Plastic properties affect the composition of prokaryotic and eukaryotic communities and further regulate the ARGs in their surface biofilms.

Plastic wastes are ubiquitous in the offshore and oceans with an increasing quantity, and inevitably, microbial communities colonized the plastics to form biofilms, which have become dispersal vectors for antibiotic resistance genes (ARGs). This study focused on the impact of plastic properties including hardness, wettability, and zeta-potential on the biomass, prokaryotic and eukaryotic communities and ARGs in biofilms formed on specific plastics (polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET)) in an estuarine environment. The results showed that, in comparison to PP, more biomass characterized by more dry weight, chlorophyll a (Chl a) and total organic carbon (TOC) was found in biofilms formed on PE and PET, which may be related to their lower surface wettability. Proteobacteria were the dominant prokaryotic phyla, and they accounted for 53.06%, 81.90%, 37.06%, 76.25%, and 54.27% of the total sequences in biofilms on PE, PP, PET, water and sediment, respectively. Ascomycota were the predominant eukaryotic phyla in biofilms, water, and sediment, and their abundances were elevated in biofilms on PP, which accounted for 34.73%. The biofilms on PP had a higher relative abundance of ARGs (3.13) compared to those on PE (2.59) and PET (0.23). Furthermore, both the plastic-biofilm properties (e.g. dry weight, Chl a, and TOC) and microbial communities (e.g., Fungi and Proteobacteria) may be involved in regulating the abundance of ARGs. Moreover, mobile genetic elements (MGEs) were significantly correlated to both the absolute and relative abundance of ARGs, indicating that MGEs may regulate the migration of ARGs in biofilms. Taken together, this investigation provides the significance of the plastic type, surface properties, and surrounding environments in shaping microbial communities and ARGs in biofilms formed on plastics.

Extraction and quantification of metal-containing nanoparticles in marine shellfish based on single particle inductively coupled plasma-mass spectrometry technique.

Quantitative characterization of nanoparticles (NPs) in marine shellfish is critical to understanding the risks of bio-accumulation. Based on single particle (sp)ICP-MS and electron microscopy, a standardized protocol was developed to extract Ag, Au, and indigenous Ti-containing NPs from mussels. The optimal parameters are: dry sample extraction with tetramethylammonium hydroxide (TMAH), 5% (v/v) final concentration of TMAH, extraction at 25 â„ƒ for 12 h, and separation by centrifugation (3000 rpm for 5 min). The particle number recoveries of spiked Ag and Au NPs were 88 ± 0.9% and 95 ± 1.1%, respectively, while Ti-containing NPs had a particle number concentration of 8.2 × 106 particles/mg and an average size of 70 nm in tested mussels. Furthermore, titanium oxide NPs, including rutile, anatase, and Magnéli phases (TixO2x-1) were found ubiquitously in 10 shellfish based on the optimal method. The particle number concentrations and average sizes of the Ti-containing NPs were 2.1 × 106-8.4 × 106 particles/mg and 70-80 nm, respectively. These Ti-containing NPs, such as TiO2, accounted for about half of the Ti mass in shellfish, indicating that marine shellfish may be a significant sink for Ti-containing NPs.

Human activities can drive sulfate-reducing bacteria community in Chinese intertidal sediments by affecting metal distribution.

Sulfate-reducing bacteria (SRB), which are ubiquitous in intertidal sediments, play an important role in global sulfur and carbon cycles, and in the bioremediation of toxic metalloids/metals. Pollution from human activities is now a major challenge to the sustainable development of the intertidal zone, but little is known about how and to what extent various anthropic and/or natural factors affect the SRB community. In the current study, based on the dsrB gene, we investigated the SRB community in intertidal sediment along China's coastline. The results showed that dsrB gene abundances varied among different sampling sites, with the highest average abundance of SRB at XHR (near the Bohai Sea). The SRB community structures showed obvious spatial distribution patterns with latitude along the coastal areas of China, with Desulfobulbus generally being the dominant genus. Correlation analysis and redundancy discriminant analysis revealed that total organic carbon (TOC) and pH were significantly correlated with the richness of the SRB community, and salinity, pH, sulfate and climatic parameters could be the important natural factors influencing the composition of the SRB community. Moreover, metals, especially bioavailable metals, could regulate the diversity and composition of the SRB communities. Importantly, according to structural equation model (SEM) analysis, anthropic factors (e.g., population, economy and industrial activities) could drive SRB community diversity directly or by significantly affecting the concentrations of metals. This study provides the first comprehensive investigation of the direct and indirect anthropic factors on the SRB community in intertidal sediments on a continental scale.

