Bioemulsifier from sponge-associated bacteria reduces staphylococcal biofilm.

Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Marine Bacillus pumilus substances exhibit antimicrobial effect on multidrug-resistant Staphylococcus aureus.

AIMS: This study aimed to assess the antimicrobial potential of Bp1-AdE, produced by Bacillus pumilus 64-1, and to investigate its mode of action against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). METHODS AND RESULTS: Bp-1AdE, derived from sponge-associated B. pumilus, exhibited bactericidal activity at 1 550 µg ml-1 against S. aureus ATCC29213 and MRSA strains. Light and fluorescence microscopy revealed drastic cell lysis of S. aureus treated with Bp-1AdE. Scanning and transmission electron microscopy suggested that Bp-1AdE disrupts the cytoplasmic membrane. Toxicity assays showed that Bp-1AdE was non-toxic to Tenebrio molitor larvae. Liquid chromatography-mass spectrometry and Global Natural Product Social spectral libraries identified four substances within Bp-1AdE, including aliphatic alcohols [3,4-dipentylhexane-2,5-diol and 1,1'-(4,5-dibutyl-3,6-dimethylcyclohexane-1,2-diyl)bis(ethan-1-one)] and terpenoids (cholic acid and canrenone). CONCLUSIONS: Bp-1AdE demonstrated selective toxicity and bactericidal activity, highlighting its potential for controlling infections caused by multidrug-resistant S. aureus strains.

Marine Pseudomonas: diving into the waves of blue biotechnology.

From marine to terrestrial environments, Pseudomonas spp. exhibit a remarkable ability not only to adapt but also thrive even amidst adverse conditions. This fact turns Pseudomonas spp. into one of the most prominent candidates for novel biotechnological solutions. Even though terrestrial isolates have been extensively studied, there is still an almost untapped source to be explored in marine Pseudomonas. Harnessing such strains offers an opportunity to discover novel bioactive compounds that could address current global challenges in healthcare and sustainable development. Therefore, this minireview aimed to provide an overview of the main recent discoveries regarding antimicrobials, antifouling, enzymes, pigments, and bioremediation strategies derived from marine isolates of Pseudomonas spp. Future research perspectives will also be discussed to foster forthcoming endeavors to explore the marine counterparts of such a prolific bacterial genus.

In treacherous waters: detection of colistin-resistant bacteria in water and plastic litter from a recreational estuary.

Colistin resistance poses a major therapeutic challenge and resistant strains have now been reported worldwide. However, the occurrence of such bacteria in aquatic environments is considerably less understood. This study aimed to isolate and characterize colistin-resistant strains from water and plastic litter collected in an urban recreational estuary. Altogether, 64 strains with acquired colistin resistance were identified, mainly Acinetobacter spp. and Enterobacter spp. From these, 40.6% were positive for at least one mcr variant (1-9), 26.5% harbored, extended-spectrum beta-lactamases, 23.4% harbored, sulfonamide resistance genes, and 9.3% harbored, quinolone resistance genes. merA, encoding mercury resistance, was detected in 10.5% of these strains, most of which were also strong biofilm producers. The minimum inhibitory concentration toward colistin was determined for the mcr-positive strains and ranged from 2 to ≥512 µg ml-1. Our findings suggest that Gram-negative bacteria highly resistant to a last-resort antimicrobial can be found in recreational waters and plastic litter, thereby evidencing the urgency of the One Health approach to mitigate the antimicrobial resistance crisis.

Occurrence of extended-spectrum ß-lactamases-producing Escherichia coli isolates over gradient pollution in an urban tropical estuary.

Bacterial resistance to antimicrobials is a global public health problem that surpasses the human context and can be increased by pollution. However, the lack of systematic monitoring of resistance in some aquatic matrices, such as tropical estuaries, makes it unknown whether its occurrence is associated with anthropogenic pollution in these environments. Therefore, we investigated the occurrence of extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli as a resistance indicator for 12 consecutive months at three representative points of a pollution gradient in Guanabara Bay (GB), Brazil. Sixty-six E. coli strains were selected from 72 samples of GB waters in the presence of ceftriaxone (8 µg mL-1 ) and identified by MALDI-TOF MS. Of the 66, 55 (83.3%) strains were ESBL producers. They carried beta-lactamase/ESBL genes, with the predominance of blaCTX-M (54, 98.2%), especially the blaCTX-M-1,2 allele (49.1%). These strains were detected frequently (81.8%) from the point with the highest pollution levels. Furthermore, the marker for Class 1 integron, intI1 gene, was detected in 54.5% of ESBL producers. These data suggest an association between antimicrobial-resistant E. coli and sewage pollution in aquatic environments raising concerns about the possible risks of human exposure to these waters and fish consumption.

Antimicrobial resistance and biotechnological potential of plastic-associated bacteria isolated from an urban estuary.

Plastics have quickly become one of the major pollutants in aquatic environments worldwide and solving the plastic pollution crisis is considered a central goal of modern society. In this study, 10 different plastic samples, including high- and low-density polyethylene and polypropylene, were collected from a deeply polluted urban estuary in Brazil. By employing different isolation and analysis approaches to investigate plastic-associated bacteria, a predominance of potentially pathogenic bacteria such as Acinetobacter, Aeromonas, and Vibrio was observed throughout all plastic samples. Bacteria typically found in the aquatic environment harboured clinically relevant genes encoding resistance to carbapenems (blaKPC ) and colistin (such as mcr-3 and mcr-4), along with genetic determinants associated with potentially active gene mobilization. Whole genome sequencing and annotation of three plastic-associated Vibrio strains further demonstrated the carriage of mobile genetic elements and antimicrobial resistance and virulence genes. On the other hand, bacteria isolated from the same samples were also able to produce esterases, lipases, and bioemulsifiers, thus highlighting that the plastisphere could also be of special interest from a biotechnological perspective.

The biotechnological potential of Aeromonas: a bird's eye view.

The genus Aeromonas comprises Gram-negative bacilli widely distributed in aquatic habitats that can also be found in the terrestrial environment and in close association with humans and animals. Aeromonas spp. are particularly versatile bacteria, with high genomic plasticity and notable capacity to adapt to different environments and extreme conditions. On account of being mostly associated with their pathogenic potential, research on the biotechnological potentialities of Aeromonas spp. is considerably scarce when compared to other bacterial groups. Nonetheless, studies over the years have been hinting at several interesting hidden potentialities in this bacterial group, especially with the recent advances in whole-genome sequencing, unveiling Aeromonas spp. as interesting candidates for the discovery of novel industrial biocatalysts, bioremediation strategies, and biopolyester production. In this context, the present study aims to provide an overview of the main biotechnological applications reported in the genus Aeromonas and provide new insights into the further exploration of these frequently overlooked, yet fascinating, bacteria.

Unravelling the sponge microbiome as a promising source of biosurfactants.

Microbial surfactants are particularly useful in bioremediation and heavy metal removal from soil and aquatic environments, amongst other highly valued uses in different economic and biomedical sectors. Marine sponge-associated bacteria are well-known producers of bioactive compounds with a wide array of potential applications. However, little progress has been made on investigating biosurfactants produced by these bacteria, especially when compared with other groups of biologically active molecules harnessed from the sponge microbiome. Using a thorough literature search in eight databases, the purpose of the review was to compile the current knowledge on biosurfactants from sponge-associated bacteria, with a focus on their relevant biotechnological applications. From the publications between the years 1995 and 2021, lipopeptides and glycolipids were the most identified chemical classes of biosurfactants. Firmicutes was the dominant phylum of biosurfactant-producing strains, followed by Actinobacteria and Proteobacteria. Bioremediation led as the most promising application field for the studied surface-active molecules in sponge-derived bacteria, despite the reports endorsed their use as antimicrobial and antibiofilm agents. Finally, we appoint some key strategies to instigate the research appetite on the isolation and characterization of novel biosurfactants from the poriferan microbiome.

Uncovering the Microbial Diversity of Two Exotic Calcareous Sponges.

Sponges-associated microorganisms play important roles in their health and ecology; consequently, they may be crucial in the successful adaptation of exotic species to novel environments. However, few studies have focused on the microbial diversity of exotic sponges, especially those with calcium carbonate spicules (class Calcarea). Therefore, this is the first in situ characterization of the microbiota of the exotic calcareous sponges Sycettusa hastifera and Paraleucilla magna. Our results suggest that S. hastifera has a more stable microbiota than P. magna, as there were no differences in its beta diversity among sampling sites. Conversely, P. magna showed significant differences in its microbial communities, perhaps related to its adhesion to artificial substrate and/or shellfish mariculture activities. Each sponge species presented a single dominant proteobacterial OTU potentially active in the nitrogen cycle, which could help sponge detoxification, especially in polluted areas where exotic species usually establish. Our results show the importance of assessing the microbial diversity to unveil host-microorganism relationships and suggest that these associated nitrogen-cycling microorganisms could favor the success of exotic sponges in new environments.

