Parity in bacterial communities and resistomes: Microplastic and natural organic particles in the Tyrrhenian Sea.

Petroleum-based microplastic particles (MPs) are carriers of antimicrobial resistance genes (ARGs) in aquatic environments, influencing the selection and spread of antimicrobial resistance. This research characterized MP and natural organic particle (NOP) bacterial communities and resistomes in the Tyrrhenian Sea, a region impacted by plastic pollution and climate change. MP and NOP bacterial communities were similar but different from the free-living planktonic communities. Likewise, MP and NOP ARG abundances were similar but different (higher) from the planktonic communities. MP and NOP metagenome-assembled genomes contained ARGs associated with mobile genetic elements and exhibited co-occurrence with metal resistance genes. Overall, these findings show that MPs and NOPs harbor potential pathogenic and antimicrobial resistant bacteria, which can aid in the spread of antimicrobial resistance. Further, petroleum-based MPs do not represent novel ecological niches for allochthonous bacteria; rather, they synergize with NOPs, collectively facilitating the spread of antimicrobial resistance in marine ecosystems.

No widespread signature of the COVID-19 quarantine period on water quality across a spectrum of coastal systems in the United States of America.

During the recent COVID-19 related quarantine period, anecdotal evidence emerged pointing to a rapid, sharp improvement in water quality in some localities. Here we present results from an analysis of the impacts of the COVID-19 quarantine period using two long-term coastal water quality datasets. These datasets rely on sampling that operates at appropriate timescales to quantify the influence of reduced human activity on coastal water quality and span coastal ecosystems ranging from low human influence to highly urbanized systems. We tested two hypotheses: 1) reduced tourism during the COVID-19 quarantine period would lead to improved coastal water quality, and 2) water quality improvements would scale to the level of human influence, meaning that highly urbanized or tourist-centric watersheds would see greater improvement than more rural watersheds. A localized reduction in fecal indicator bacteria was observed in four highly impacted regions of the Texas (USA) coast, but this pattern was not widespread. In less impacted regions, the signature of natural, decadal environmental variability (e.g., dissolved oxygen and turbidity) overwhelmed any potential signature of reduced human activity. Results from this study add to the growing body of literature on the environmental impacts of the COVID-19 quarantine period, and when considered with existing literature, emphasize that coastal water quality improvements appear to be ephemeral and reserved for the most severely affected (by human activity) systems. Furthermore, results show the importance of assessing COVID-19 signatures against long-term, decadal datasets that adequately reveal a system's natural variation.

Bioplastic accumulates antibiotic and metal resistance genes in coastal marine sediments.

The oceans are increasingly polluted with plastic debris, and several studies have implicated plastic as a reservoir for antibiotic resistance genes and a potential vector for antibiotic-resistant bacteria. Bioplastic is widely regarded as an environmentally friendly replacement to conventional petroleum-based plastic, but the effects of bioplastic pollution on marine environments remain largely unknown. Here, we present the first evidence that bioplastic accumulates antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in marine sediments. Biofilms fouling ceramic, polyethylene terephthalate (PET), and polyhydroxyalkanoate (PHA) were investigated by shotgun metagenomic sequencing. Four ARG groups were more abundant in PHA: trimethoprim resistance (TMP), multidrug resistance (MDR), macrolide-lincosamide-streptogramin resistance (MLS), and polymyxin resistance (PMR). One MRG group was more abundant in PHA: multimetal resistance (MMR). The relative abundance of ARGs and MRGs were strongly correlated based on a Mantel test between the Bray-Curtis dissimilarity matrices (R = 0.97, p < 0.05) and a Pearson's analysis (R = 0.96, p < 0.05). ARGs were detected in more than 40% of the 57 metagenome-assembled genomes (MAGs) while MRGs were detected in more than 90% of the MAGs. Further investigation (e.g., culturing, genome sequencing, antibiotic susceptibility testing) revealed that PHA biofilms were colonized by hemolytic Bacillus cereus group bacteria that were resistant to beta-lactams, vancomycin, and bacitracin. Taken together, our findings indicate that bioplastic, like conventional petroleum-based plastic, is a reservoir for resistance genes and a potential vector for antibiotic-resistant bacteria in coastal marine sediments.

