The first use of LC-MS/MS proteomic approach in the brown mussel Perna perna after bacterial challenge: Searching for key proteins on immune response.

The brown mussel Perna perna is a valuable fishing resource, primarily in tropical and subtropical coastal regions. Because of their filter-feeding habits, mussels are directly exposed to bacteria in the water column. Escherichia coli (EC) and Salmonella enterica (SE) inhabit human guts and reach the marine environment through anthropogenic sources, such as sewage. Vibrio parahaemolyticus (VP) is indigenous to coastal ecosystems but can be harmful to shellfish. In this study, we aimed to assess the protein profile of the hepatopancreas of P. perna mussel challenged by introduced - E. coli and S. enterica - and indigenous marine bacteria - V. parahaemolyticus. Bacterial-challenge groups were compared with non-injected (NC) and injected control (IC) - that consisted in mussels not challenged and mussels injected with sterile PBS-NaCl, respectively. Through LC-MS/MS proteomic analysis, 3805 proteins were found in the hepatopancreas of P. perna. From the total, 597 were significantly different among conditions. Mussels injected with VP presented 343 proteins downregulated compared with all the other conditions, suggesting that VP suppresses their immune response. Particularly, 31 altered proteins - upregulated or downregulated - for one or more challenge groups (EC, SE, and VP) compared with controls (NC and IC) are discussed in detail in the paper. For the three tested bacteria, significantly different proteins were found to perform critical roles in immune response at all levels, namely: recognition and signal transduction; transcription; RNA processing; translation and protein processing; secretion; and humoral effectors. This is the first shotgun proteomic study in P. perna mussel, therefore providing an overview of the protein profile of the mussel hepatopancreas, focused on the immune response against bacteria. Hence, it is possible to understand the immune-bacteria relationship at molecular levels better. This knowledge can support the development of strategies and tools to be applied to coastal marine resource management and contribute to the sustainability of coastal systems.

How does the brown mussel Perna perna respond to environmental pollution? A review on pollution biomarkers.

The brown mussel Perna perna (Linnaeus, 1758) is a valuable resource for aquaculture in tropical and subtropical coastal regions. It presents desirable characteristics for biomonitoring, including being sessile, widely distributed and abundant, and is a filter-feeder able to accumulate several classes of pollutants (e.g., metals, hydrocarbons, among others). Mussels' biological responses to pollution exposure can be measured as biomarkers, which include alterations ranging from molecular to physiological levels, to estimate the degree of environmental contamination and its effects on biota. This full review compiles two decades (2000-2020) of literature concerning biological effects on P. perna mussel caused by environmental pollutants (i.e., metals, hydrocarbons, and emerging pollutants), considering environmental and farm-based biomonitoring. Biochemical markers related to mussels' oxidative status were efficient for the biomonitoring of metals (i.e., antioxidant enzymes associated with oxidative damage in biomolecules). Genotoxicity and cytotoxicity indicators (i.e., comet, micronucleus, and neutral red assays) provided a depiction of hydrocarbon contamination. The neutral red assay gave a time-concentration cytotoxic response to a wide range of pollutants, including emerging pollutants (e.g., pharmaceuticals and biocides) and hydrocarbons. Perna perna hemocyte parameters provided a useful approach for biocide biomonitoring. This paper summarizes useful biomarkers from molecular to physiological levels in this mussel species used to identify and quantify the degree of coastal pollution. An integrated biomarker analysis may provide a way to overcome possible biomarker variations and assess multi-polluted sites. Nevertheless, it is necessary to investigate biomarker variations according to natural factors (e.g., season and gonad maturation stage) to standardize them for trustworthy biomonitoring.

Effects of aeration on the suspended matter from a tropical and eutrophic estuary.