Antibiotic resistance genes in biofilms on plastic wastes in an estuarine environment.

Plastic wastes are ubiquitous in aquatic environment. Biofilms, which are often formed on the surface of plastic waste, may contain antibiotic resistance genes (ARGs). This study focused on the occurrence and distribution of ARGs, metal resistance genes (MRGs) and their associated microbial communities in biofilms formed on different types of plastic, in comparison to associated sediment and water samples taken from the Yangtze Estuary. The results showed that polypropylene (PP) and polyethylene (PE) with visible biofilms were highly abundant, and the average absolute abundance of most tested ARGs in the biofilms was higher than that in the sediment and water, indicating that biofilms on plastics can act as a reservoir for ARGs. Moreover, the biofilms on PE had a higher relative abundance of ARGs, compared to those on other plastics, and Firmicutes on PE may be potential hosts for these ARGs. Furthermore, Bacillus, Mycobacterium and Pseudomonas may be multi-resistance genera on plastics, and tetA and tetW may have more potential hosts on PET and PP. Metals, total phosphorus and salinity may be the major environmental factors regulating ARGs in biofilms formed on plastics. The results provide new insights into evaluating the risks caused by plastic wastes and ARGs in biofilms formed on plastics in estuarine environment.

Antibiotic resistance genes in sediments of the Yangtze Estuary: From 2007 to 2019.

To better understand the occurrence and succession of antibiotic resistance genes (ARGs) in the environment, the investigation of ARGs in sediment for a long time scale is urgently needed. In this study, sediment samples were taken in the Yangtze Estuarine area from 2007 to 2019, and the interannual variations in ARGs and their possible physicochemical and socioeconomic influencing factors were analyzed. The results showed that the abundance of ARGs, including sul1, sul2, tetM, tetW, aac(6')-Ib and qnrS, was higher in recent years (from 2015 to 2019) than that in earlier years (from 2007 to 2011), and heavier ARG pollution was found in Wusongkou (WSK) samples than in Liuhekou (LHK) samples. According to the redundancy discriminant analysis (RDA) and correlation analysis, the antibiotics (especially individual antibiotic categories, including oxytetracycline, doxycycline hyclate and norfloxacin), metals and a metal resistance gene (zntA) and total organic carbon (TOC) showed significant correlations to ARGs. In addition, antibiotics, metals, TOC and ARGs were also significantly correlated with several socioeconomic indices. Furthermore, the extended STIRPAT model analysis revealed that the second industry product and the first industry product were the major socioeconomic driver factors for the ARG distribution at WSK and LHK, respectively. Overall, with socioeconomic development, antibiotics, metals, TOC and ARGs increased in sediment. In addition, antibiotics, metals and TOC may participate in the regulation of the occurrence and distribution of ARGs in the Yangtze Estuary for the long time scale.

Antibiotic resistance genes (ARGs) and their associated environmental factors in the Yangtze Estuary, China: From inlet to outlet.

