Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release.

The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.

Aerobic and anaerobic methane oxidation in a seasonally anoxic basin.

Shallow coastal waters are dynamic environments that dominate global marine methane emissions. Particularly high methane concentrations are found in seasonally anoxic waters, which are spreading in eutrophic coastal systems, potentially leading to increased methane emissions to the atmosphere. Here we explore how the seasonal development of anoxia influenced methane concentrations, rates of methane oxidation, and the community composition of methanotrophs in the shallow eutrophic water column of Mariager Fjord, Denmark. Our results show the development of steep concentration gradients toward the oxic-anoxic interface as methane accumulated to 1.4 µM in anoxic bottom waters. Yet, the fjord possessed an efficient microbial methane filter near the oxic-anoxic interface that responded to the increasing methane flux. In experimental incubations, methane oxidation near the oxic-anoxic interface proceeded both aerobically and anaerobically with nearly equal efficiency reaching turnover rates as high as 0.6 and 0.8 d-1, respectively, and was seemingly mediated by members of the Methylococcales belonging to the Deep Sea-1 clade. Throughout the period, both aerobic and anaerobic methane oxidation rates were high enough to consume the estimated methane flux. Thus, our results indicate that seasonal anoxia did not increase methane emissions.

The Isolation and Characterization of Rare Mycobiome Associated With Spacecraft Assembly Cleanrooms.

Ensuring biological cleanliness while assembling and launching spacecraft is critical for robotic exploration of the solar system. To date, when preventing forward contamination of other celestial bodies, NASA Planetary Protection policies have focused on endospore-forming bacteria while fungi were neglected. In this study, for the first time the mycobiome of two spacecraft assembly facilities at Jet Propulsion Laboratory (JPL) and Kennedy Space Center (KSC) was assessed using both cultivation and sequencing techniques. To facilitate enumeration of viable fungal populations and downstream molecular analyses, collected samples were first treated with chloramphenicol for 24 h and then with propidium monoazide (PMA). Among cultivable fungi, 28 distinct species were observed, 16 at JPL and 16 at KSC facilities, while 13 isolates were potentially novel species. Only four isolated species Aureobasidium melanogenum, Penicillium fuscoglaucum, Penicillium decumbens, and Zalaria obscura were present in both cleanroom facilities, which suggests that mycobiomes differ significantly between distant locations. To better visualize the biogeography of all isolated strains the network analysis was undertaken and confirmed higher abundance of Malassezia globosa and Cyberlindnera jadinii. When amplicon sequencing was performed, JPL-SAF and KSC-PHSF showed differing mycobiomes. Metagenomic fungal reads were dominated by Ascomycota (91%) and Basidiomycota (7.15%). Similar to amplicon sequencing, the number of fungal reads changed following antibiotic treatment in both cleanrooms; however, the opposite trends were observed. Alas, treatment with the antibiotic did not allow for definitive ascribing changes observed in fungal populations between treated and untreated samples in both cleanrooms. Rather, these substantial differences in fungal abundance might be attributed to several factors, including the geographical location, climate and the in-house cleaning procedures used to maintain the cleanrooms. This study is a first step in characterizing cultivable and viable fungal populations in cleanrooms to assess fungal potential as biocontaminants during interplanetary explorations. The outcomes of this and future studies could be implemented in other cleanrooms that require to reduce microbial burden, like intensive care units, operating rooms, or cleanrooms in the semiconducting and pharmaceutical industries.

Anaerobic methane oxidation in a coastal oxygen minimum zone: spatial and temporal dynamics.

Coastal waters are a major source of marine methane to the atmosphere. Particularly high concentrations of this potent greenhouse gas are found in anoxic waters, but it remains unclear if and to what extent anaerobic methanotrophs mitigate the methane flux. Here we investigate the long-term dynamics in methanotrophic activity and the methanotroph community in the coastal oxygen minimum zone (OMZ) of Golfo Dulce, Costa Rica, combining biogeochemical analyses, experimental incubations and 16S rRNA gene sequencing over 3 consecutive years. Our results demonstrate a stable redox zonation across the years with high concentrations of methane (up to 1.7 µmol L-1 ) in anoxic bottom waters. However, we also measured high activities of anaerobic methane oxidation in the OMZ core (rate constant, k, averaging 30 yr-1 in 2018 and 8 yr-1 in 2019-2020). The OPU3 and Deep Sea-1 clades of the Methylococcales were implicated as conveyors of the activity, peaking in relative abundance 5-25 m below the oxic-anoxic interface and in the deep anoxic water respectively. Although their genetic capacity for anaerobic methane oxidation remains unexplored, their sustained high relative abundance indicates an adaptation of these clades to the anoxic, methane-rich OMZ environment, allowing them to play major roles in mitigating methane fluxes.

