobacteria.

Methylotrophic bacteria (non-methanotrophic methanol oxidizers) consuming reduced carbon compounds containing no carbon-carbon bonds as their sole carbon and energy source have been found in a great variety of environments. Here, we report a unique moderately thermophilic methanol-oxidising bacterium (strain LS7-MT) that grows optimally at 55 °C (with a growth range spanning 30 to 60 °C). The pure isolate was recovered from a methane-utilizing mixed culture enrichment from an alkaline thermal spring in the Ethiopia Rift Valley, and utilized methanol, methylamine, glucose and a variety of multi-carbon compounds. Phylogenetic analysis of the 16S rRNA gene sequences demonstrated that strain LS7-MT represented a new facultatively methylotrophic bacterium within the order Hyphomicrobiales of the class Alphaproteobacteria. This new strain showed 94 to 96% 16S rRNA gene identity to the two methylotroph genera, Methyloceanibacter and Methyloligella. Analysis of the draft genome of strain LS7-MT revealed genes for methanol dehydrogenase, essential for methanol oxidation. Functional and comparative genomics of this new isolate revealed genomic and physiological divergence from extant methylotrophs. Strain LS7-MT contained a complete mxaF gene cluster and xoxF1 encoding the lanthanide-dependent methanol dehydrogenase (XoxF). This is the first report of methanol oxidation at 55 °C by a moderately thermophilic bacterium within the class Alphaproteobacteria. These findings expand our knowledge of methylotrophy by the phylum Proteobacteria in thermal ecosystems and their contribution to global carbon and nitrogen cycles.

A Novel Cold-adapted Methylovulum species, with a High C16:1ω5c Content, Isolated from an Arctic Thermal Spring in Spitsbergen.

A novel cold-adapted methane-oxidizing bacterium, termed TFB, was isolated from the thermoglacial Arctic karst spring, Trollosen, located in the South Spitsbergen National Park (Norway). The source water is cold and extremely low in phosphate and nitrate. The isolate belongs to the Methylovulum genus of gammaproteobacterial methanotrophs, with the closest phylogenetic affiliation with Methylovulum miyakonense and Methylovulum psychrotolerans (96.2 and 96.1% 16S rRNA gene sequence similarities, respectively). TFB is a strict aerobe that only grows in the presence of methane or methanol. It fixes atmospheric nitrogen and contains Type I intracellular membranes. The growth temperature range was 2-22°C, with an optimum at 13-18°C. The functional genes pmoA, mxaF, and nifH were identified by PCR, whereas mmoX and cbbL were not. C16:1ω5c was identified as the major fatty acid constituent, at an amount (>49%) not previously found in any methanotrophs, and is likely to play a major role in cold adaptation. Strain TFB may be regarded as a new psychrotolerant or psychrophilic species within the genus Methylovulum. The recovery of this cold-adapted bacterium from a neutral Arctic thermal spring increases our knowledge of the diversity and adaptation of extremophilic gammaproteobacterial methanotrophs in the candidate family "Methylomonadaceae".

A Novel Moderately Thermophilic Type Ib Methanotroph Isolated from an Alkaline Thermal Spring in the Ethiopian Rift Valley.

Aerobic moderately thermophilic and thermophilic methane-oxidizing bacteria make a substantial contribution in the control of global warming through biological reduction of methane emissions and have a unique capability of utilizing methane as their sole carbon and energy source. Here, we report a novel moderately thermophilic Methylococcus-like Type Ib methanotroph recovered from an alkaline thermal spring (55.4 °C and pH 8.82) in the Ethiopian Rift Valley. The isolate, designated LS7-MC, most probably represents a novel species of a new genus in the family Methylococcaceae of the class Gammaproteobacteria. The 16S rRNA gene phylogeny indicated that strain LS7-MC is distantly related to the closest described relative, Methylococcus capsulatus (92.7% sequence identity). Growth was observed at temperatures of 30-60 °C (optimal, 51-55 °C), and the cells possessed Type I intracellular membrane (ICM). The comparison of the pmoA gene sequences showed that the strain was most closely related to M. capsulatus (87.8%). Soluble methane monooxygenase (sMMO) was not detected, signifying the biological oxidation process from methane to methanol by the particulate methane monooxygenase (pMMO). The other functional genes mxaF, cbbL and nifH were detected by PCR. To our knowledge, the new strain is the first isolated moderately thermophilic methanotroph from an alkaline thermal spring of the family Methylococcaceae. Furthermore, LS7-MC represents a previously unrecognized biological methane sink in thermal habitats, expanding our knowledge of its ecological role in methane cycling and aerobic methanotrophy.

High-quality draft genome of the methanotroph Methylovulum psychrotolerans Str. HV10-M2 isolated from plant material at a high-altitude environment.

Here we present the genome of Methylovulum psychrotolerans strain HV10-M2, a methanotroph isolated from Hardangervidda national park (Norway). This strain represents the second of the two validly published species genus with a sequenced genome. The other is M. miyakonense HT12, which is the type strain of the species and the type species of the genus Methylovulum. We present the genome of M. psychrotolerants str. HV10-M2 and discuss the differences between M. psychrotolerans and M. miyakonense. The genome size of M. psychrotolerans str. HV10-M2 is 4,923,400 bp and contains 4415 protein-coding genes, 50 RNA genes and an average GC content of 50.88%.

A 3-Year Review of the Outcome of Pars Plana Vitrectomy for Dropped Lens Fragments after Cataract Surgery in a Tertiary Eye Hospital in Dhaka, Bangladesh.