A comprehensive understanding of the complex biogeochemical interactions between organic matter and persistent contaminants in the suspended matter is vital for eco-efficient estuary recovery. However, little is known regarding aeration effects in suspended particulate aggregates. Therefore, this study aimed to investigate the effects of aeration on the suspended matter from a Tropical and Eutrophic estuarine environment. Anoxic water with 60 g/L of suspended particulate matter (SPM) was collected from Guanabara Bay, Rio de Janeiro, Brazil, transferred to experimental boxes and aerated for 61 days. SPM aggregates monitoring included abiotic variables measurements and, determination of total organic matter (TOM), biopolymers composition, bacterial activity, trace metals, and polycyclic aromatic hydrocarbons (PAHs) concentrations. The aeration enhanced dissolved oxygen (DO) concentration and the redox potential (Eh). However, from days 0 to 61 the predominant bacterial activities were denitrification and fermentation. Electron transport system activity increased after day 10, and aerobic activity was detected after day 19. In summary, aeration increased aerobic bacterial activity, lipids (LIP) and trace metal concentrations, although diminished protein/carbohydrate ratio and PAH concentration. Trace metals concentration (Ni, Pb, Cu, Cr, Mn, and Fe) were the highest on day 19 when the pH was 5.9. Copper presented toxic values (Cu > 20.0 µg/g). The pH showed a strong negative correlation with Eh (r = -0.94; p < 0.001). Acidic environment (pH ≤ 5.9) in marine ecosystems with high loads of toxic trace metals is unsafe for biota. Therefore, managers must be aware of the environmental and biological risks of introducing the aeration technique into a eutrophic marine environment.

Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution.

The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL-1), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL-1). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in aquatic environments and the immune response of mussel hemocytes. Immune-related parameters may therefore be useful as indicators of bivalve health and environmental quality. Our flow cytometric analysis of P. perna hemocytes represents a new approach for studying Perna perna biology and might represent a novel tool for measuring organic pollution and water quality.

Bacterial degradation of bisphenol analogues: an overview.

Bisphenol A (BPA) is one of the most produced synthetic monomers in the world and is widespread in the environment. BPA was replaced by bisphenol analogues (BP) because of its adverse effects on life. Bacteria can degrade BPA and other bisphenol analogues (BP), diminishing their environmental concentrations. This study aimed to summarize the knowledge and contribute to future studies. In this review, we surveyed papers on bacterial degradation of twelve different bisphenol analogues published between 1987 and June 2022. A total of 102 original papers from PubMed and Google Scholar were selected for this review. Most of the studies (94.1%, n = 96) on bacterial degradation of bisphenol analogues focused on BPA, and then on bisphenol F (BPF), and bisphenol S (BPS). The number of studies on bacterial degradation of bisphenol analogues increased more than six times from 2000 (n = 2) to 2021 (n = 13). Indigenous microorganisms and the genera Sphingomonas, Sphingobium, and Cupriavidus could degrade several BP. However, few studies focussed on Cupriavidus. The acknowledgement of various aspects of BP bacterial biodegradation is vital for choosing the most suitable microorganisms for the bioremediation of a single BP or a mixture of BP.

Faecal bacteria density in tropical seawater: The Itanemas' cove case study, Angra dos Reis, Brazil.

Angra dos Reis can receive up to 1.3 million tourists in the summer season. The lack of an adequate sanitary system makes sewage contamination a growing concern in Ribeira Bay, Angra dos Reis, Brazil. This study aims to investigate the seasonal variation of faecal indicator bacteria (FIB) and abiotic variables in Itanema cove situated within Ribeira Bay. Despite the seasonal population increase (>80 individuals) and the absence of an integrated sanitary system, our results indicate that Itanema is still a stable estuarine environment. From 2017 to 2019, the mean salinity was 27.91, pH was 7.82, and water temperature was 25.01 °C. However, the FIB number was 105, suggesting the absence of sewage treatment in Itanema's outfall. Following current conservation status, baseline studies are mandatory for background references of endangered coastal areas such as Itanema cove. These studies are crucial for future governance decisions and sustainable tourism implementation in Angra dos Reis.

The micro from mega: Dental calculus description and the first record of fossilized oral bacteria from an extinct proboscidean.

OBJECTIVE: This study explores the chemical composition and surface aspects of fossilized dental calculus from the South American Quaternary proboscidean Notiomastodon platensis and the first record of fossilized oral bacteria from extinct megafauna. MATERIALS: Blocks of dental calculus removed from the third molar of five specimens of Notiomastodon platensis collected from Brazil, Argentina, and Ecuador. METHODS: We analyzed five samples of dental calculus by SEM and SEM-EDS, following a rigid protocol to avoid bacterial contamination. RESULTS: The dental calculus surface is homogeneous, porous, with various crystals, and composed mainly by oxygen and calcium. One sample revealed a well-preserved mineralized biofilm, with several rods and cocci bacteria. CONCLUSIONS: This is the first fossilized record of oral bacterial communities associated with extinct proboscideans. SIGNIFICANCE: This record confirms the parasitism between oral bacteria and Notiomastodon platensis and will enable the study of paleogenomic aspects of oral microbiota of proboscideans. LIMITATIONS: Fossilization conditions of proboscidean teeth with dental calculus are variable among specimens. Although rare, the preservation of oral bacteria is expected because of the oral biofilm composition.

Are invasive species always negative to aquatic ecosystem services? The role of dark false mussel for water quality improvement in a multi-impacted urban coastal lagoon.

The dark false mussel Mytilopsis leucophaeata occurs as non-native species in Asia, Europe and South America. Despite the low population density usually found in its native range, M. leucophaeata forms dense clusters in newly invaded areas which lead to impacts on local organisms and environment. Some of the impacts of non-native species on newly colonized areas may be positive. However, despite the empirical evidences of increase in water transparency by freshwater dresseinids, the hypothesis that dark false mussel enhances the quality of brackish water has never been tested so far. Thus, the present study aimed to determine M. leucophaeata impacts on the water quality of a nutrient-enriched coastal lagoon by (1) analyzing a historical water dataset for pre- and post-invasion at Rodrigo de Freitas Lagoon (Brazil) and (2) experimentally testing dark false mussel direct effects on water quality. Historical field data evidenced significant lower phytoplankton abundance and chlorophyll a, and higher water transparency for the post-invasion period. These pattens were also supported by time-series analyses, which detected a decreasing trend for total coliforms density and an increased trend for dissolved oxygen over time. Moreover, time series indicated a gradual increase in lagoon water level with time, but none or minor changes were evidenced for floodgates operation routine, meteorological variables, and the frequency of sewage input. In microcosms, M. leucophaeata was effective to increase water transparency and decrease E. coli densities. The combination of field and laboratory data partially supported the hypothesis that M. leucophaeata invasion promoted an improvement in lagoon water quality, but increased phosphorus availability through excretion in microcosm assays. Management of dark false mussel populations seems to be a potential strategy for water quality improvement within already invaded systems where high sewage-enriched effluents are released. However, as non-native species often unbalance ecological relationships and functioning of the invaded ecosystems, new introductions of M. leucophaeata must be avoided.

Simultaneous detection of the mecA and ileS-2 genes in coagulase-negative staphylococci isolated from Brazilian hospitals by multiplex PCR.

Coagulase-negative Staphylococcus spp. (CNS) has been associated with primary bloodstream infections and implanted medical devices. Its importance is increasing due to the acquisition of resistance to oxacillin (Oxa) and, recently, resistance to mupirocin (Mup). Mupirocin, a topical antimicrobial, has been used in the prevention of staphylococci catheter colonization. Susceptibility to Oxa and Mup was analyzed by different testing methods in clinical CNS isolates. Among 112 CNS strains, 69 (61.6%) were Oxa(R) by the disk diffusion (DD) method and 72 (64.2%) grew on the oxacillin agar screen plate. S. epidermidis and S. haemolyticus presented high rates of oxacillin resistance, 75.4% and 96.1%, respectively. Twenty four (21.4%) strains were Mup(R) by the DD test and 21 of them (87.5%) were identified as S. epidermidis. The detection of the mecA and ileS-2 genes, determined by multiplex-PCR, showed that 72 (64.2%) CNS strains possessed the mecA gene, while 16 (14.3%) possessed the ileS-2 gene. Fifteen of these strains presented the two resistance genes simultaneously. The isolates containing the ileS-2 gene presented a minimum inhibitory concentration (MIC) >1024 microg/mL in the E-test, while low-level mupirocin resistance (MICs of 12-16 microg/mL) was observed in those strains without ileS-2. The resistances to high and low levels of mupirocin could not be distinguished when the DD test was used. The analysis of the Mup(R) S. epidermidis strains by Pulsed Field Gel Electrophoresis showed that 17 (80.9%) strains belonged to one of two patterns (A and B), which have been shown to be prevalent in hospitals in Rio de Janeiro. This report showed that the PCR method for detection of oxacillin and mupirocin resistance in CNS is necessary to determine accurate rates of these resistance, and will can help in the staphylococcal infections prevention and control policies in Brazil.

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates.

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.