Assessing the Risk of Transfer of Microorganisms at the International Space Station Due to Cargo Delivery by Commercial Resupply Vehicles.

BACKGROUND: With increasing numbers of interplanetary missions, there is a need to establish robust protocols to ensure the protection of extraterrestrial planets being visited from contamination by terrestrial life forms. The current study is the first report comparing the commercial resupply vehicle (CRV) microbiome with the International Space Station (ISS) microbiome to understand the risks of contamination, thus serving as a model system for future planetary missions. RESULTS: Samples obtained from the internal surfaces and ground support equipment of three CRV missions were subjected to various molecular techniques for microbial diversity analysis. In total, 25 samples were collected with eight defined locations from each CRV mission prior to launch. In general, the internal surfaces of vehicles were clean, with an order of magnitude fewer microbes compared to ground support equipment. The first CRV mission had a larger microbial population than subsequent CRV missions, which were clean as compared to the initial CRV locations sampled. Cultivation assays showed the presence of Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes and members of Ascomycota and Basidiomycota. As expected, shotgun metagenome analyses revealed the presence of more microbial taxa compared to cultivation-based assays. The internal locations of the CRV microbiome reportedly showed the presence of microorganisms capable of tolerating ultraviolet radiation (e.g., Bacillus firmus) and clustered separately from the ISS microbiome. CONCLUSIONS: The metagenome sequence comparison of the CRV microbiome with the ISS microbiome revealed significant differences showing that CRV microbiomes were a negligible part of the ISS environmental microbiome. These findings suggest that the maintenance protocols in cleaning CRV surfaces are highly effective in controlling the contaminating microbial population during cargo transfer to the ISS via the CRV route.

Validation of the International Space Station Smart Sample Concentrator for Microbial Monitoring of Low Biomass Water Samples.

Microbial monitoring on the International Space Station (ISS) is essential to keep astronauts healthy. Current practice involves culture-based methods, but future directives by the National Aeronautics and Space Administration (NASA) will require the use of molecular-based approaches, such as quantitative PCR (qPCR). However, in order to successfully and reliably detect the allowable limit of 5 × 104 colony forming units (CFUs) of bacteria per liter in potable water on the ISS with qPCR, water concentration must first be performed. This report presents the data from a validation study of a NASA-sponsored small business research initiative to develop a microgravity-compatible, automated water concentrator to be used on the ISS, which has been named the ISS Smart Sample Concentrator (iSSC). Efficiency and reproducibility of the iSSC were compared with a ground-based automated water concentrator and the standard Millipore manual filtration. Using 104 CFU/L of Sphingomonas paucimobilis, Ralstonia pickettii, and Cupriavidus basilensis and a mixed microbial community, we have shown, through culture and qPCR, that the iSSC is comparable, if not better, at recovering and concentrating bacteria from large volumes of water, with good reproducibility.

Description of Chloramphenicol Resistant Kineococcus rubinsiae sp. nov. Isolated From a Spacecraft Assembly Facility.