A Holistic Assessment of Polyethylene Fiber Ingestion in Larval and Juvenile Japanese Medaka Fish.

Microplastic pollution is of public concern for global environmental health, aquaculture, and fisheries. Toxicity studies have shown that microplastic ingestion may cause intestinal damage, microbiota dysbiosis, and disturb the lipid and energy metabolism in fish. To determine the impact of environmentally relevant, chronic, low dose microplastic fibers on fish health, medaka larvae, and juveniles were exposed to five concentrations of polyethylene (PE) fibers for 21 days through the feed. Fish growth and condition were assessed to determine the overall impact on fish health. To identify impaired energy intake, the gastrointestinal tract (GIT) integrity was evaluated at the molecular and cellular levels. Microbiota analysis was performed by comparing the top seven most abundant phyla present in both larval and juvenile fish exposed to 0, 1.5, and 3 PE fibers/fish/day. A shift in the phyla Proteobacteria and Bacteroidetes were observed. Larval samples demonstrated decreased proteobacteria abundance, while juvenile samples displayed an increase in abundance. Relative gene expression of key digestive genes from GIT tissue was quantified using real time-quantitative polymerase chain reaction. An effect on digestive gene expression potentially affecting nutrient absorption and antioxidant production was indicated via a significant decrease of solute carrier family 6 member 6 expression in larvae exposed to 6 fibers/fish/day. No significant molecular changes were observed in juvenile GIT tissue, although a non-monotonous dose-response was observed. GIT morphology was analyzed using histomorphological observations of the GIT mucus and cell types. No significant impairment of the GIT epithelial layers was observed in larvae or juveniles. To assess growth and condition, Fulton's condition factor was measured. No differences were observed in larval or juvenile growth. Comparisons of different developmental stages allowed for identifying vulnerable developmental stages for microplastic exposure; larvae were more susceptible to molecular changes, while shifts in juvenile microbial communities were similar to changes reported post-polystyrene microplastic sphere exposure. This study is one of the first to provide toxicological data on the risk of PE fiber ingestion during fish development stages. Results indicate no imminent threat to fish condition at current measured environmental levels of microplastics; however, close monitoring of vital spawning grounds for commercially important fishes is recommended.

Draft Genome Sequences of 13 Vibrio cholerae Strains from the Rio Grande Delta.

Vibrio cholerae is the etiologic agent of cholera, an acute and often fatal diarrheal disease that affects millions globally. We report the draft genome sequences of 13 non-O1/O139 V. cholerae strains isolated from the Rio Grande Delta in Texas. These genomes will aid future analyses of environmental serovars.

Long-term water quality analysis reveals correlation between bacterial pollution and sea level rise in the northwestern Gulf of Mexico.

Long-term assessments are needed to identify water quality trends and their socio-environmental drivers for coastal management and watershed restoration. This study provides the first long-term assessment of fecal bacterial pollution in the northwestern Gulf of Mexico using enterococci data spanning the Texas coast from 2009 to 2020. The data were representative of 66 beaches, 169 stations, and over 75,000 samples. Findings demonstrate that 22 beaches are 'hotspots' of pollution and experienced enterococci levels that frequently exceeded the USEPA beach action value. Further, enterococci were correlated with time, population size, and sea level. Weak correlations detected in some counties highlight the multifactorial nature of water quality; additional factors are likely influencing enterococci levels. The correlation with sea level is concerning, as counties vulnerable to sea level rise frequently reported enterococci concentrations exceeding the beach action value. In consideration of sea level rise predictions, targeted studies are needed to pinpoint drivers of fecal pollution.

Temporal changes in water temperature and salinity drive the formation of a reversible plastic-specific microbial community.