Delving into the Mechanisms of Sponge-Associated Enterobacter against Staphylococcal Biofilms.

Staphylococci are one of the most common causes of biofilm-related infections. Such infections are hard to treat with conventional antimicrobials, which often lead to bacterial resistance, thus being associated with higher mortality rates while imposing a heavy economic burden on the healthcare system. Investigating antibiofilm strategies is an area of interest in the fight against biofilm-associated infections. Previously, a cell-free supernatant from marine-sponge-associated Enterobacter sp. inhibited staphylococcal biofilm formation and dissociated the mature biofilm. This study aimed to identify the chemical components responsible for the antibiofilm activity of Enterobacter sp. Scanning electron microscopy confirmed that the aqueous extract at the concentration of 32 µg/mL could dissociate the mature biofilm. Liquid chromatography coupled with high-resolution mass spectrometry revealed seven potential compounds in the aqueous extract, including alkaloids, macrolides, steroids, and triterpenes. This study also suggests a possible mode of action on staphylococcal biofilms and supports the potential of sponge-derived Enterobacter as a source of antibiofilm compounds.

Homoscleromorpha-derived Bacillus spp. as potential sources of biotechnologically-relevant hydrolases and biosurfactants.

Despite hydrolytic exoenzymes and biosurfactants having been gradually reported from the poriferan microbiome, little is known about these bioproducts in microorganisms inhabiting Homoscleromorpha sponges. Here, we investigated the production of hydrolases and biosurfactants in bacteria isolated from three shallow-water homoscleromorph species, Oscarella sp., Plakina cyanorosea, and Plakina cabofriense. A total of 99 of 107 sponge-associated bacterial isolates exhibited activity for at least one of the analyzed hydrolases. Following fermentation in Luria-Bertani (LB) and Tryptic Soy Broth (TSB), two isolates, 80BH11 and 80B1:1010b, showed higher lipase and peptidase activities. Both of them belonged to the Bacillus genus and were isolated from Oscarella. Central composite design leveraged up the peptidase activity in 280% by Bacillus sp. 80BH11 in the TSB medium for 48 h at 30 °C. The optimized model also revealed that pH 6.5 and 45 °C were the best conditions for peptidase reaction. In addition, Bacillus sp. 80BH11 was able to release highly emulsifying and remarkably stable surfactants in the LB medium. Surfactin was finally elucidated as the biosurfactant generated by this sponge-derived Bacillus. In conclusion, we hope to have set the scenery for further prospecting of industrial enzymes and biosurfactants in Homoscleromorpha microbiomes.

Antimicrobial and antibiofilm activities of marine sponge-associated bacteria against multidrug-resistant Staphylococcus spp. isolated from canine skin.

Dogs play important roles in our society, thus the concern for their health becomes imperative. Staphylococcus spp. are commensal bacterium frequently isolated from canine skin and recognized as zoonotic agents. These bacteria have been becoming increasingly resistant to antimicrobials used to treat infections and to produce biofilm, which further increases their virulence capability and resistance. In this context, sponges-associated bacteria are known as prolific sources of substances with antimicrobial activities, representing a potential to integrate the arsenal of drugs for clinical use. In this study, 121 strains of Staphylococcus isolated from healthy or infected dogs were characterized according to their resistance to antimicrobials, as well as to their biofilm production ability. From the total of strains, 82 were resistant to at least one antimicrobial and 40 were multidrug-resistant (MDR). Furthermore, 117 out of 121 were capable to produce biofilm, and within those 36 were classified as strong biofilm producers. A set of fifteen bacterial strains previously isolated from marine sponges were also evaluated for antimicrobial and antibiofilm activities. Among the marine bacteria with antimicrobial activity, eight inhibited the growth of more than 50% of the MDR Staphylococcus. In addition, the cell-free supernatant obtained from five sponge-associated bacteria cultures was able to disaggregate more than 50% of the mature biofilm staphylococcal cells. The organic extracts (256 µg/mL) from two potential strains, Pseudomonas fluorescens H40 and H41, dissociated the biofilm of a strain classified as MDR and strong biofilm producer in 88.5% and 91.3%, respectively. These marine Pseudomonas strains also exhibited a strong activity of antimicrobial and antibiofilm substances. The results suggest that the sponge-associated bacteria analyzed could be potential sources of antimicrobial and antibiofilm substances against MDR and biofilm producers Staphylococcus isolated from canine skin.