A Gram-positive, coccoid, motile, aerobic bacterium, designated strain B12T was isolated from a Jet Propulsion Laboratory spacecraft assembly cleanroom, Pasadena, CA, United States. Strain B12T was resistant to chloramphenicol (100 µg/mL), and is a relatively slow grower (3-5 days optimal). Strain B12T was found to grow optimally at 28 to 32°C, pH 7 to 8, and 0.5% NaCl. Fatty acid methyl ester analysis showed that the major fatty acid of the strain B12T was anteiso C15 : 0 (66.3%), which is also produced by other Kineococcus species. However, arachidonic acid (C20 : 4 ω6,9,12,16c) was present in strain B12T and Kineococcus glutinatus YIM 75677T but absent in all other Kineococcus species. 16S rRNA analysis revealed that strain B12T was 97.9% similar to Kineococcus radiotolerans and falls within the Kineococcus clade. Low 16S rRNA gene sequence similarities (<94%) with other genera in the family Kineosporiaceae, including Angustibacter (93%), Kineosporia (94% to 95%), Pseudokineococcus (93%), Quadrisphaera (93%), and Thalassiella (94%) demonstrated that the strain B12T does not belong to these genera. Phylogenetic analysis of the gyrB gene show that all known Kineococcus species exhibited <86% sequence similarity with B12T. Multi-locus sequence and whole genome sequence analyses confirmed that B12T clades with other Kineococcus species. Average nucleotide identity of strain B12T were 75-78% with other Kineococcus species, while values ranged from 72-75% with species from other genera within family Kineosporiaceae. Average amino-acid identities were 66-72% with other Kineococcus species, while they ranged from 50-58% with species from other genera. The dDDH comparison of strain B12T genome with members of genera Kineococcus showed 20-22% similarity, again demonstrating that B12T is distantly related to other members of the genus. Furthermore, analysis of whole proteome deduced from WGS places strain B12T in order Kineosporiales, confirming that strain B12T is a novel member of family Kineosporiaceae. Based on these analyses and other genome characteristics, strain B12T is assigned to a novel species within the genus Kineococcus, and the name Kineococcus rubinsiae sp. nov., is proposed. The type strain is B12T (=FJII-L1-CM-PAB2T; NRRL B-65556T, DSM 110506T).

Correction to: Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station.

In our original published manuscript entitled "Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station" (Singh et al. 2019), we found a taxonomic description format error: As per the Rule 27.

Microbial biomass turnover times and clues to cellular protein repair in energy-limited deep Baltic Sea sediments.

The discovery of active microbial life deeply buried beneath the seafloor has opened important questions: how do microorganisms cope with extreme energy limitation, what is their metabolic activity, and how do they repair damages to essential biomolecules? We used a D:L-amino acid model to calculate microbial biomass turnover times. We used a metagenome and metatranscriptome analysis to investigate the distribution of the gene that encodes Protein-L-iso aspartate(D-aspartate) O-methyltransferase (PCMT), an enzyme which recognizes damaged L-isoapartyl and D-aspartyl residues in proteins and catalyzes their repair. Sediment was retrieved during the Integrated Ocean Drilling Program (IODP) Expedition 347 from Landsort Deep and the Little Belt in the Baltic Sea. The study covers the period from the Baltic Ice Lake ca. 13 000 years ago to the present. Our results provide new knowledge on microbial biomass turnover times and protein repair in relation to different regimes of organic matter input. For the first time, we show that the PCMT gene was widely distributed and expressed among phylogenetically diverse groups of microorganisms. Our findings suggest that microbial communities are capable of repairing D-amino acids within proteins using energy obtained from the degradation of a mixture of labile compounds in microbial necromass and more recalcitrant organic matter.

Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station.

Several evolutionarily distinct, near full-length draft metagenome-resolved genomes (MRG), were assembled from sequences recovered from the International Space Station (ISS) environments. The retrieval of MRGs facilitated the exploration of a large collection of archived strains (~ 500 isolates) and assisted in isolating seven related strains. The whole genome sequences (WGS) of seven ISS strains exhibited 100% identity to the 4.85 × 106 bp of four MRGs. The "metagenome to phenome" approach led to the description of a novel bacterial genus from the ISS samples. The phylogenomics and traditional taxonomic approaches suggested that these seven ISS strains and four MRGs were not phylogenetically affiliated to any validly described genera of the family Erwiniaceae, but belong to a novel genus with the proposed name Kalamiella. Comparative genomic analyses of Kalamiella piersonii strains and MRGs showed genes associated with carbohydrate (348 genes), amino acid (384), RNA (59), and protein (214) metabolisms; membrane transport systems (108), pathways for biosynthesis of cofactors, vitamins, prosthetic groups, and pigments (179); as well as mechanisms for virulence, disease, and defense (50). Even though Kalamiella genome annotation and disc diffusion tests revealed multidrug resistance, the PathogenFinder algorithm predicted that K. piersonii strains are not human pathogens. This approach to isolating microbes allows for the characterization of functional pathways and their potential virulence properties that can directly affect human health. The isolation of novel strains from the ISS has broad applications in microbiology, not only because of concern for astronaut health but it might have a great potential for biotechnological relevance. The metagenome to phenome approach will help to improve our understanding of complex metabolic networks that control fundamental life processes under microgravity and in deep space.

