The role of quorum sensing mediated developmental traits in the resistance of Serratia marcescens biofilms against protozoan grazing

Resistance against protozoan grazers is a crucial factor that is important for the survival of many bacteria in their natural environment. However, the basis of resistance to protozoans and how resistance factors are regulated is poorly understood. In part, resistance may be due to biofilm formation, which is known to protect bacteria from environmental stress conditions. The ubiquitous organism Serratia marcescens uses quorum sensing (QS) control to regulate virulence factor expression and biofilm formation. We hypothesized that the QS system of S. marcescens also regulates mechanisms that protect biofilms against protozoan grazing. To investigate this hypothesis, we compared the interactions of wild-type and QS mutant strains of S. marcescens biofilms with two protozoans having different feeding types under batch and flow conditions. Under batch conditions, S. marcescens forms microcolony biofilms, and filamentous biofilms are formed under flow conditions. The microcolony-type biofilms were protected from grazing by the suspension feeder, flagellate Bodo saltans, but were not protected from the surface feeder, Acanthamoeba polyphaga. In contrast, the filamentous biofilm provided protection against A. polyphaga. The main findings presented in this study suggest that (i) the QS system is not involved in grazing resistance of S. marcescens microcolony-type biofilms; (ii) QS in S. marcescens regulates antiprotozoan factor(s) that do not interfere with the grazing efficiency of the protozoans; and (iii) QS-controlled, biofilm-specific differentiation of filaments and cell chains in biofilms of S. marcescens provides an efficient mechanism against protozoan grazing.     

Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.     

Natural genetic transformation of Streptococcus mutans growing in biofilms

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

A novel compound from the marine bacterium Bacillus pumilus S6-15 inhibits biofilm formation in gram-positive and gram-negative species

Biofilm formation is a critical problem in nosocomial infections and in the aquaculture industries and biofilms show high resistance to antibiotics. The aim of the present study was to reveal a novel anti-biofilm compound from marine bacteria against antibiotic resistant gram-positive and gram-negative biofilms. The bacterial extract (50 μg ml(-1)) of S6-01 (Bacillus indicus = MTCC 5559) showed 80-90% biofilm inhibition against Escherichia coli, Shigella flexneri, Proteus mirabilis and S6-15 (Bacillus pumilus = MTCC 5560) showed 80-95% biofilm inhibition against all the 10 tested organisms. Furthermore, they also reduced the hydrophobicity index and extracellular polymeric substances (EPS) production. Structural elucidation of the active principle in S6-15 using GC-MS, (1)H NMR, and (13)C NMR spectral data revealed it to be 4-phenylbutanoic acid. This is the first report of 4-phenylbutanoic acid as a natural product. The purified compound (10-15 μg ml(-1)) showed potential activity against a wide range of biofilms. This study for the first time, reports a novel anti-biofilm compound from a marine bacterium with wide application in medicine and the aquaculture industry.     

Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans

The abilities of Streptococcus mutans to form biofilms and to survive acidic pH are regarded as two important virulence determinants in the pathogenesis of dental caries. Environmental stimuli are thought to regulate the expression of several genes associated with virulence factors through the activity of two-component signal transduction systems. Yet, little is known of the involvement of these systems in the physiology and pathogenicity of S. mutans. In this study, we describe a two-component regulatory system and its involvement in biofilm formation and acid resistance in S. mutans. By searching the S. mutans genome database with tblastn with the HK03 and RR03 protein sequences from S. pneumoniae as queries, we identified two genes, designated hk11 and rr11, that encode a putative histidine kinase and its cognate response regulator. To gain insight into their function, a PCR-mediated allelic-exchange mutagenesis strategy was used to create the hk11 (Em(r)) and rr11 (Em(r)) deletion mutants from S. mutans wild-type NG8 named SMHK11 and SMRR11, respectively. The mutants were examined for their growth rates, genetic competence, ability to form biofilms, and resistance to low-pH challenge. The results showed that deletion of hk11 or rr11 resulted in defects in biofilm formation and resistance to acidic pH. Both mutants formed biofilms with reduced biomass (50 to 70% of the density of the parent strain). Scanning electron microscopy revealed that the biofilms formed by the mutants had sponge-like architecture with what appeared to be large gaps that resembled water channel-like structures. The mutant biofilms were composed of longer chains of cells than those of the parent biofilm. Deletion of hk11 also resulted in greatly diminished resistance to low pH, although we did not observe the same effect when rr11 was deleted. Genetic competence was not affected in either mutant. The results suggested that the gene product of hk11 in S. mutans might act as a pH sensor that could cross talk with one or more response regulators. We conclude that the two-component signal transduction system encoded by hk11 and rr11 represents a new regulatory system involved in biofilm formation and acid resistance in S. mutans.     