Characterization of biofilms and antimicrobial resistance of coagulase-negative Staphylococcus species involved with subclinical mastitis.

Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.

Harnessing the sponge microbiome for industrial biocatalysts.

Within the marine sphere, host-associated microbiomes are receiving growing attention as prolific sources of novel biocatalysts. Given the known biocatalytic potential of poriferan microbial inhabitants, this review focuses on enzymes from the sponge microbiome, with special attention on their relevant properties and the wide range of their potential biotechnological applications within various industries. Cultivable bacterial and filamentous fungal isolates account for the majority of the enzymatic sources. Hydrolases, mainly glycoside hydrolases and carboxylesterases, are the predominant reported group of enzymes, with varying degrees of tolerance to alkaline pH and growing salt concentrations being common. Prospective areas for the application of these microbial enzymes include biorefinery, detergent, food and effluent treatment industries. Finally, alternative strategies to identify novel biocatalysts from the sponge microbiome are addressed, with an emphasis on modern -omics-based approaches that are currently available in the enzyme research arena. By providing this current overview of the field, we hope to not only increase the appetite of researchers to instigate forthcoming studies but also to stress how basic and applied research can pave the way for new biocatalysts from these symbiotic microbial communities in a productive fashion. KEY POINTS: • The sponge microbiome is a burgeoning source of industrial biocatalysts. • Sponge microbial enzymes have useful habitat-related traits for several industries. • Strategies are provided for the future discovery of microbial enzymes from sponges.

Paraclostridium is the Main Genus of Anaerobic Bacteria Isolated from New Species of the Marine Sponge Plakina in the Brazilian Southeast Coast.

Despite the broad assessment of sponge bacterial diversity through cultivation-independent and dependent strategies, the knowledge focusing on cultivable anaerobes from this holobiont is still incipient. Plakina is a genus with the highest number of described species from the smallest of poriferan classes, Homoscleromorpha. The Brazilian Atlantic coast has been presenting itself as a hotspot for the discovery of new plakinidae species, with initial surveys just now concerning to characterize their microbiome. The current study aimed to isolate and identify strict anaerobes from recently described species of Plakina collected at the coast of Cabo Frio, RJ. Samples of four sympatric morphotypes of Plakina cyanorosea and Plakina cabofriense were collected on the coast of Cabo Frio, RJ. Using five different culture media, a total of 93 bacterial isolates were recovered, among which 60 were strict anaerobes and, ultimately, 34 remaining viable. A total of 76.5% from these strains were mostly identified as Clostridium bifermentans by mass spectrometry and 82.4% identified by 16S rRNA sequencing, almost all of them affiliated to the genus Paraclostridium, and with one isolate identified as Clostridium butyricum by both techniques. None of the anaerobic bacteria exhibited antimicrobial activity by the adopted screening test. The present work highlights not only the need for cultivation and characterization of the anaerobic microbiota from marine sponges but also adds the existing scarce knowledge of culturable bacterial communities from Homoscleromorph sponges from Brazilian coast.

Phylogeny and Antagonistic Activities of Culturable Bacteria Associated with the Gut Microbiota of the Sea Urchin (Paracentrotus lividus).

In this study, we have investigated the phylogeny and the antagonistic interactions of culturable bacteria isolated from the sea urchin Paracentrotus lividus collected from Aber and Morgat, both located in Crozon peninsula, France. Bacteria were isolated from the gastrointestinal tracts of ten specimens by using conventional culture-dependent method and then investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. Assays for antagonistic interactions among the bacterial strains were performed; bacteria (including at least one strain representative of each OTU identified) were screened for antimicrobial substance production. So, 367 bacterial strains were isolated on marine-agar. On the basis of morphological characteristics, 180 strains were sequenced and 94 OTUs were classified. The dominant phyla were Proteobacteria, Firmicutes and Actinobacteria, with a high abundance of the strains belonging to the genus Psychrobacter. From the antagonistic interactions assays, it could be determined that 22.7% strains were positive for at least one antagonism interaction, 18.3% of them isolated from the sea urchins collected in Morgat. We hypothesize that the bacteria isolated in this study may represent the transitory microbiota of the gastrointestinal tract of P. lividus, and that this microbiota may be related to the diet of this marine invertebrate. Furthermore, our results suggest that chemical antagonism could play a significant role in shaping the bacterial communities within gastrointestinal tract of the sea urchins. In addition, most isolated bacteria may have promising biotechnology applications.