Microbial turnover times in the deep seabed studied by amino acid racemization modelling.

The study of active microbial populations in deep, energy-limited marine sediments has extended our knowledge of the limits of life on Earth. Typically, microbial activity in the deep biosphere is calculated by transport-reaction modelling of pore water solutes or from experimental measurements involving radiotracers. Here we modelled microbial activity from the degree of D:L-aspartic acid racemization in microbial necromass (remains of dead microbial biomass) in sediments up to ten million years old. This recently developed approach (D:L-amino acid modelling) does not require incubation experiments and is highly sensitive in stable, low-activity environments. We applied for the first time newly established constraints on several important input parameters of the D:L-amino acid model, such as a higher aspartic acid racemization rate constant and a lower cell-specific carbon content of sub-seafloor microorganisms. Our model results show that the pool of necromass amino acids is turned over by microbial activity every few thousand years, while the turnover times of vegetative cells are in the order of years to decades. Notably, microbial turnover times in million-year-old sediment from the Peru Margin are up to 100-fold shorter than previous estimates, highlighting the influence of microbial activities on element cycling over geologic time scales.

Exploration of Microbial Diversity and Community Structure of Lonar Lake: The Only Hypersaline Meteorite Crater Lake within Basalt Rock.

Lonar Lake is a hypersaline and hyperalkaline soda lake and the only meteorite impact crater in the world situated in basalt rocks. Although culture-dependent studies have been reported, a comprehensive understanding of microbial community composition and structure in Lonar Lake remains elusive. In the present study, microbial community structure associated with Lonar Lake sediment and water samples was investigated using high-throughput sequencing. Microbial diversity analysis revealed the existence of diverse, yet largely consistent communities. Proteobacteria (30%), Actinobacteria (24%), Firmicutes (11%), and Cyanobacteria (5%) predominated in the sequencing survey, whereas Bacteroidetes (1.12%), BD1-5 (0.5%), Nitrospirae (0.41%), and Verrucomicrobia (0.28%) were detected in relatively minor abundances in the Lonar Lake ecosystem. Within the Proteobacteria phylum, the Gammaproteobacteria represented the most abundantly detected class (21-47%) within sediment samples, but only a minor population in the water samples. Proteobacteria and Firmicutes were found at significantly higher abundance (p ≥ 0.05) in sediment samples, whereas members of Actinobacteria, Candidate division TM7 and Cyanobacteria (p ≥ 0.05) were significantly abundant in water samples. Compared to the microbial communities of other hypersaline soda lakes, those of Lonar Lake formed a distinct cluster, suggesting a different microbial community composition and structure. Here we report for the first time, the difference in composition of indigenous microbial communities between the sediment and water samples of Lonar Lake. An improved census of microbial community structure in this Lake ecosystem provides a foundation for exploring microbial biogeochemical cycling and microbial function in hypersaline lake environments.

Biological synthesis of metallic nanoparticles.

The synthesis of metallic nanoparticles is an active area of academic and, more importantly, "application research" in nanotechnology. A variety of chemical and physical procedures could be used for synthesis of metallic nanoparticles. However, these methods are fraught with many problems including use of toxic solvents, generation of hazardous by-products, and high energy consumption. Accordingly, there is an essential need to develop environmentally benign procedures for synthesis of metallic nanoparticles. A promising approach to achieve this objective is to exploit the array of biological resources in nature. Indeed, over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles. In this review, we provide an overview of various reports of synthesis of metallic nanoparticles by biological means. FROM THE CLINICAL EDITOR: This review provides an overview of various methods of synthesis of metallic nanoparticles by biological means. Many chemical and physical procedures used for synthesis of metallic nanoparticles are fraught with major problems: toxic solvents, hazardous by-products, high energy consumption. Over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles.