Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Locating and Quantifying Carbon Steel Corrosion Rates Linked to Fungal B20 Biodiesel Degradation.

Fungi that degrade B20 biodiesel in storage tanks have also been linked to microbiologically influenced corrosion (MIC). A member of the filamentous fungal genus Paecilomyces and a yeast from the genus Wickerhamomyces were isolated from heavily contaminated B20 storage tanks from multiple Air Force bases. Although these taxa were linked to microbiologically influenced corrosion in situ, precise measurement of their corrosion rates and pitting severity on carbon steel was not available. In the experiments described here, we directly link fungal growth on B20 biodiesel to higher corrosion rates and pitting corrosion of carbon steel under controlled conditions. When these fungi were growing solely on B20 biodiesel for carbon and energy, consumption of FAME and n-alkanes was observed. The corrosion rates for both fungi were highest at the interface between the B20 biodiesel and the aqueous medium, where they acidified the medium and produced deeper pits than abiotic controls. Paecilomyces produced the most corrosion of carbon steel and produced the greatest pitting damage. This study characterizes and quantifies the corrosion of carbon steel by fungi that are common in fouled B20 biodiesel through their metabolism of the fuel, providing valuable insight for assessing MIC associated with storing and dispensing B20 biodiesel. IMPORTANCE Biodiesel is widely used across the United States and worldwide, blended with ultra-low-sulfur diesel in various concentrations. In this study, we were able to demonstrate that the filamentous fungus Paecilomyces AF001 and the yeast Wickerhamomyces SE3 were able to degrade fatty acid methyl esters and alkanes in biodiesel, causing increases in acidity. Both fungi also accelerated the corrosion of carbon steel, especially at the interface of the fuel and water, where their biofilms were located. This research provides controlled, quantified measurements and the localization of microbiologically influenced corrosion caused by common fungal contaminants in biodiesel fuels.

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides.

Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several ß-alanines that occurred in a repeating ααß motif, causing the compound to adopt a unique right-handed 311 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI50 0.55 ± 0.04 µM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.

Cross-resistance profiles of malaria mosquito P450s associated with pyrethroid resistance against WHO insecticides.

Extensive use of pyrethroids for malaria control in Africa has led to widespread pyrethroid resistance in the two major African vectors of malaria An. gambiae and An. funestus. This is often associated with constitutively elevated levels of cytochrome P450s involved with pyrethroid metabolism and detoxification. P450s have the capacity to metabolise diverse substrates, which raises concerns about their potential to cause cross-resistance. A bank of seven recombinant P450s from An. gambiae (CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 9J5) and An. funestus (CYP6P9a) commonly associated with pyrethroid resistance were screened against twelve insecticides representing the five major classes of insecticides recommended by WHO for malaria control; permethrin, etofenprox and bifenthrin (type I pyrethroids), deltamethrin, lambda cyhalothrin and cypermethrin (type II pyrethroids), DDT (organochlorine), bendiocarb (carbamate), malathion, pirimiphos methyl and fenitrothion (organophosphates) and pyriproxyfen (juvenile hormone analogue). DDT was not metabolised by the P450 panel, while bendiocarb was only metabolised by CYP6P3. Pyrethroids and pyriproxyfen were largely susceptible to metabolism by the P450 panel, as were organophosphates, which are activated by P450s. Primiphos-methyl is increasingly used for malaria control. Examination of the pirimiphos-methyl metabolites generated by CYP6P3 revealed both the active pirimiphos-methyl-oxon form and the inactive oxidative cleavage product 2-diethylamino-6-hydroxy-4-methylpyrimidine. The inhibition profile of CYPs 6M2, 6P2, 6P3, 6P9a and 9J5 was also examined using diethoxyfluorescein (DEF) as the probe substrate. Bendiocarb was the weakest inhibitor with IC50 > 100 µM across the P450 panel, while CYP6M2 showed strongest inhibition by malathion (IC50 0.7 µM). The results suggest that P450s present at elevated levels in two major Anopheline vectors of malaria in Africa have the capacity to metabolise a diverse range of pyrethroid and organophosphate insecticides as well as pyriproxyfen that could impact vector control.

Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML.