The occurrence of antibiotic resistance genes (ARGs) and their associated environmental factors in estuaries are poorly understood. In this study, we comprehensively analyzed ARGs in both water and sediments from inlet to outlet of the Yangtze Estuary, China. The relative abundances of ARGs were higher in the turbidity maximum zone (TMZ) than other sites, implying that suspended particulate matter (SPM) was the major reservoir for ARGs in water. ARGs showed an increasing trend from inlet to outlet in sediments. Positively correlation between intI1 and sul1 in both water and sediments indicated that sul1 may be regulated by intI1. Correlation analysis and redundancy analysis showed that the spatial variations of estuarine ARGs were positively correlated with sample properties (e.g., temperature, SPM, pH) and chemical pollutants (e.g., heavy metals and antibiotic residues), among which chemical pollutants were the major drivers for the ARG distribution in both water and sediments.

Time-dependent effects of ZnO nanoparticles on bacteria in an estuarine aquatic environment.

Many studies have examined the acute toxicity of nanoparticles (NPs) towards model bacteria. In this study, we report the time-dependent effects of ZnO NPs on native, selected Zn-resistant and dominant bacteria in estuarine waters. An initial inhibition of bacterial growth followed by a recovery at 24 h was observed, and this rebound phenomenon was particularly notable when the raw water samples were treated with relatively high ZnO NP concentrations (1 and 10 mg/L).By comparing the groups treated with Zn2+, Zn2+ was shown to largely explain the acute cytotoxic effect of ZnO NPs on bacteria in raw waters. Furthermore, similar to the native bacteria, especially the dominant bacteria, the viability of Escherichia coli (E. coli) decreased with the increasing treatments time and the concentrations of ZnO NPs in water with different salinities. Moreover, the expression of Zn-resistance genes including zntA and zntR in E. coli suggested that the Zn-resistance system in E. coli can be activated to defend against the stress of Zn2+ released from ZnO NPs, and salinity may promote this process in estuarine aquatic systems. Thus, the effect of ZnO NPs on bacteria in estuarine water bodies is likely determined by the synergistic effect of environmental salinity and dissolved Zn ions. As such, our findings are of high relevance and importance for understanding the ecological disturbances caused by anthropogenic NPs in estuarine environments.

Characteristics of microbial community indicate anthropogenic impact on the sediments along the Yangtze Estuary and its coastal area, China.

In the contaminated coastal sediments, variations of microbial community can reflect the impact of anthropogenic activities. The identification, evaluation and monitoring of the potential bio-indicator species and biomarker communities are vital for the ecological studies in sedimentary environments. Based on the high-throughput sequencing, the microbial communities were characterized in the sediments along the Yangtze Estuary and its coastal area. The results showed that the structure and composition of microbial communities varied greatly among different sampling sites at the phyla level, especially for Euryarchaeota. Metabolic pathway and quantitative PCR analyses suggested that the methane metabolism-related microbes were mainly included in the phylum of Euryarchaeota. Elevated abundances of methane metabolism-related microbes were found at Shidongkou (SDK) and Wusongkou (WSK), where microbes were seriously impacted by the wastewater treatment plant (WWTP) effluent and urban runoff. By comparing with the Euryarchaeota in WWTP sludge, the relatively high abundance of Euryarchaeota in sediment at SDK may be mainly related to the massive growth of indigenous species, promoted by anthropogenic nutrients. Moreover, redundancy discriminant analysis and correlation analysis revealed that methanogens and methanotrophs mainly respond to the nutrients and metals, such as total organic carbon, total phosphorus, total nitrogen, SO42-, NO2-, NH4+, Cr, and Zn, which were often related to human activities. Network analyses showed that the species related to the metabolism of methane may play a vital role in the interassociation among different microbial communities. Therefore, methanogens, methanotrophs and their community compositions could be considered as potential bio-indicator species and biomarker communities, indicating anthropogenic activities in the sediments along the Yangtze Estuary and its coastal area.

Polyurethane, epoxy resin and polydimethylsiloxane altered biofilm formation and mussel settlement.