Culturable bacterial communities associated to Brazilian Oscarella species (Porifera: Homoscleromorpha) and their antagonistic interactions.

Sponges offer an excellent model to investigate invertebrate-microorganism interactions. Furthermore, bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to characterize the bacteria inhabiting a genus of sponges, Oscarella, and their potentiality for antimicrobial production. Bacterial isolates were recovered from different Oscarella specimens, among which 337 were phylogenetically identified. The culturable community was dominated by Proteobacteria and Firmicutes, and Vibrio was the most frequently isolated genus, followed by Shewanella. When tested for antimicrobial production, bacteria of the 12 genera isolated were capable of producing antimicrobial substances. The majority of strains were involved in antagonistic interactions and inhibitory activities were also observed against bacteria of medical importance. It was more pronounced in some isolated genera (Acinetobacter, Bacillus, Photobacterium, Shewanella and Vibrio). These findings suggest that chemical antagonism could play a significant role in shaping bacterial communities within Oscarella, a genus classified as low-microbial abundance sponge. Moreover, the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing therapies to treat infections caused by multidrug-resistant bacteria. This study was the first to investigate the diversity and antagonistic activity of bacteria isolated from Oscarella spp. It highlights the biotechnological potential of sponge-associated bacteria.

Expression of the stress-response regulators CtsR and HrcA in the uropathogen Staphylococcus saprophyticus during heat shock.

The uropathogen Staphylococcus saprophyticus is an ubiquitous bacterium but little is known about mechanisms that allow its persistence in diverse environments. Here we evaluated S. saprophyticus growth and survival during heat shock, the expression of stress response regulators ctsR and hrcA through qRT-PCR and heat shock protein synthesis through 35S-Met metabolic labeling. S. saprophyticus does not tolerate temperatures much higher than the optimal 37 °C, as its growth is greatly affected at 42 °C, though viability is maintained up to 48 °C. At 42 °C, the expression of ctsR and hrcA repressor genes approximately triple when compared to 37 °C and continue to increase together with temperature till 48 °C. Expression of hrcA peaks after 20 min of heat shock and decreases significantly after 30 min, indicating that heat stress response regulated by this gene may last 20-30 min. An increase in temperature is accompanied by the synthesis of at least eight proteins, three of which are likely the chaperones DnaK, GroEL and ClpB. In silico analysis indicate that the groEL gene may be regulated by HrcA, clpB by CtsR and dnaK by both repressors. This is the first work to discuss heat stress response in S. saprophyticus and a step forward in the understanding of mechanisms that make this a widespread and emergent pathogen.

The CtsR regulator controls the expression of clpC, clpE and clpP and is required for the virulence of Enterococcus faecalis in an invertebrate model.

The intrinsic ruggedness of Enterococcus faecalis is responsible for its widespread distribution in nature and is often viewed as an important virulence determinant. Previously, we showed that the ClpB ATPase is negatively regulated by CtsR and is required for thermotolerance and virulence in a Galleria mellonella invertebrate model. Here, we used in silico, Northern blot and quantitative real-time PCR analyses to identify additional members of the CtsR regulon, namely the clpP peptidase and the clpC and clpE ATPases. When compared to the parent strain, virulence of the ΔctsR strain in G. mellonella was significantly attenuated.

Polyphasic identification of Vibrio species from aquatic sources using mass spectrometry, housekeeping gene sequencing and whole genome analysis.

Accurate bacterial identification is essential for determining the causative agent of an infection, thus facilitating appropriate treatment and management strategies in both human and animal health contexts. Some species in the Vibrio genus are recognized pathogens, associated with a variety of infections. However, identification of these bacteria is oftentimes controversial. Therefore, we aimed to evaluate different identification approaches in terms of their reliability in distinguishing Vibrio species. To achieve this, we selected a set of 40 Vibrio isolates previously recovered from water and floating plastic samples in a large bay environment and identified them employing MALDI-TOF mass spectrometry, and rrs and pyrH gene sequencing. A subset of isolates was also submitted to whole genome sequencing. Overall, MALDI-TOF was found to be a fast-screening methodology for identification, notably at genus-level. However, for better species discrimination, pyrH gene sequencing stood out as a more reliable tool in contrast to rrs gene sequencing and MALDI-TOF, as corroborated by whole genome sequencing analysis.