Plastic is a ubiquitous pollutant in the marine environment. Here, we investigated how temporal changes in environmental factors affect the microbial communities formed on plastic (polyethylene terephthalate; PET) versus a ceramic substrate. In situ mesocosms (N = 90 replicates) were deployed at the sediment-water interface of a coastal lagoon and sampled every 4 weeks for 424 days. Sequencing data (16S rRNA) was parsed based on variation in temperature with the exposure starting in fall 2016 and remaining in situ through the next four seasons (winter, spring, summer and fall 2017). PET biofilms were distinct during the summer when salinity and temperature were highest. In particular, a significant shift in the relative abundance of Ignavibacteriales and Cytophagales was observed during the summer, but PET and ceramic communities were again indistinguishable the following fall. Water temperature, salinity and pH were significant drivers of PET biofilm diversity as well as the relative abundance of plastic-discriminant taxa. This study illustrates the temporal and successional dynamics of PET biofilms and clearly demonstrates that increased water temperature, salinity, pH and exposure length play a role in the formation of a plastic-specific microbial community, but this specificity can be lost with a change in environmental conditions.

Preliminary Assessment of Microbial Community Structure of Wind-Tidal Flats in the Laguna Madre, Texas, USA.

Aside from two samples collected nearly 50 years ago, little is known about the microbial composition of wind tidal flats in the hypersaline Laguna Madre, Texas. These mats account for ~42% of the lagoon's area. These microbial communities were sampled at four locations that historically had mats in the Laguna Madre, including Laguna Madre Field Station (LMFS), Nighthawk Bay (NH), and two locations in Kenedy Ranch (KRN and KRS). Amplicon sequencing of 16S genes determined the presence of 51 prokaryotic phyla dominated by Bacteroidota, Chloroflexi, Cyanobacteria, Desulfobacteria, Firmicutes, Halobacteria, and Proteobacteria. The microbial community structure of NH and KR is significantly different to LMFS, in which Bacteroidota and Proteobacteria were most abundant. Twenty-three cyanobacterial taxa were identified via genomic analysis, whereas 45 cyanobacterial taxa were identified using morphological analysis, containing large filamentous forms on the surface, and smaller, motile filamentous and coccoid forms in subsurface mat layers. Sample sites were dominated by species in Oscillatoriaceae (i.e., Lyngbya) and Coleofasciculaceae (i.e., Coleofasciculus). Most cyanobacterial sequences (~35%) could not be assigned to any established taxa at the family/genus level, given the limited knowledge of hypersaline cyanobacteria. A total of 73 cyanobacterial bioactive metabolites were identified using ultra performance liquid chromatography-Orbitrap MS analysis from these commu nities. Laguna Madre seems unique compared to other sabkhas in terms of its microbiology.

Relationship between Rainfall, Fecal Pollution, Antimicrobial Resistance, and Microbial Diversity in an Urbanized Subtropical Bay.

Urbanized bays are vulnerable to fecal bacterial pollution, and the extent of this pollution, in marine recreational waters, is commonly assessed by quantifying enterococcus concentrations. Recent reports have questioned the utility of enterococci as an indicator of fecal bacterial pollution in subtropical bays impaired by non-point source pollution, and enterococcus data alone cannot identify fecal bacterial sources (i.e., hosts). The purpose of this study was to assess relationships between rainfall, fecal bacterial pollution, antimicrobial resistance, and microbial diversity in an urbanized subtropical bay. Thus, a comprehensive bacterial source tracking (BST) study was conducted using a combination of traditional and modern BST methods. Findings show that rainfall was directly correlated with elevated enterococcus concentrations, including the increased prevalence of Enterococcus faecium, although it was not correlated with an increase in the prevalence of antimicrobial-resistant strains. Rainfall was also correlated with decreased microbial diversity. In contrast, neither rainfall nor enterococcus concentrations were directly correlated with the concentrations of three omnipresent host-associated fecal markers (i.e., human, canine, and gull). Notably, the human fecal marker (HF183) was inversely correlated with enterococcus concentrations, signifying that traditional enterococcus data alone are not an accurate proxy for human fecal waste in urbanized subtropical bays.IMPORTANCE The presence of human enteric pathogens, stemming from fecal pollution, is a serious environmental and public health concern in recreational waters. Accurate assessments of fecal pollution are therefore needed to properly assess exposure risks and guide water quality policies and practices. In this study, the absence of a direct correlation between enterococci and source-specific human and animal markers disputes the utility of enterococci as an indicator of fecal pollution in urbanized subtropical bays. Moreover, the inverse correlation between enterococci and the human-specific marker HF183 indicates that recreational beach advisories, triggered by elevated enterococcus concentrations, are a misleading practice. This study clearly demonstrates that a multiparameter approach that includes the quantitation of host-specific markers, as well as analyses of microbial diversity, is a more effective means of assessing water quality in urbanized subtropical bays.