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1)) and high (10(7) PFU mL(-1)) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 μg mL(-1), respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.     

TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa

Type VI secretion systems (T6SSs) contribute to interactions of bacterial pathogens and symbionts with their hosts. Previously, we showed that Pseudomonas aeruginosa T6S is posttranslationally activated upon phosphorylation of Fha1, an FHA domain protein, by PpkA, a membrane-spanning threonine kinase. Herein, additional structural, enzymatic and genetic requirements for PpkA-catalysed T6SS activation are identified. We found that PpkA plays an essential structural role in the T6SS, and that this role is intimately linked to its ability to promote secretion and phosphorylate Fha1. Protein localization and protein–protein interaction studies show that a complex containing Fha1 and the T6S ATPase, ClpV1 is recruited to the T6S apparatus in a phosphorylation-dependent manner. The mechanism of PpkA activation was also investigated. We identified critical PpkA autophosphorylation sites and showed that small molecule-induced dimerization of the extracellular domains of PpkA is sufficient to activate the T6SS. Finally, we discovered TagR, a component of the T6S posttranslational regulatory pathway that functions upstream of PpkA to promote kinase activity. We present a model whereby an unknown cue causes dimerization of the extracellular domains of PpkA, leading to its autophosphorylation, recruitment of the Fha1-ClpV1 complex, phosphorylation of Fha1, and T6SS activation. Our findings should facilitate approaches for identifying physiological activators of T6S.     

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.     

In Silico Design,Synthesis, and In Vitro Evaluation of Novel Amphipathic Short Linear Peptides Against Clinically Relevant Bacterial Biofilms

Microbial biofilms are organized communities of cells that are associated with a wide spectrum of resistant and chronic infections that lead to the treatment failure. Accordingly, there is an urgent demand to create novel effective therapeutic drugs that can inhibit biofilm formation with new mechanisms of action to surmount the current escalating resistance. In this study, in silico hybrid model was utilized to develop three novel short linear peptides (4, 5, and 6) with potential biofilm inhibiting activities (scores?>?1.0). The peptides were composed of cationic and hydrophobic residues. They were synthesized using solid-phase strategy. Synthesized peptides were purified and characterized by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization spectroscopy, respectively. They were evaluated using in vitro assay as potential inhibitors of clinically relevant Gram-positive and Gram-negative biofilms. Peptide (4) with five positive charges at physiological pH (4 cationic moieties and W:R?=?1:4) showed activity against biofilms of Gram-positive strains (Staphylococcus epidermidis and Listeria monocytogenes). On the other hand, peptide (5) with six positive charges (5 cationic moieties and W:R?=?2:2) demonstrated activity against Gram-positive (S. epidermidis) and Gram-negative (Escherichia coli) biofilms. Interestingly, peptide (6), with seven positive charges (6 cationic moieties and W:R?=?2:5) revealed higher and broader spectrum of activity against biofilms of Gram-positive (S. epidermidis, S. aureus, L. monocytogenes) and Gram-negative (E. coli).

     

Involvement of a novel efflux system in biofilm-specific resistance to antibiotics

Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.     

Can viruses form biofilms?

The recent finding that the human T-cell leukemia virus type 1 (HTLV-1) encases itself in a carbohydrate-rich adhesive extracellular 'cocoon', which enables its efficient and protected transfer between cells, unveiled a new infectious entity and a novel mechanism of viral transmission. These HTLV-1 structures are observed at the surface of T cells from HTLV-1-infected patients and are reminiscent of bacterial biofilms. The virus controls the synthesis of the matrix, which surrounds the virions and attaches them to the T cell surface. We propose that, similar to bacterial biofilms, viral biofilms could represent 'viral communities' with enhanced infectious capacity and improved spread compared with 'free' viral particles, and might constitute a key reservoir for chronic infections.     

Metal resistance in Candida biofilms

Yeasts are often successful in metal-polluted environments; therefore, the ability of biofilm and planktonic cell Candida tropicalis to endure metal toxicity was investigated. Fifteen water-soluble metal ions, chosen to represent groups 6A to 6B of the periodic table, were tested against this organism. With in vitro exposures as long as 24 h, biofilms were up to 65 times more tolerant to killing by metals than corresponding planktonic cultures. Of the most toxic heavy metals tested, only very high concentrations of Hg2+, CrO4 (2-) or Cu2+ killed surface-adherent Candida. Metal-chelator precipitates could be formed in biofilms following exposure to the heavy metals Cu2+ and Ni2+. This suggests that Candida biofilms may adsorb metal cations from their surroundings and that sequestration in the extracellular matrix may contribute to resistance. We concluded that biofilm formation may be a strategy for metal resistance and/or tolerance in yeasts.     

Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis

The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics

Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of P. aeruginosa UCBPP-PA14 (ndvB, PA1875–1877 and tssC1) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents.