Simulated lunar microgravity transiently arrests growth and induces osteocyte-chondrocyte lineage differentiation in human Wharton's jelly stem cells.

Human Wharton's jelly stem cells (hWJSCs) are multipotent stem cells that are extensively employed in biotechnology applications. However, the impact of simulated lunar microgravity (sµG) on the growth, differentiation, and viability of this cell population is incompletely characterized. We aimed to determine whether acute (72 h) exposure to sµG elicited changes in growth and lineage differentiation in hWJSCs and if putative changes were maintained once exposure to terrestrial gravity (1.0 G) was restored. hWJSCs were cultured under standard 1.0 G conditions prior to being passaged and cultured under sµG (0.16 G) using a random positioning machine. Relative to control, hWJSCs cultured under sµG exhibited marked reductions in growth but not viability. Cell population expression of characteristic stemness markers (CD 73, 90, 105) was significantly reduced under sµG conditions. hWJSCs had 308 significantly upregulated and 328 significantly downregulated genes when compared to 1.0 G culture conditions. Key markers of cell replication, including MKI67, were inhibited. Significant upregulation of osteocyte-chondrocyte lineage markers, including SERPINI1, MSX2, TFPI2, BMP6, COMP, TMEM119, LUM, HGF, CHI3L1 and SPP1, and downregulation of cell fate regulators, including DNMT1 and EZH2, were detected in sµG-exposed hWJSCs. When returned to 1.0 G for 3 days, sµG-exposed hWJSCs had accelerated growth, and expression of stemness markers increased, approaching normal (i.e. 95%) levels. Our data support earlier findings that acute sµG significantly reduces the cell division potential of hWJSCs and suggest that acute sµG-exposure induces reversible changes in cell growth accompanied by osteocyte-chondrocyte changes in lineage differentiation.

Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.

Machine learning identification of Pseudomonas aeruginosa strains from colony image data.

When grown on agar surfaces, microbes can produce distinct multicellular spatial structures called colonies, which contain characteristic sizes, shapes, edges, textures, and degrees of opacity and color. For over one hundred years, researchers have used these morphology cues to classify bacteria and guide more targeted treatment of pathogens. Advances in genome sequencing technology have revolutionized our ability to classify bacterial isolates and while genomic methods are in the ascendancy, morphological characterization of bacterial species has made a resurgence due to increased computing capacities and widespread application of machine learning tools. In this paper, we revisit the topic of colony morphotype on the within-species scale and apply concepts from image processing, computer vision, and deep learning to a dataset of 69 environmental and clinical Pseudomonas aeruginosa strains. We find that colony morphology and complexity under common laboratory conditions is a robust, repeatable phenotype on the level of individual strains, and therefore forms a potential basis for strain classification. We then use a deep convolutional neural network approach with a combination of data augmentation and transfer learning to overcome the typical data starvation problem in biological applications of deep learning. Using a train/validation/test split, our results achieve an average validation accuracy of 92.9% and an average test accuracy of 90.7% for the classification of individual strains. These results indicate that bacterial strains have characteristic visual 'fingerprints' that can serve as the basis of classification on a sub-species level. Our work illustrates the potential of image-based classification of bacterial pathogens and highlights the potential to use similar approaches to predict medically relevant strain characteristics like antibiotic resistance and virulence from colony data.

Soil Microbiomes Associated with a Novel Perennial Grain Cultivated under Temperate Agricultural Conditions.

A perennial wheatgrass called Kernza perennial grains was developed by the Land Institute to harness the benefits of perenniality on soil health in a commercial farming system. This study compared bacterial and fungal soil microbiomes surrounding 1-year-old Kernza, 4-year-old Kernza, and 6-week-old winter wheat in Hudson Valley, New York.

A mobile target.

The global spread of antibiotic resistance could be due to a number of factors, and not just the overuse of antibiotics in agriculture and medicine as previously thought.

Metagenomes from Soils along an Agricultural Transect in Ulster County, New York.

Many modern farming practices negatively impact ecosystems on the local and global scales. Here, we assessed the taxonomic structures of 48 soil microbial communities along an agricultural transect using 16S rRNA and internal transcribed spacer (ITS) amplicon sequencing. We further characterized the functional structures of a subsample of 12 microbiomes using whole-genome sequencing.

Bacteria communities and water quality parameters in riverine water and sediments near wastewater discharges.