Isolation, characterization, and comparative genomic analysis of a phage infecting high-level aminoglycoside-resistant (HLAR) Enterococcus faecalis.

Enterococcus is a genus of Gram-positive bacteria that are commensal to the gastrointestinal tracts of humans but some species have been increasingly implicated as agents of nosocomial infections. The increase in infections and the spread of antibiotic-resistant strains have contributed to renewed interest in the discovery of Enterococcus phages. The aims of this study were (1) the isolation, characterization, and genome sequencing of a phage capable of infecting an antibiotic-resistant E. faecalis strain, and (2) the comparative genomic analysis of publicly-available Enterococcus phages. For this purpose, multiple phages were isolated from wastewater treatment plant (WWTP) influent using a high-level aminoglycoside-resistant (HLAR) E. faecalis strain as the host. One phage, phiNASRA1, demonstrated a high lytic efficiency (∼97.52%). Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) showed that phiNASRA1 belongs to the Siphoviridae family of double-stranded DNA viruses. The phage was approximately 250 nm in length and its complete genome (40,139 bp, 34.7% GC) contained 62 open reading frames (ORFs). Phylogenetic comparisons of phiNASRA1 and 31 publicly-available Enterococcus phages, based on the large subunit terminase and portal proteins, grouped phage by provenance, size, and GC content. In particular, both phylogenies grouped phages larger than 100 kbp into distinct clades. A phylogeny based on a pangenome analysis of the same 32 phages also grouped phages by provenance, size, and GC content although agreement between the two single-locus phylogenies was higher. Per the pangenome phylogeny, phiNASRA1 was most closely related to phage LY0322 that was similar in size, GC content, and number of ORFs (40,139 and 40,934 bp, 34.77 and 34.80%, and 60 and 64 ORFs, respectively). The pangenome analysis did illustrate the high degree of sequence diversity and genome plasticity as no coding sequence was homologous across all 32 phages, and even 'conserved' structural proteins (e.g., the large subunit terminase and portal proteins) were homologous in no more than half of the 32 phage genomes. These findings contribute to a growing body of literature devoted to understanding phage biology and diversity. We propose that this high degree of diversity limited the value of the single-locus and pangenome phylogenies. By contrast, the high degree of homology between phages larger than 100 kbp suggests that pangenome analyses of more similar phages is a viable method for assessing subclade diversity. Future work is focused on validating phiNASRA1 as a potential therapeutic agent to eradicate antibiotic-resistant E. faecalis infections in an animal model.

Draft Genome Sequences of 42 Environmental Vibrio vulnificus Strains Isolated from the Northern Gulf of Mexico.

Vibrio vulnificus is a Gram-negative bacterium and an opportunistic pathogen that can cause septicemia or necrotizing fasciitis. Here, we report the draft genome sequences of 42 environmental V. vulnificus strains collected from the northern Gulf of Mexico. These data will allow for more robust comparisons between clinical and environmental strains.

Shotgun Metagenomics Reveals the Benthic Microbial Community Response to Plastic and Bioplastic in a Coastal Marine Environment.

Plastic is incredibly abundant in marine environments but little is known about its effects on benthic microbiota and biogeochemical cycling. This study reports the shotgun metagenomic sequencing of biofilms fouling plastic and bioplastic microcosms staged at the sediment-water interface of a coastal lagoon. Community composition analysis revealed that plastic biofilms were indistinguishable in comparison to a ceramic biofilm control. By contrast, bioplastic biofilms were distinct and dominated by sulfate-reducing microorganisms (SRM). Analysis of bioplastic gene pools revealed the enrichment of esterases, depolymerases, adenylyl sulfate reductases (aprBA), and dissimilatory sulfite reductases (dsrAB). The nearly 20-fold enrichment of a phylogenetically diverse polyhydroxybutyrate (PHB) depolymerase suggests this gene was distributed across a mixed microbial assemblage. The metagenomic reconstruction of genomes identified novel species of Desulfovibrio, Desulfobacteraceae, and Desulfobulbaceae among the abundant SRM, and these genomes contained genes integral to both bioplastic degradation and sulfate reduction. Findings indicate that bioplastic promoted a rapid and significant shift in benthic microbial diversity and gene pools, selecting for microbes that participate in bioplastic degradation and sulfate reduction. If plastic pollution is traded for bioplastic pollution and sedimentary inputs are large, the microbial response could unintentionally affect benthic biogeochemical activities through the stimulation of sulfate reducers.

Draft Genome Sequences of 38 Serratia marcescens Isolates Associated with Acroporid Serratiosis.

Serratia marcescens is a Gram-negative bacterium causally linked to acroporid serratiosis, a form of white pox disease implicated in the decline of elkhorn corals. We report draft genomes of 38 S. marcescens isolates collected from host and nonhost sources. The availability of these genomes will aid future analyses of acroporid serratiosis.

Comparative Genomic Analysis of Vibrio diabolicus and Six Taxonomic Synonyms: A First Look at the Distribution and Diversity of the Expanded Species.

Vibrio is a diverse genus of Gammaproteobacteria autochthonous to marine environments worldwide. Vibrio diabolicus and V. antiquarius were originally isolated from deep-sea hydrothermal fields in the East Pacific Rise. These species are closely related to members of the Harveyi clade (e.g., V. alginolyticus and V. parahaemolyticus) that are commonly isolated from coastal systems. This study reports the discovery and draft genome sequence of a novel isolate (Vibrio sp. 939) cultured from Pacific oysters (Crassostrea gigas). Questions surrounding the identity of Vibrio sp. 939 motivated a genome-scale taxonomic analysis of the Harveyi clade. A 49-genome phylogeny based on 1,109 conserved coding sequences and a comparison of average nucleotide identity (ANI) values revealed a clear case of synonymy between Vibrio sp. 939, V. diabolicus Art-Gut C1 and CNCM I-1629, V. antiquarius EX25 and four V. alginolyticus strains (E0666, FF273, TS13, and V2). This discovery expands the V. diabolicus species and makes available six additional genomes for comparative genomic analyses. The distribution of the expanded species is thought to be global given the range of isolation sources (horse mackerel, seawater, sediment, dentex, oyster, artemia and polycheate) and origins (China, India, Greece, United States, East Pacific Rise, and Chile). A subsequent comparative genomic analysis of this new eight-genome subclade revealed a high degree of individual genome plasticity and a large repertoire of genes related to virulence and defense. These findings represent a significant revision to the understanding of V. diabolicus and V. antiquarius as both have long been regarded as distinct species. This first look at the expanded V. diabolicus subclade suggests that the distribution and diversity of this species mirrors that of other Harveyi clade species, which are notable for their ubiquity and diversity.

Are We Underestimating Microplastic Contamination in Aquatic Environments?