Algal blooms in lakes are often associated with anthropogenic eutrophication; however, they can occur without the human introduction of nutrients to a lake. A rare bloom of the alga Picocystis sp. strain ML occurred in the spring of 2016 at Mono Lake, a hyperalkaline lake in California, which was also at the apex of a multiyear-long drought. These conditions presented a unique sampling opportunity to investigate microbiological dynamics and potential metabolic function during an intense natural algal bloom. We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from the surface to a depth of 25 m in June 2016. Across sampled depths, rRNA gene sequencing revealed that Picocystis-associated chloroplasts were found at 40 to 50% relative abundance, greater than values recorded previously. Despite high relative abundances of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectable limits below a depth of 15 m, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, both metagenomic and metatranscriptomic data suggested a depletion of anaerobic sulfate-reducing microorganisms throughout the lake's water column. Transcripts associated with photosystem I and II were expressed at both 2 m and 25 m, suggesting that limited oxygen production could occur at extremely low light levels at depth within the lake. Blooms of Picocystis appear to correspond with a loss of microbial activity such as sulfate reduction within Mono Lake, yet microorganisms may survive within the sediment to repopulate the lake water column as the bloom subsides.IMPORTANCE Mono Lake, California, provides a habitat to a unique ecological community that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists from Mono Lake to understand how the microbial community responds to human-influenced drought or algal bloom or what metabolisms are lost in the water column as a consequence of such environmental pressures. While previously identified anaerobic members of the microbial community disappear from the water column during drought and bloom, sediment samples suggest that these microorganisms survive at the lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank that could restore the microbial community as a bloom subsides. Our work sheds light on the potential photosynthetic activity of the halotolerant alga Picocystis sp. strain ML and how the function and activity of the remainder of the microbial community responds during a bloom at Mono Lake.

Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes.

Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.

Design features of offshore oil production platforms influence their susceptibility to biocorrosion.

Offshore oil-producing platforms are designed for efficient and cost-effective separation of oil from water. However, design features and operating practices may create conditions that promote the proliferation and spread of biocorrosive microorganisms. The microbial communities and their potential for metal corrosion were characterized for three oil production platforms that varied in their oil-water separation processes, fluid recycling practices, and history of microbially influenced corrosion (MIC). Microbial diversity was evaluated by 16S rRNA gene sequencing, and numbers of total bacteria, archaea, and sulfate-reducing bacteria (SRB) were estimated by qPCR. The rates of 35S sulfate reduction assay (SRA) were measured as a proxy for metal biocorrosion potential. A variety of microorganisms common to oil production facilities were found, but distinct communities were associated with the design of the platform and varied with different locations in the processing stream. Stagnant, lower temperature (<37 °C) sites in all platforms had more SRB and higher SRA compared to samples from sites with higher temperatures and flow rates. However, high (5 mmol L-1) levels of hydrogen sulfide and high numbers (107 mL-1) of SRB were found in only one platform. This platform alone contained large separation tanks with long retention times and recycled fluids from stagnant sites to the beginning of the oil separation train, thus promoting distribution of biocorrosive microorganisms. These findings tell us that tracking microbial sulfate-reducing activity and community composition on off-shore oil production platforms can be used to identify operational practices that inadvertently promote the proliferation, distribution, and activity of biocorrosive microorganisms.

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana.

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Nutrient enrichment increased species richness of leaf litter fungal assemblages in a tropical forest.

Microbial communities play a major role in terrestrial ecosystem functioning, but the determinates of their diversity and functional interactions are not well known. In this study, we explored leaf litter fungal diversity in a diverse Panama lowland tropical forest in which a replicated factorial N, P, K and micronutrient fertilization experiment of 40 × 40 m plots had been ongoing for nine years. We extracted DNA from leaf litter samples and used fungal-specific amplification and a 454 pyrosequencing approach to sequence two loci, the nuclear ribosomal internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) D1 region. Using a 95% sequence similarity threshold for ITS1 spacer recovered a total of 2523 OTUs, and the number of unique ITS1 OTUs per 0.5-1.0 g leaf litter sample ranged from 55 to 177. Ascomycota were the dominant phylum among the leaf litter fungi (71% of the OTUs), followed by Basidiomycota (26% of the OTUs). In contrast to our expectations based on temperate ecosystems, long-term addition of nutrients increased, rather than decreased, species richness relative to controls. Effect of individual nutrients was more subtle and seen primarily as changes in community compositions especially at lower taxonomic levels, rather than as significant changes in species richness. For example, plots receiving P tended to show a greater similarity in community composition compared to the other nutrient treatments, the +PK, +NK and +NPK plots appeared to be more dominated by the Nectriaceae than other treatments, and indicator species for particular nutrient combinations were identified.

Thermoanaerobaculum aquaticum gen. nov., sp. nov., the first cultivated member of Acidobacteria subdivision 23, isolated from a hot spring.