In many environments, biofilms are a major mode and an emergent form of microbial life. Biofilms play crucial roles in biogeochemical cycling and invertebrate recruitment in marine environments. However, relatively little is known about how marine biofilms form on different substrata and about how these biofilms impact invertebrate recruitment. Here, we performed a comparative analysis of a 28-day-old biofilm community on non-coated (a control glass) and coated substrata (polyurethane (PU), epoxy resin (EP) and polydimethylsiloxane (PDMS)) and examined the settlement of Mytilus coruscus plantigrades on these biofilms. PU, EP and PDMS deterred the development of marine biofilms by reducing the biofilm biomass including the biofilm dry weight, cell density of the bacteria and diatoms and chlorophyll a concentrations. Further analysis of bacterial community revealed that EP altered the bacterial community composition compared with that on the glass substrata by reducing the relative abundance of Ruegeria (Alphaproteobacteria) and by increasing the relative abundance of Methylotenera (Betaproteobacteria) and Cyanobacteria in the biofilms. However, bacterial communities developed on PU and PDMS, as well as glass and PU, EP and PDMS did not exhibit differences from each other. The M. coruscus settlement rates on biofilms on PU, EP and PDMS were reduced by 20-41% compared with those on the glass after 28 days. Thus, the tested coatings impacted the development of marine biofilms by altering the biofilm biomass and/or the bacterial community composition. The mussel settlements decreased in the biofilms that formed on the coatings compared with those on non-coated glass.

Seasonal and spatial distribution of antibiotic resistance genes in the sediments along the Yangtze Estuary, China.

Antibiotics resistance genes (ARGs) are considered as an emerging pollutant among various environments. As a sink of ARGs, a comprehensive study on the spatial and temporal distribution of ARGs in the estuarine sediments is needed. In the present study, six ARGs were determined in sediments taken along the Yangtze Estuary temporally and spatially. The sulfonamides, tetracyclines and fluoroquinolones resistance genes including sul1, sul2, tetA, tetW, aac(6')-Ib, and qnrS, were ubiquitous, and the average abundances of most ARGs showed significant seasonal differences, with relative low abundances in winter and high abundances in summer. Moreover, the relative high abundances of ARGs were found at Shidongkou (SDK) and Wusongkou (WSK), which indicated that the effluents from the wastewater treatment plant upstream and inland river discharge could influence the abundance of ARGs in sediments. The positive correlation between intI1 and sul1 implied intI1 may be related to the occurrence and propagation of sulfonamides resistance genes. Correlation analysis and redundancy discriminant analysis showed that antibiotic concentrations had no significant correlation to their corresponding ARGs, while the total extractable metal, especially the bioavailable metals, as well as other environmental factors including temperature, clay, total organic carbon and total nitrogen, could regulate the occurrence and distribution of ARGs temporally and spatially. Our findings suggested the comprehensive effects of multiple pressures on the distribution of ARGs in the sediments, providing new insight into the distribution and dissemination of ARGs in estuarine sediments, spatially and temporally.

Sulphate-reducing bacteria (SRB) in the Yangtze Estuary sediments: Abundance, distribution and implications for the bioavailibility of metals.

Ubiquitous in the aquatic environment, sulphate-reducing bacteria (SRB) are considered one of the dominant microorganisms involved in the degradation of sulphate. This study focused on the spatial and temporal distributions of SRB in the Scirpus triquter rhizosphere sediments with a comparison to non-rhizosphere sediments and evaluated the implication of SRB to the bioavailability of metals in the Yangtze Estuary. The results showed that taking dsrB as the target gene, SRB abundances in rhizosphere sediments were significantly higher than those in non-rhizosphere sediments (P<0.01). SRB abundances were relatively higher in April and January than other seasons. Moreover, redundancy discriminate analysis (RDA) results indicated that sulphate, pH and TOC were the major environmental factors affecting the SRB abundance in rhizosphere sediments. The concentrations of most metals were significantly related to SRB abundance, and sulphide concentrations showed a significantly positive correlation to metal concentrations, indicating metal sulphide/metal associated sulphide could be regulated by SRB. Furthermore, electron microscope analysis found that nano-sized metal sulphide particles were ubiquitous in rhizosphere sediments and could be further taken up by plants. This study provides new insights into the immobilization and removal of heavy metals and the ecological value of the sulphate-reducing bacteria in the Yangtze Estuary.