Wastewater treatment plant (WWTP) discharges alter water quality and microbial communities by introducing human-associated bacteria in the environment and by altering microbial communities. To fully understand this impact, it is crucial to study whether WWTP discharges affect water and sediments microbial communities in comparable ways and whether such effects depend on specific environmental variables. Here, we present a dataset investigating the impact of a WWTP on water quality and bacterial communities by comparing samples collected directly from the WWTP outflow to surface waters and sediments at two sites above and two sites below it over a period of five months. When possible, we measured five physicochemical variables (e.g., temperature, turbidity, conductivity, dissolved oxygen, and salinity), four bioindicators (e.g., Escherichia coli, total coliforms, Enterococcus sp., and endotoxins), and two molecular indicators (e.g., intI1's relative abundance, and 16S rRNA gene profiling). Preliminary results suggest that bioindicators correlate with environmental variables and that bacterial communities present in the water tables, sediments, and treated water differ greatly in composition and structure.

Mobility of antimicrobial resistance across serovars and disease presentations in non-typhoidal Salmonella from animals and humans in Vietnam.

Non-typhoidal Salmonella (NTS) is a major cause of bacterial enterocolitis globally but also causes invasive bloodstream infections. Antimicrobial resistance (AMR) hampers the treatment of these infections and understanding how AMR spreads between NTS may help in developing effective strategies. We investigated NTS isolates associated with invasive disease, diarrhoeal disease and asymptomatic carriage in animals and humans from Vietnam. Isolates included multiple serovars and both common and rare phenotypic AMR profiles; long- and short-read sequencing was used to investigate the genetic mechanisms and genomic backgrounds associated with phenotypic AMR profiles. We demonstrate concordance between most AMR genotypes and phenotypes but identified large genotypic diversity in clinically relevant phenotypes and the high mobility potential of AMR genes (ARGs) in this setting. We found that 84 % of ARGs identified were located on plasmids, most commonly those containing IncHI1A_1 and IncHI1B(R27)_1_R27 replicons (33%), and those containing IncHI2_1 and IncHI2A_1 replicons (31%). The vast majority (95%) of ARGS were found within 10 kbp of IS6/IS26 elements, which provide plasmids with a mechanism to exchange ARGs between plasmids and other parts of the genome. Whole genome sequencing with targeted long-read sequencing applied in a One Health context identified a comparatively limited number of insertion sequences and plasmid replicons associated with AMR. Therefore, in the context of NTS from Vietnam and likely for other settings as well, the mechanisms by which ARGs move contribute to a more successful AMR profile than the specific ARGs, facilitating the adaptation of bacteria to different environments or selection pressures.

Passive exposure to cannabidiol oil does not cause microbiome dysbiosis in larval zebrafish.

The use of cannabidiol oil derived products has dramatically increased in popularity and is predicted to grow steadily over the next decade. Given its relative stability, cannabidiol is likely to accumulate in the environment and affect aquatic animals and their host-associated microbiomes. Here, using zebrafish larvae, a model system in environmental toxicology, we show that passive exposure to a concentration as high as 200 µg/L cannabidiol oil did not affect larvae survival and had limited effects on their host-associated microbial communities. We found that the changes in community structure were limited to a decrease in two sequence variants identified as Methylobacterium-Methylorubrum sp. and one ASV identified as Staphylococcus sp., as well as the increase of one sequence variant identified as Chryseobacterium sp., a bacterium commensal to zebrafish. More importantly, we found that cannabidiol oil did not affect the overall richness and diversity of the exposed fish microbiomes. These results suggest that passive exposure to cannabidiol oil is unlikely to impact aquatic organisms in significant ways.

Changes in toxin production of environmental Pseudomonas aeruginosa isolates exposed to sub-inhibitory concentrations of three common antibiotics.

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P. aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P. aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.

Draft Genome Sequences of Bacillus subtilis Strains TNC1(2019), TNC3(2019), and TNW1(2019), as Well as Bacillus velezensis Strains TNC2(2019) and TNW2(2019), Isolated from Cabbage Kimchee.

We report the draft genome sequences of five novel Bacillus strains isolated from five different batches of fermented Napa cabbage kimchee. Strains TNC1(2019), TNC3(2019), and TNW1(2019) were identified as Bacillus subtilis, while TNC2(2019) and TNW2(2019) were identified as Bacillus velezensis.

Draft Genome Sequences of Bacillus sp. Strains RM1(2019), RM2(2019), RM9(2019), RM11(2019), and RM15(2019), Isolated from Temperate Soils in the Hudson River Valley of New York.

We report the draft genome sequences of five novel Bacillus strains isolated from temperate soils in Annandale-on-Hudson, NY. Strains RM1(2019), RM9(2019), and RM15(2019) were identified as Bacillus toyonensis, while RM11(2019) was identified as Bacillus thuringiensis The draft genome of strain RM2(2019) was unclassified and likely represents a new species.

Plant-driven changes in soil microbial communities influence seed germination through negative feedbacks.

Plant-soil feedbacks (PSFs) drive plant community diversity via interactions between plants and soil microbes. However, we know little about how frequently PSFs affect plants at the seed stage, and the compositional shifts in fungi that accompany PSFs on germination.We conducted a pairwise PSF experiment to test whether seed germination was differentially impacted by conspecific versus heterospecific soils for seven grassland species. We used metagenomics to characterize shifts in fungal community composition in soils conditioned by each plant species. To investigate whether changes in the abundance of certain fungal taxa were associated with multiple PSFs, we assigned taxonomy to soil fungi and identified putative pathogens that were significantly more abundant in soils conditioned by plant species that experienced negative or positive PSFs.We observed negative, positive, and neutral PSFs on seed germination. Although conspecific and heterospecific soils for pairs with significant PSFs contained host-specialized soil fungal communities, soils with specialized microbial communities did not always lead to PSFs. The identity of host-specialized pathogens, that is, taxa uniquely present or significantly more abundant in soils conditioned by plant species experiencing negative PSFs, overlapped among plant species, while putative pathogens within a single host plant species differed depending on the identity of the heterospecific plant partner. Finally, the magnitude of feedback on germination was not related to the degree of fungal community differentiation between species pairs involved in negative PSFs. Synthesis. Our findings reveal the potential importance of PSFs at the seed stage. Although plant species developed specialized fungal communities in rhizosphere soil, pathogens were not strictly host-specific and varied not just between plant species, but according to the identity of plant partner. These results illustrate the complexity of microbe-mediated interactions between plants at different life stages that next-generation sequencing can begin to unravel.

Antibiotic Pollution in the Environment: From Microbial Ecology to Public Policy.

The ability to fight bacterial infections with antibiotics has been a longstanding cornerstone of modern medicine. However, wide-spread overuse and misuse of antibiotics has led to unintended consequences, which in turn require large-scale changes of policy for mitigation. In this review, we address two broad classes of corollaries of antibiotics overuse and misuse. Firstly, we discuss the spread of antibiotic resistance from hotspots of resistance evolution to the environment, with special concerns given to potential vectors of resistance transmission. Secondly, we outline the effects of antibiotic pollution independent of resistance evolution on natural microbial populations, as well as invertebrates and vertebrates. We close with an overview of current regional policies tasked with curbing the effects of antibiotics pollution and outline areas in which such policies are still under development.

New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.IMPORTANCESalmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections.

Dysbiosis and early mortality in zebrafish larvae exposed to subclinical concentrations of streptomycin.

Exposure to low concentrations of antibiotics found in aquatic environments can increase susceptibility to infection in adult fish due to microbiome disruption. However, little is known regarding the effect of antibiotic pollution on fish larvae. Here, we show that exposure to streptomycin, a common antibiotic used in medicine and aquaculture, disrupts the normal composition of zebrafish larvae microbiomes, significantly reducing the microbial diversity found in the fish. Exposure to streptomycin also significantly increased early mortality among fish larvae, causing full mortality within a few days of exposure at 10 µg/mL. Finally, we found that subclinical concentrations of streptomycin also increased the abundance of class 1 integrons, an integrase-dependent genetic system associated to the horizontal transfer of antibiotic resistance genes, in the larvae microbiomes. These results suggest that even low concentrations of streptomycin associated with environmental pollution could impact fish populations and lead to the creation of antibiotic resistance reservoirs.

Exposure to Arsenic Alters the Microbiome of Larval Zebrafish.

Exposure to environmental toxins such as heavy metals can perturb the development and stability of microbial communities associated with human or animal hosts. Widespread arsenic contamination in rivers and riparian habitats therefore presents environmental and health concerns for populations living near sources of contamination. To investigate how arsenic affects host microbiomes, we sequenced and characterized the microbiomes of twenty larval zebrafish exposed to three concentrations of arsenic that are found in contaminated water-low (10 ppb), medium (50 ppb), and high (100 ppb) for 20 days. We found that even a small concentration of arsenic changed the overall microbial composition, structure and diversity of microbial communities, causing dysbiosis in developing larval zebrafish microbiota. In addition, we found that a high concentration of arsenic also increased the abundance of a class 1 integron, an integrase-dependent system facilitating the horizontal transfer of genes conferring resistance to heavy metals and antibiotics.

Draft Genome Sequences of Phenotypically Distinct Janthinobacterium sp. Isolates Cultured from the Hudson Valley Watershed.

Investigation of the Hudson Valley watershed reveals many violacein-producing bacteria. These are of interest for their biotherapeutic potential in treating chytrid infections of amphibians. The draft whole-genome sequences for seven Janthinobacterium isolates with a variety of phenotypes are provided in this study.

Draft Genome Sequence of a Violacein-Producing Iodobacter sp. from the Hudson Valley Watershed.

Iodobacter species are among a number of freshwater Gram-negative violacein-producing bacteria. Janthinobacterium lividum and Chromobacterium violaceum have had their whole genomes sequenced and annotated. This is the first report of a draft whole-genome sequence of a violacein-producing Iodobacter strain that was isolated from the Hudson Valley watershed.