A novel bacterium was isolated from a freshwater hot spring, the Hale House Spring, located at Hot Springs National Park, Hot Springs, AR, USA. Cells of strain MP-01(T) stained Gram-negative, were rod-shaped, non-motile, strictly anaerobic and chemo-organotrophic and did not form spores. Growth occurred at 50-65 °C, with an optimum at 60 °C, at pH 6.0-8.0, with an optimum at pH 6.5-7.0, and at NaCl concentrations up to 0.5 % (w/v), with optimum growth in the absence of NaCl. Strain MP-01(T) was capable of fermentative growth on pyruvate or proteinaceous substrates as well as reducing Fe(III) and Mn(IV). Major fatty acids were iso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0 and iso-C17 : 0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine and the major isoprenoid quinone was MK-10. In the polyamine pattern, sym-homospermidine was the predominant compound. The DNA G+C content was 62.7 mol%. Analysis of the 16S rRNA gene sequence of the isolate indicated that strain MP-01(T) represents the first reported cultivated member of subdivision 23 of the Acidobacteria. It is proposed that strain MP-01(T) represents a novel genus and species, for which the name Thermoanaerobaculum aquaticum gen. nov., sp. nov. is proposed. The type strain of Thermoanaerobaculum aquaticum is MP-01(T) ( = DSM 24856(T) = JCM 18256(T)).

Fontimonas thermophila gen. nov., sp. nov., a moderately thermophilic bacterium isolated from a freshwater hot spring, and proposal of Solimonadaceae fam. nov. to replace Sinobacteraceae Zhou et al. 2008.

A novel bacterial strain designated HA-01(T) was isolated from a freshwater terrestrial hot spring located at Hot Springs National Park, Arkansas, USA. Cells were Gram-negative-staining, rod-shaped, aerobic, chemo-organotrophic, oxidase- and catalase-positive, non-spore-forming and motile by means of a single polar flagellum. Growth occurred at 37-60 °C, with an optimum between 45 and 50 °C, and at pH 6.5-8.5, with an optimum between pH 6.5 and 7.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the closest relatives of strain HA-01(T) were Solimonas aquatica NAA-16(T) (93.8 %), Solimonas flava CW-KD 4(T) (94.1 %), Solimonas soli DCY12(T) (93.1 %), Solimonas variicoloris MN28(T) (94.0 %), Nevskia ramosa Soe1(T) (91.2 %) and Hydrocarboniphaga effusa AP103(T) (91.1 %). Major fatty acids consisted of C(16 : 0), iso-C(16 : 0), C(16 : 1)ω5c and summed feature 8 (C(18 : 1)ω9c, C(18 : 1)ω7c and C(18 : 1)ω6c). Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the major isoprenoid quinone was Q-8. The DNA G+C content was 64.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, it is proposed that strain HA-01(T) represents a novel species in a new genus for which the name Fontimonas thermophila gen. nov., sp. nov. is proposed. The type strain of the type species is HA-01(T) (= DSM 23609(T) = CCUG 59713(T)). A new family, Solimonadaceae fam. nov., is also proposed to replace Sinobacteriaceae Zhou et al. 2008.

Impact of organosulfur content on diesel fuel stability and implications for carbon steel corrosion.

Ultralow sulfur diesel (ULSD) fuel has been integrated into the worldwide fuel infrastructure to help meet a variety of environmental regulations. However, desulfurization alters the properties of diesel fuel in ways that could potentially impact its biological stability. Fuel desulfurization might predispose ULSD to biodeterioration relative to sulfur-rich fuels and in marine systems accelerate rates of sulfate reduction, sulfide production, and carbon steel biocorrosion. To test such prospects, an inoculum from a seawater-compensated ballast tank was amended with fuel from the same ship or with refinery fractions of ULSD, low- (LSD), and high sulfur diesel (HSD) and monitored for sulfate depletion. The rates of sulfate removal in incubations amended with the refinery fuels were elevated relative to the fuel-unamended controls but statistically indistinguishable (∼50 µM SO4/day), but they were found to be roughly twice as fast (∼100 µM SO4/day) when the ship's own diesel was used as a source of carbon and energy. Thus, anaerobic hydrocarbon metabolism likely occurred in these incubations regardless of fuel sulfur content. Microbial community structure from each incubation was also largely independent of the fuel amendment type, based on molecular analysis of 16S rRNA sequences. Two other inocula known to catalyze anaerobic hydrocarbon metabolism showed no differences in fuel-associated sulfate reduction or methanogenesis rates between ULSD, LSD, and HSD. These findings suggest that the stability of diesel is independent of the fuel organosulfur compound status and reasons for the accelerated biocorrosion associated with the use of ULSD should be sought elsewhere.