Bacterial community structure in response to environmental impacts in the intertidal sediments along the Yangtze Estuary, China.

This study was designed to investigate the characteristics of bacterial communities in intertidal sediments along the Yangtze Estuary and their responses to environmental factors. The results showed that bacterial abundance was significantly correlated with salinity, SO42- and total organic carbon, while bacterial diversity was significantly correlated with SO42- and total nitrogen. At different taxonomic levels, both the dominant taxa and their abundances varied among the eight samples, with Proteobacteria being the most dominant phylum in general. Cluster analysis revealed that the bacterial community structure was influenced by river runoff and sewerage discharge. Moreover, SO42-, salinity and total phosphorus were the vital environmental factors that influenced the bacterial community structure. Quantitative PCR and sequencing of sulphate-reducing bacteria indicated that the sulphate reduction process occurs frequently in intertidal sediments. These findings are important to understand the microbial ecology and biogeochemical cycles in estuarine environments.

Biofilms as a sink for antibiotic resistance genes (ARGs) in the Yangtze Estuary.

Biofilms are ubiquitous throughout aquatic environments and they are thought to promote the acquisition and dissemination of antibiotic resistant genes (ARGs). This study focused on the occurrence and distribution of five types of ARG in naturally-occurring biofilms, in comparison to associated sediment and water samples, from the Yangtze Estuary, which borders the meta-city of Shanghai, China. The detection frequency and abundances of most ARGs showed the following order: biofilm > sediment > water, which can be attributed to a high level of antibiotics and metals that can accelerate the generation and propagation of ARGs in biofilms. Most of ARG abundances were contributed by extracellular DNA (eDNA) in biofilm and sediment samples. ARGs (sul1, sul2, tetA and tetW) in eDNA were significantly correlated with TOC in both biofilm and sediment samples. Furthermore, both intracellular DNA-associated ARGs per gram of microbial biomass carbon (MBC) and eDNA-associated ARGs per gram of non-MBC and were higher in biofilms than sediments, and the partitioning coefficients of ARGs in eDNA between biofilm and water were higher than those between sediment and water. Our results provide new insight for evaluating the occurrence and abundance of ARGs in aquatic environments, confirming that biofilms are a significant sink for ARGs in the estuarine environment.

Pyomelanin from Pseudoalteromonas lipolytica reduces biofouling.

Members of the marine bacterial genus Pseudoalteromonas are efficient producers of antifouling agents that exert inhibitory effects on the settlement of invertebrate larvae. The production of pigmented secondary metabolites by Pseudoalteromonas has been suggested to play a role in surface colonization. However, the physiological characteristics of the pigments produced by Pseudoalteromonas remain largely unknown. In this study, we identified and characterized a genetic variant that hyperproduces a dark-brown pigment and was generated during Pseudoalteromonas lipolytica biofilm formation. Through whole-genome resequencing combined with targeted gene deletion and complementation, we found that a point mutation within the hmgA gene, which encodes homogentisate 1,2-dioxygenase, is solely responsible for the overproduction of the dark-brown pigment pyomelanin. In P. lipolytica, inactivation of the hmgA gene led to the formation of extracellular pyomelanin and greatly reduced larval settlement and metamorphosis of the mussel Mytilus coruscus. Additionally, the extracted pyomelanin from the hmgA deletion mutant and the in vitro-synthesized pyomelanin also reduced larval settlement and metamorphosis of M. coruscus, suggesting that extracellular pyomelanin released from marine Pseudoalteromonas biofilm can inhibit the settlement of fouling organisms.

Bacterial community structure in the intertidal biofilm along the Yangtze Estuary, China.

