Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species (“public goods”), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities.     

Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Facilitation and inhibition of larval attachment of the bryozoan Bugula neritina in association with mono-species and multi-species biofilms

In this study, we investigated the effect of mono-species and multi-species biofilms on larval attachment of the bryozoan Bugula neritina. The effect of biofilms was examined through a double-dish choice bioassay in which larvae were given the choice of attaching either to a clean surface of a container or to surfaces covered with biofilms. Larvae attached in response to mono-species biofilms of 5 out of 7 bacterial isolates from a subtidal region, but they avoided surfaces covered by biofilms of 7 out of 8 isolates obtained from an intertidal region. In the follow-up choice experiments with multi-species biofilms developed for 2 days, 7 days, 14 days, 28 days and 30 days, larvae preferentially attached to filmed surfaces over the unfilmed surfaces. When biofilms from 2 different tidal regions (intertidal and subtidal) were offered as choices in the double-dish bioassay, larvae in all cases attached on the subtidal biofilms. Two-day-old subtidal biofilms with low densities of bacteria induced significantly higher (p < 0.05) attachment than did 30- day-old intertidal biofilms, which had high bacterial density. Terminal Restriction Fragment Polymorphism (T-RFLP) analysis revealed that the bacterial communities were substantially different in the subtidal and intertidal regions during all periods of the experiment. Attachment of B. neritina on subtidal biofilms did not depend on the bacterial density but rather was negatively correlated with diatom density, thickness of the exopolysaccharide layer and biofilm age. Our results suggest that the larvae of B. neritina can discriminate between biofilmed and clean surfaces and between biofilms developed under different tidal zones.     

Development of a small-scale bioreactor method to monitor the molecular diversity and environmental impacts of bacterial biofilm communities from an acid mine drainage impacted creek

Shamokin Creek is a tributary of the Susquehanna River in central Pennsylvania that is heavily impacted by the acid mine drainage (AMD) caused by the oxidation of pyrite from the region's extensive anthracite coal mining industry. Recent studies have begun to characterize the microbial communities present in this and other AMD-impacted waters, but varying environmental conditions have complicated attempts to determine the ecological impacts of individual bacterial species within these communities. This study developed a small-scale biofilm reactor protocol that allowed us to simultaneously monitor the development of bacterial biofilm communities in AMD-impacted creek collected water using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes, while assessing the impacts that the developing biofilms were having on water quality. Our analysis confirmed that the diversity and composition of these small in situ biofilm communities could be monitored using molecular methods, and indicated the possible presence of many taxa frequently found in AMD environments, including Sulfobacillus, Nitrospira, Desulfovibrio, Geobacter, and Leptothrix species. A significant increase in the total sulfate was observed in the bioreactor, and as most likely due to the accumulation of sulfur-oxidizing bacteria such as Sulfobacillus in the biofilms. This system will allow us to study the microbial ecology of Shamokin Creek through controlled experiments that will ultimately integrate microscopic, molecular, physiological and chemical analyses, and that can be utilized to develop more effective and cost-efficient environmental remediation techniques for AMD-impacted areas.     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.     

The potential of lactic acid bacteria to colonize biotic and abiotic surfaces and the investigation of their interactions and mechanisms

Lactic acid bacteria (LAB) are a heterogeneous group of Gram-positive bacteria that comprise several species which have evolved in close association with humans (food and lifestyle). While their use to ferment food dates back to very ancient times, in the last decades, LAB have attracted much attention for their documented beneficial properties and for potential biomedical applications. Some LAB are commensal that colonize, stably or transiently, host mucosal surfaces, inlcuding the gut, where they may contribute to host health. In this review, we present and discuss the main factors enabling LAB adaptation to such lifestyle, including the gene reprogramming accompanying gut colonization, the specific bacterial components involved in adhesion and interaction with host, and how the gut niche has shaped the genome of intestine-adapted species. Moreover, the capacity of LAB to colonize abiotic surfaces by forming structured communities, i.e., biofilms, is briefly discussed, taking into account the main bacterial and environmental factors involved, particularly in relation to food-related environments. The vast spread of LAB surface-associated communities and the ability to control their occurrence hold great potentials for human health and food safety biotechnologies.

     

Identification of Bacterial Strains Isolated from the Mediterranean Sea Exhibiting Different Abilities of Biofilm Formation

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.     