Using Wastewater Surveillance to Monitor Gastrointestinal Pathogen Infections in the State of Oklahoma.

During the COVID-19 pandemic, wastewater surveillance was widely used to monitor temporal and geographical infection trends. Using this as a foundation, a statewide program for routine wastewater monitoring of gastrointestinal pathogens was established in Oklahoma. The results from 18 months of surveillance showed that wastewater concentrations of Salmonella, Campylobacter, and norovirus exhibit similar seasonal patterns to those observed in reported human cases (F = 4-29, p < 0.05) and that wastewater can serve as an early warning tool for increases in cases, offering between one- and two-weeks lead time. Approximately one third of outbreak alerts in wastewater correlated in time with confirmed outbreaks of Salmonella or Campylobacter and our results further indicated that several outbreaks are likely to go undetected through the traditional surveillance approach currently in place. Better understanding of the true distribution and burden of gastrointestinal infections ultimately facilitates better disease prevention and control and reduces the overall socioeconomic and healthcare related impact of these pathogens. In this respect, wastewater represents a unique opportunity for monitoring infections in real-time, without the need for individual human testing. With increasing demands for sustainable and low-cost disease surveillance, the usefulness of wastewater as a long-term method for tracking infectious disease transmission is likely to become even more pronounced.

Physiological response of Clostridium carboxidivorans during conversion of synthesis gas to solvents in a gas-fed bioreactor.

Clostridium carboxidivorans P7 is one of three microbial catalysts capable of fermenting synthesis gas (mainly CO, CO(2) , and H(2) ) to produce the liquid biofuels ethanol and butanol. Gasification of feedstocks to produce synthesis gas (syngas), followed by microbial conversion to solvents, greatly expands the diversity of suitable feedstocks that can be used for biofuel production beyond commonly used food and energy crops to include agricultural, industrial, and municipal waste streams. C. carboxidivorans P7 uses a variation of the classic Wood-Ljungdahl pathway, identified through genome sequence-enabled approaches but only limited direct metabolic analyses. As a result, little is known about gene expression and enzyme activities during solvent production. In this study, we measured cell growth, gene expression, enzyme activity, and product formation in autotrophic batch cultures continuously fed a synthetic syngas mixture. These cultures exhibited an initial phase of growth, followed by acidogenesis that resulted in a reduction in pH. After cessation of growth, solventogenesis occurred, pH increased and maximum concentrations of acetate (41 mM), butyrate (1.4 mM), ethanol (61 mM), and butanol (7.1 mM) were achieved. Enzyme activities were highest during the growth phase, but expression of carbon monoxide dehydrogenase (CODH), Fe-only hydrogenases and two tandem bi-functional acetaldehyde/alcohol dehydrogenases were highest during specific stages of solventogenesis. Several amino acid substitutions between the tandem acetaldehyde/alcohol dehydrogenases and the differential expression of their genes suggest that they may have different roles during solvent formation. The data presented here provide a link between the expression of key enzymes, their measured activities and solvent production by C. carboxidivorans P7. This research also identifies potential targets for metabolic engineering efforts designed to produce higher amounts of ethanol or butanol from syngas. .

Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates.

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of ß-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

Environmental structure impacts microbial composition and secondary metabolism.

Determining the drivers of microbial community assembly is a central theme of microbial ecology, and chemical ecologists seek to characterize how secondary metabolites mediate these assembly patterns. Environmental structure affects how communities assemble and what metabolic pathways aid in that assembly. Here, we bridged these two perspectives by addressing the chemical drivers of community assembly within a spatially structured landscape with varying oxygen availability. We hypothesized that structured environments would favor higher microbial diversity and metabolite diversity. We anticipated that the production of a compound would be more advantageous in a structured environment (less mixing) compared to an unstructured environment (more mixing), where the molecule would have a diminished local effect. We observed this to be partially true in our experiments: structured environments had similar microbial diversity compared to unstructured environments but differed significantly in the metabolites produced. We also found that structured environments selected for communities with higher evenness, rather than communities with higher richness. This supports the idea that when characterizing the drivers of community assembly, it matters less about who is there and more about what they are doing. Overall, these data contribute to a growing effort to approach microbial community assembly with interdisciplinary tools and perspectives.

Predicting COVID-19 cases in diverse population groups using SARS-CoV-2 wastewater monitoring across Oklahoma City.