In this study, the 16S rRNA-based Illumina MiSeq sequencing was used to investigate the bacterial community structure and composition of intertidal biofilm taken along the Yangtze Estuary. The results showed that 680,721 valid sequences of seven samples were assigned to 147,239 operational taxonomic units, which belonged to 49 phyla, 246 family and 314 genera. Compared to other studies on water and sediments in the study area, biofilms showed highest index of bacterial diversity and abundances. At different taxonomic levels, both dominant taxa and their abundances varied among the seven samples, with Proteobacteria as the dominant phylum in general. Principal component analysis and cluster analysis revealed that bacterial communities at WSK differed from those at other sampling sites. Salinity, dissolved oxygen, pH and nutrients were the vital environmental factors to influence the bacterial community structure of biofilms. These results may provide a new insight into the microbial ecology in estuarine environments.

Silver Nanoparticles Impact Biofilm Communities and Mussel Settlement.

Silver nanoparticles (AgNPs) demonstrating good antimicrobial activity are widely used in many fields. However, the impact of AgNPs on the community structures of marine biofilms that drive biogeochemical cycling processes and the recruitment of marine invertebrate larvae remains unknown. Here, we employed MiSeq sequencing technology to evaluate the bacterial communities of 28-day-old marine biofilms formed on glass, polydimethylsiloxane (PDMS), and PDMS filled with AgNPs and subsequently tested the influence of these marine biofilms on plantigrade settlement by the mussel Mytilus coruscus. AgNP-filled PDMS significantly reduced the dry weight and bacterial density of biofilms compared with the glass and PDMS controls. AgNP incorporation impacted bacterial communities by reducing the relative abundance of Flavobacteriaceae (phylum: Bacteroidetes) and increasing the relative abundance of Vibrionaceae (phylum: Proteobacteria) in 28-day-old biofilms compared to PDMS. The settlement rate of M. coruscus on 28-day-old biofilms developed on AgNPs was lower by >30% compared to settlement on control biofilms. Thus, the incorporation of AgNPs influences biofilm bacterial communities in the marine environment and subsequently inhibits mussel settlement.

The effect of carbon nanotubes and titanium dioxide incorporated in PDMS on biofilm community composition and subsequent mussel plantigrade settlement.

This study investigated the effect of carbon nanotubes (CNTs) and titanium dioxide (TiO2) incorporated in PDMS on biofilm formation and plantigrade settlement of Mytilus coruscus. TiO2 increased bacterial density, and CNTs also increased bacterial density but reduced diatom density in biofilms after 28 days. Further analysis was conducted between bacterial communities on glass, PDMS, CNTs (0.5 wt%) and TiO2 (7.5 wt%). ANOSIM analysis revealed significant differences (R > 0.9) between seven, 14, 21 and 28 day-old bacterial communities. MiSeq sequencing showed that CNTs and TiO2 impacted the composition of 28 day-old bacterial communities by increasing the abundance of Proteobacteria and decreasing the abundance of Bacteroidetes. The maximum decreased settlement rate in 28 day-old biofilms on CNTs and TiO2 was > 50% in comparison to those on glass and PDMS. Thus, CNTs and TiO2 incorporated in PDMS altered the biomass and community composition of biofilms, and subsequently decreased mussel settlement.

Characterization of self-generated variants in Pseudoalteromonas lipolytica biofilm with increased antifouling activities.

Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12±5%) and translucent (frequency of 5±3%), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.

Draft Genome Sequence of Shewanella sp. ECSMB14102, a Mussel Recruitment-Promoting Bacterium Isolated from the East China Sea.

Shewanella sp. ECSMB14102, which promotes recruitment of the mussel Mytilus coruscus, was isolated from natural biofilms formed on glass slides submerged in the East China Sea. Here, we present the draft genome sequence, which comprises 4.41 Mb with a G+C content of 52.2%. The genomic information in this strain will contribute to deepening our understanding of bacteria-animal interaction.

Draft Genome Sequence of Pseudoalteromonas sp. Strain ECSMB14103, Isolated from the East China Sea.

Pseudoalteromonas sp. strain ECSMB14103 was isolated from marine biofilms formed on the East China Sea. The draft genome sequence comprises 4.11 Mp with a G+C content of 39.7%. The information from the draft genome will contribute to an understanding of bacteria-animal interaction.