BACKGROUND: Dropped lens fragments into the vitreous are uncommon, but potentially sight threatening complications of cataract surgery. The recommended approach for removal of dropped lens matter is pars plana vitrectomy with or without ultrasonic emulsification with the fragmatome. This study aimed to determine the visual outcome following pars plana vitrectomy for dropped lens fragments complicating cataract surgery at 6 weeks, 3 months and 6 months. METHODS: This study is a retrospective review of patients who had PPV as treatment for dropped lens fragments complicating cataract surgery at Ispahani Islamia Eye hospital and Institute. The case notes of all patients who had PPV for dropped nucleus from January 2013 to December 2015 were reviewed. Information retrieved included the bio-data of the patients, clinical features such as visual acuity, intraocular pressure, cornea clarity, presence of anterior chamber activity, Lens status and fundus findings at presentation, 6 weeks, 3 months and 6 months post surgery. Details of intra ocular procedure (Cryotherapy, Endo laser, Silicon oil or gas tamponade) in addition to PPV were noted. RESULT: Thirty-two cases were reviewed within the 36-month time period. There were 8(25%) females and 24(75%) males. Male to female ratio was 1:3. The best corrected visual acuity of patients at presentation was better than or equal to 6/60 in 56.3% of the patients. Dropped nucleus occurred during phacoemulsification cataract surgery in the majority (59.6%) of the patients. The best corrected visual acuity was 6/18 or better in 10(31.3%) eyes at 6 months post-surgery. Worsening of visual acuity to light perception occurred in 3(9.4%) eyes at 6 months, and 3 eyes (9.4%) developed phthisis bulbi. CONCLUSION: Modest improvement in visual acuity can be achieved after dropped nucleus complicating cataract surgery by Pars plana vitrectomy and removal of dislocated lens fragment.

Acid-Tolerant Moderately Thermophilic Methanotrophs of the Class Gammaproteobacteria Isolated From Tropical Topsoil with Methane Seeps.

Terrestrial tropical methane seep habitats are important ecosystems in the methane cycle. Methane oxidizing bacteria play a key role in these ecosystems as they reduce methane emissions to the atmosphere. Here, we describe the isolation and initial characterization of two novel moderately thermophilic and acid-tolerant obligate methanotrophs, assigned BFH1 and BFH2 recovered from a tropical methane seep topsoil habitat. The new isolates were strictly aerobic, non-motile, coccus-shaped and utilized methane and methanol as sole carbon and energy source. Isolates grew at pH range 4.2-7.5 (optimal 5.5-6.0) and at a temperature range of 30-60°C (optimal 51-55°C). 16S rRNA gene phylogeny placed them in a well-separated branch forming a cluster together with the genus Methylocaldum as the closest relatives (93.1-94.1% sequence similarity). The genes pmoA, mxaF, and cbbL were detected, but mmoX was absent. Strains BFH1 and BFH2 are, to our knowledge, the first isolated acid-tolerant moderately thermophilic methane oxidizers of the class Gammaproteobacteria. Each strain probably denotes a novel species and they most likely represent a novel genus within the family Methylococcaceae of type I methanotrophs. Furthermore, the isolates increase our knowledge of acid-tolerant aerobic methanotrophs and signify a previously unrecognized biological methane sink in tropical ecosystems.

Novel Methanotrophs of the Family Methylococcaceae from Different Geographical Regions and Habitats.

Terrestrial methane seeps and rice paddy fields are important ecosystems in the methane cycle. Methanotrophic bacteria in these ecosystems play a key role in reducing methane emission into the atmosphere. Here, we describe three novel methanotrophs, designated BRS-K6, GFS-K6 and AK-K6, which were recovered from three different habitats in contrasting geographic regions and ecosystems: waterlogged rice-field soil and methane seep pond sediments from Bangladesh; and warm spring sediments from Armenia. All isolates had a temperature range for growth of 8-35 °C (optimal 25-28 °C) and a pH range of 5.0-7.5 (optimal 6.4-7.0). 16S rRNA gene sequences showed that they were new gammaproteobacterial methanotrophs, which form a separate clade in the family Methylococcaceae. They fell into a cluster with thermotolerant and mesophilic growth tendency, comprising the genera Methylocaldum-Methylococcus-Methyloparacoccus-Methylogaea. So far, growth below 15 °C of methanotrophs from this cluster has not been reported. The strains possessed type I intracytoplasmic membranes. The genes pmoA, mxaF, cbbL, nifH were detected, but no mmoX gene was found. Each strain probably represents a novel species either belonging to the same novel genus or each may even represent separate genera. These isolates extend our knowledge of methanotrophic Gammaproteobacteria and their physiology and adaptation to different ecosystems.

Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia.

Aerobic methanotrophic bacteria are capable of utilizing methane as their sole energy source. They are commonly found at the oxic/anoxic interfaces of environments such as wetlands, aquatic sediments, and landfills, where they feed on methane produced in anoxic zones of these environments. Until recently, all known species of aerobic methanotrophs belonged to the phylum Proteobacteria, in the classes Gammaproteobacteria and Alphaproteobacteria. However, in 2007-2008 three research groups independently described the isolation of thermoacidophilic methanotrophs that represented a distinct lineage within the bacterial phylum Verrucomicrobia. Isolates were obtained from geothermal areas in Italy, New Zealand and Russia. They are by far the most acidophilic methanotrophs known, with a lower growth limit below pH 1. Here we summarize the properties of these novel methanotrophic Verrucomicrobia, compare them with the proteobacterial methanotrophs, propose a unified taxonomic framework for them and speculate on their potential environmental significance. New genomic and physiological data are combined with existing information to allow detailed comparison of the three strains. We propose the new genus Methylacidiphilum to encompass all three newly discovered bacteria.