SARS-CoV-2 was discovered among humans in late 2019 and rapidly spread across the world. Although the virus is transmitted by respiratory droplets, most infected persons also excrete viral particles in their feces. This fact prompted a range of studies assessing the usefulness of wastewater surveillance to determine levels of infection and transmission and produce early warnings of outbreaks in local communities, independently of human testing. In this study, we collected samples of wastewater from 13 locations across Oklahoma City, representing different population types, twice per week from November 2020 to end of March 2021. Wastewater samples were collected and analyzed for the presence and concentration of SARS-CoV-2 RNA using RT-qPCR. The concentration of SARS-CoV-2 in the wastewater showed notable peaks, preceding the number of reported COVID-19 cases by an average of one week (ranging between 4 and 10 days). The early warning lead-time for an outbreak or increase in cases was significantly higher in areas with larger Hispanic populations and lower in areas with a higher household income or higher proportion of persons aged 65 years or older. Using this relationship, we predicted the number of cases with an accuracy of 81-92% compared to reported cases. These results confirm the validity and timeliness of using wastewater surveillance for monitoring local disease transmission and highlight the importance of differences in population structures when interpreting surveillance outputs and planning preventive action.

Microbial communities in bulk fluids and biofilms of an oil facility have similar composition but different structure.

The oil-water-gas environments of oil production facilities harbour abundant and diverse microbial communities that can participate in deleterious processes such as biocorrosion. Several molecular methods, including pyrosequencing of 16S rRNA libraries, were used to characterize the microbial communities from an oil production facility on the Alaskan North Slope. The communities in produced water and a sample from a 'pig envelope' were compared in order to identify specific populations or communities associated with biocorrosion. The 'pigs' are used for physical mitigation of pipeline corrosion and fouling and the samples are enriched in surface-associated solids (i.e. paraffins, minerals and biofilm) and coincidentally, microorganisms (over 10(5) -fold). Throughout the oil production facility, bacteria were more abundant (10- to 150-fold) than archaea, with thermophilic members of the phyla Firmicutes (Thermoanaerobacter and Thermacetogenium) and Synergistes (Thermovirga) dominating the community. However, the structure (relative abundances of taxa) of the microbial community in the pig envelope was distinct due to the increased relative abundances of the genera Thermacetogenium and Thermovirga. The data presented here suggest that bulk fluid is representative of the biofilm communities associated with biocorrosion but that certain populations are more abundant in biofilms, which should be the focus of monitoring and mitigation strategies.

Hoeflea anabaenae sp. nov., an epiphytic symbiont that attaches to the heterocysts of a strain of Anabaena.

The heterotrophic, epiphytic, symbiotic bacterial strain WH2K(T) was previously isolated from a two-member culture in which it was attached to the heterocysts of a strain of Anabaena (SSM-00). Analysis of its 16S rRNA gene sequence demonstrated that the symbiont was most closely related to the type strain of Hoeflea marina (96.9 % similarity), which belongs to the family Phyllobacteriaceae within the order Rhizobiales of the class Alphaproteobacteria. A polyphasic taxonomic study was performed on strain WH2K(T), which consisted of irregular rods (2-5 µm long, 0.2 µm wide) that appeared to be narrower at one pole. Optimal growth was obtained in complex media with 15 g sea salts l(-1), at 18-34 °C (30 °C optimum) and at pH 6.0-8.0 (optimum pH 6.5). Unknown growth requirements were provided by small amounts of yeast extract but not by standard vitamin and trace metal solutions. Of the substrates tested, WH2K(T) was able to utilize only acetate, pyruvate, malate and fumarate. Growth was observed only under aerobic and microaerobic conditions, and nitrate was not reduced. No photosynthetic pigments were detected under any of the growth conditions tested. The predominant fatty acids were a summed feature that comprises C(18 : 1)ω7c, C(18 : 1)ω9t, C(18 : 1)ω12t or any combination of these (64.0 %) and an unidentified fatty acid of equivalent chain length 17.603 (13.5 %). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphoglycolipid, unknown lipids and an unidentified aminolipid. The only respiratory ubiquinone detected was Q-10. The DNA G+C content of the strain was 58.1 mol%. The organism can form a site-specific attached symbiotic relationship with a species of Anabaena. Based on phylogenetic and phenotypic evidence, it is proposed that strain WH2K(T) be classified within a novel species of the genus Hoeflea, for which the name Hoeflea anabaenae sp. nov. is proposed. The type strain is WH2K(T) ( = CCUG 56626(T)  = NRRL B-59520(T)).