Erosion from Staphylococcus aureus Biofilms Grown under Physiologically Relevant Fluid Shear Forces Yields Bacterial Cells with Reduced Avidity to Collagen

An estimated 65% of infective diseases are associated with the presence of bacterial biofilms. Biofilm-issued planktonic cells promote blood-borne, secondary sites of infection by the inoculation of the infected sites with bacteria from the intravascular space. To investigate the potential role of early detachment events in initiating secondary infections, we studied the phenotypic attributes of Staphylococcus aureus planktonic cells eroding from biofilms with respect to expression of the collagen adhesin, CNA. The collagen-binding abilities of S. aureus have been correlated to the development of osteomyelitis and septic arthritis. In this study, we focused on the impact of CNA expression on S. aureus adhesion to immobilized collagen in vitro under physiologically relevant shear forces. In contrast to the growth phase-dependent adhesion properties characteristic of S. aureus cells grown in suspension, eroding planktonic cells expressed invariant and lower effective adhesion rates regardless of the age of the biofilm from which they originated. These results correlated directly with the surface expression level of CNA. However, subsequent analysis revealed no qualitative differences between biofilms initiated with suspension cells and secondary biofilms initiated with biofilm-shed planktonic cells. Taken together, our findings suggest that, despite their low levels of CNA expression, S. aureus planktonic cells shed from biofilms retain the capacity for metastatic spread and the initiation of secondary infection. These findings demonstrate the need for a better understanding of the phenotypic properties of eroding planktonic cells, which could lead to new therapeutic strategies to target secondary infections.     

De Novo Designed α-Sheet Peptides Inhibit Functional Amyloid Formation of Streptococcus mutans Biofilms

Streptococcus mutans is a bacterial species that predominates in the oral microbiome. S. mutans binds to the tooth surface, metabolizes sugars and produces acid, leading to cavity formation. S. mutans can also cause infectious endocarditis. Recent evidence suggests that S. mutans biofilms contain amyloid fibrils. Amyloids are insoluble fibrillar protein aggregates, and bacteria use functional amyloids to improve robustness of their biofilms. While the functional amyloids in bacteria such as Escherichia coli and Staphylococcus aureus have been heavily investigated, little is known about the mechanism of S. mutans amyloid formation. Previous results from our laboratory with the amyloidogenic proteins and peptides from the aforementioned bacteria and other mammalian amyloid systems suggest that amyloid formation progresses via an intermediate that adopts a unique secondary structure—α-sheet. De novo designed peptides with alternating l- and d-amino acid also adopt an α-sheet secondary structure and inhibit amyloid formation by binding to soluble oligomeric species during amyloidogenesis. Inhibition of fibrillization by α-sheet peptides suggests the presence of α-sheet during amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans, α-sheet peptides were compared to epigallocatechin gallate for their ability to inhibit fibril formation in S. mutans. Inhibition was demonstrated in a biofilm plate assay and on hydroxyapatite surfaces both in S. mutans alone and in bacteria from human saliva. The observed inhibition suggests that an α-sheet mediated mechanism may be operative during functional amyloid formation.     

Characterisation of the bacteria associated with barnacle,Balanus amphitrite,shell and their role in gregarious settlement of cypris larvae

The acorn barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) is a model organism to investigate pelago-benthic transitions in marine invertebrates. A driver for larval settlement in this organism is the need to attach close to conspecifics, to allow reproduction to take place. Adult barnacles are covered by microbial biofilms and the contribution of these biofilms to conspecific recognition is not fully understood. Little information is available on microbial communities associated with B. amphitrite. We compared biofilm communities from the barnacle shell surface with those from the surrounding rocks using the culture-independent methods of quantitative PCR and denaturing gradient gel electrophoresis. Quantification of the relative abundances of higher bacterial taxa showed that barnacles hosted a greater proportion of α-Proteobacteria compared to rock-associated biofilms (p < 0.01). Differences in relative abundances of other taxa were not observed but DGGE profiling suggested that differences were present at lower taxonomic levels. The capacity of these communities to influence larval settlement was assessed by growing multispecies biofilms on artificial medium, obtained by extracting nutrients from adult barnacles. Biofilms composed of shell-associated bacteria were capable of promoting conspecific settlement by 67% compared to control surfaces (p < 0.05), while rock-associated communities showed contrasting effects. A taxonomic comparison of settlement-stimulating and -inhibiting bacteria was performed by DGGE and band sequencing. All partial 16S rRNA genes sequenced were similar to members of the Vibrio and Pseudoalteromonas genera, suggesting that larvae can detect and respond to variations in the composition of microbial biofilms at low taxonomic levels. Our results indicate that barnacle larvae may be able to detect parentally-associated biofilms and use this information to settle close to members of its own species.     

Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood

Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities.     

Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.     

Effective Removal of Staphylococcal Biofilms by the Endolysin LysH5

Staphylococcal biofilms are a major concern in both clinical and food settings because they are an important source of contamination. The efficacy of established cleaning procedures is often hindered due to the ability of some antimicrobial compounds to induce biofilm formation, and to the presence of persister cells, a small bacterial subpopulation that exhibits multidrug tolerance. Phage lytic enzymes have demonstrated antimicrobial activity against planktonic and sessile bacteria. However, their ability to lyse and/or select persister cells remains largely unexplored so far. In this work, the lytic activity of the endolysin LysH5 against Staphylococcus aureus and Staphylococcus epidermidis biofilms was confirmed. LysH5 reduced staphylococcal sessile cell counts by 1–3 log units, compared with the untreated control, and sub-inhibitory concentrations of this protein did not induce biofilm formation. LysH5-surviving cells were not resistant to the lytic activity of this protein, suggesting that no persister cells were selected. Moreover, to prove the lytic ability of LysH5 against this subpopulation, both S. aureus exponential cultures and persister cells obtained after treatment with rifampicin and ciprofloxacin were subsequently treated with LysH5. The results demonstrated that besides the notable activity of endolysin LysH5 against staphylococcal biofilms, persister cells were also inhibited, which raises new opportunities as an adjuvant for some antibiotics.     

Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes

The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions.     

Microbial activity in drinking water-associated biofilms

Microbial biofilms from surfaces in contact with water may play a beneficial role in drinking water treatment as biological filters. However, detrimental effects such as biofouling (i.e., biocorrosion and water quality deterioration) may also occur. In this study microbiological processes and factors influencing the activity of bacteria in biofilms were investigated by conventional cultivation methods. The presence of bacteria belonging to different ecophysiological groups was assessed during drinking water treatment, in biofilms developed on concrete, steel and sand surfaces. Influences of the treatment process, type of immersed material and physico-chemical characteristics of raw/bulk water and biofilms upon the dynamics of bacterial communities were evaluated. Results revealed intense microbial activity in biofilms occurring in the drinking water treatment plant of Cluj. Ammonification, iron reduction and manganese oxidation were found to be the predominant processes. Multiple significant correlations were established between the evolution of biofilm bacteria and the physico-chemical parameters of raw/ bulk water. The type of immersed material proved to have no significant influence upon the evolution of microbial communities, but the treatment stage, suggesting that the processes applied restrict microbial growth not only in bulk fluid but in biofilms, too.     

Genetic diversity of phosphate-solubilizing peanut (Arachis hypogaea L.) associated bacteria and mechanisms involved in this ability

In this study, attempts were made to analyze mechanisms involved in the bacterial phosphate-solubilizing ability of peanut isolates. Bacteria were taxonomically identified by analysis of 16S rDNA sequence. Levels of soluble P released by the isolates in unbuffered or buffered with Tris–HCl or MES NBRIP-BPB medium as well as the production of D-gluconic acid were determined in their culture. Presence of two of the genes encoding the cofactor PQQ of GDH enzyme was analyzed in the genome of this bacterial collection. 16S rDNA sequence analysis indicated that isolates belong to genera Serratia, Enterobacter, Pantoea, Acinetobacter, Bacillus and Enterococcus. All bacteria showed ability to solubilize tricalcium phosphate either in unbuffered or buffered medium. Nevertheless, addition of buffer solutions reduced levels of Pi liberated by the isolates. Although almost all isolates produced detectable amounts of D-gluconic acid, no correlation with levels of P soluble released were observed. The presence of pqqE and pqqC genes was detected only in Gram negative bacteria. It was concluded from this study that the mechanism involved in phosphate solubilization is organic acids production and, presence of pqq genes in all Gram negative bacteria analyzed encourages to confirm their role in bacterial phosphate solubilizing ability as well to identify genes involved in this PGP trait in Gram positive bacteria.