Plastic debris, specifically microplastic in the aquatic environment, is an escalating environmental crisis. Efforts at national scales to reduce or ban microplastics in personal care products are starting to pay off, but this will not affect those materials already in the environment or those that result from unregulated products and materials. To better inform future microplastic research and mitigation efforts this study (1) evaluates methods currently used to quantify microplastics in the environment and (2) characterizes the concentration and size distribution of microplastics in a variety of products. In this study, 50 published aquatic surveys were reviewed and they demonstrated that most (~80%) only account for plastics ≥ 300 µm in diameter. In addition, we surveyed 770 personal care products to determine the occurrence, concentration and size distribution of polyethylene microbeads. Particle concentrations ranged from 1.9 to 71.9 mg g-1 of product or 1649 to 31,266 particles g-1 of product. The large majority ( > 95%) of particles in products surveyed were less than the 300 µm minimum diameter, indicating that previous environmental surveys could be underestimating microplastic contamination. To account for smaller particles as well as microfibers from synthetic textiles, we strongly recommend that future surveys consider methods that materials < 300 µm in diameter.

Draft Genome Sequence of a Vibrio harveyi Strain Associated with Vibriosis in Pacific White Shrimp (Litopenaeus vannamei).

Vibrio harveyi is a Gram-negative bacterium associated with vibriosis in penaeid shrimp. Here, we report the draft genome sequence of a V. harveyi strain isolated from Pacific white shrimp (Litopenaeus vannamei) during a vibriosis outbreak. The availability of this genome will aid future studies of vibriosis in shrimp aquaculture.

Draft Genome Sequences of One Marine and One Clinical Vibrio parahaemolyticus Strain, Both Isolated in Sweden.

Vibrio parahaemolyticus is the leading bacterial pathogen associated with seafood consumption. Here, we report the draft genome sequences of one marine and one clinical strain, both isolated in Sweden. These sequences will inform future comparative analysis of V. parahaemolyticus in northern Europe.

The thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus: Sequence variation and implications for detection and function.

Vibrio parahaemolyticus is a leading cause of bacterial food-related illness associated with the consumption of undercooked seafood. Only a small subset of strains is pathogenic. Most clinical strains encode for the thermostable direct hemolysin (TDH) and/or the TDH-related hemolysin (TRH). In this work, we amplify and sequence the trh gene from over 80 trh+strains of this bacterium and identify thirteen genetically distinct alleles, most of which have not been deposited in GenBank previously. Sequence data was used to design new primers for more reliable detection of trh by endpoint PCR. We also designed a new quantitative PCR assay to target a more conserved gene that is genetically-linked to trh. This gene, ureR, encodes the transcriptional regulator for the urease gene cluster immediately upstream of trh. We propose that this ureR assay can be a useful screening tool as a surrogate for direct detection of trh that circumvents challenges associated with trh sequence variation.

Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance.

Over a 1-year period, bi-monthly estuarine surface water and plankton samples (63-200 and > 200 µm fractions) were assayed by polymerase chain reaction for the prevalence of total Vibrio parahaemolyticus, V. vulnificus and V. cholerae and select genes associated with clinical strains found in each species. Neither temperature nor plankton abundance was a significant correlate of total V. parahaemolyticus; however, the prevalence of genes commonly associated with clinical strains (trh, tdh, ORF8) increased with temperature and copepod abundance (P < 0.05). The prevalence of total V. vulnificus and the siderophore-related viuB gene also increased with temperature and copepod and decapod abundance (P < 0.001). Temperature and copepod abundance also covaried with the prevalence of V. cholerae (P < 0.05), but there was no significant relationship with ctxA or other genes commonly found in clinical strains. Results show that genes commonly associated with clinical Vibrio strains were more frequently detected in association with chitinous plankton. We conclude that V. parahaemolyticus, V. vulnificus, V. cholerae and subpopulations that harbour genes common to clinical strains respond distinctly to seasonal changes in temperature as well as shifts in the taxonomic composition of discrete plankton fractions.

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.