Wild-type Biofilm Research Articles

Methicillin resistant Staphylococcus aureus mazEF expression promotes infections by influencing cellular growth, antibiotic sensitivity, and formation of biofilms

Staphylococcus aureus infections are hard to treat due to the emergence of antibiotic resistant strains, as well as their ability to form biofilms. The MazEF toxin–antitoxin system is thought play a role in bacterial biofilm phenotype as well as antibiotic resistance. In S. aureus, the physiologic function of the mazEF gene in the disease transition from acute to chronic infection is not well understood. In methicillin resistant S. aureus (MRSA), loss of mazF expression results in loss of resistance to first generation cephalosporins. mazF::tn displayed sensitivity while the isogenic wild type (WT) remained resistant. mazF::tn displayed significantly increased growth of biofilms on metal implants over 48 h compared to WT and the complemented transposon mutant. mazF::tn biofilms displayed significantly decreased antibiotic tolerance to vancomycin and cefazolin in comparison to WT and complement biofilms. Mice given mazF::tn in a sepsis model displayed less abscess burden and increased survival (100%) when treated with cefazolin compared to WT bacteremia treated with cefazolin (20%). mazF::tn periprosthetic joint infections displayed increased biofilm burden at acute time points and decreased biofilm burden at chronic time points. Our data suggests MazEF in MRSA is responsible for controlling growth of biofilms, antibiotic tolerance, and influence chronic infections in vivo.

Genetic determinants of increased sodium hypochlorite and ciprofloxacin susceptibility in Pseudomonas aeruginosa PA14 biofilms

Reactive chlorine species (RCS) like sodium hypochlorite (NaOCl) are potent oxidizing agents and widely used biocides in surface disinfection, water treatment, and biofilm elimination. Moreover, RCS are also produced by the human immune system to kill invading pathogens. However, bacteria have developed mechanisms to survive the damage caused by RCS. Using the comprehensive Pseudomonas aeruginosa PA14 transposon mutant library in a genetic screen, we identified a total of 28 P. aeruginosa PA14 mutants whose biofilms showed increased susceptibility to NaOCl in comparison to PA14 WT biofilms. Of these, ten PA14 mutants with a disrupted apaH, PA0793, acsA, PA1506, PA1547, PA3728, yajC, queA, PA3869, or PA14_32840 gene presented a 4-fold increase in NaOCl susceptibility compared to wild-type biofilms. While none of these mutants showed a defect in biofilm formation or attenuated susceptibility of biofilms toward the oxidant hydrogen peroxide (H2O2), all but PA14_32840 also exhibited a 2–4-fold increase in susceptibility toward the antibiotic ciprofloxacin. Further analyses revealed attenuated levels of intracellular ROS and catalase activity only for the apaH and PA1547 mutant, providing insights into the oxidative stress response in P. aeruginosa biofilms. The findings of this paper highlight the complexity of biofilm resistance and the intricate interplay between different mechanisms to survive oxidative stress. Understanding resistance strategies adopted by biofilms is crucial for developing more effective ways to fight resistant bacteria, ultimately contributing to better management of bacterial growth and resistance in clinical and environmental settings.

A New Convenient Method to Assess Antibiotic Resistance and Antimicrobial Efficacy against Pathogenic Clostridioides difficile Biofilms.

Clostridioides difficile is a widely distributed anaerobic pathogen. C. difficile infection is a serious problem in healthcare. Its biofilms have been found to exhibit biocorrosivity, albeit very little, but sufficient for it to correlate with biofilm growth/health. This work demonstrated the use of a disposable electrochemical biofilm test kit using two solid-state electrodes (a 304 stainless steel working electrode, and a graphite counter electrode, which also served as the reference electrode) in a 10 mL serum vial. It was found that the C. difficile 630∆erm Adp-4 mutant had a minimum inhibitory concentration (MIC) for vancomycin twice that of the 630∆erm wild type strain in biofilm prevention (2 ppm vs. 1 ppm by mass) on 304 stainless steel. Glutaraldehyde, a commonly used hospital disinfectant, was found ineffective at 2% (w/w) for the prevention of C. difficile 630∆erm wild type biofilm formation, while tetrakis(hydroxymethyl)phosphonium sulfate (THPS) disinfectant was very effective at 100 ppm for both biofilm prevention and biofilm killing. These antimicrobial efficacy data were consistent with sessile cell count and biofilm imaging results. Furthermore, the test kit provided additional transient biocide treatment information. It showed that vancomycin killed C. difficile 630∆erm wild type biofilms in 2 d, while THPS only required minutes.

Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1- mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1- mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1- mutant. In the sloughing assay, nuc1- mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.

Biofilms: A developmental niche for vancomycin-intermediate Staphylococcus aureus

Staphylococcus aureus are gram-positive bacteria responsible for a wide array of diseases, ranging from skin and soft tissue infections to more chronic illnesses such as toxic shock syndrome, osteomyelitis, and endocarditis. Vancomycin is currently one of the most effective antibiotics available in treating patients infected with methicillin-resistant S. aureus (MRSA), however the emergence of vancomycin-resistant S. aureus (VRSA), and more commonly vancomycin-intermediate S. aureus (VISA), threaten the future efficacy of vancomycin. Intermediate resistance to vancomycin occurs due to mutations within the loci of Staphylococcal genes involved in cell wall formation such as rpoB, graS, and yycG. We hypothesized the VISA phenotype may also arise as a result of the natural stress occurring within S. aureus biofilms, and that this phenomenon is mediated by the RecA/SOS response. Wildtype and recA null mutant/lexAG94E strains of S. aureus biofilms were established in biofilm microtiter assays or planktonic cultures with or without the addition of sub-inhibitory concentrations of vancomycin (0.063 mg/l - 0.25 mg/L ciprofloxacin, 0.5 mg/l vancomycin). Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin-intermediate resistance. The results indicated that a greater subpopulation of cells from wildtype biofilms (4.16 × 102 CFUs) emerged from intermediate-resistant concentrations of vancomycin (4 μg/ml) compared with the planktonic counterpart (1.53 × 101 CFUs). Wildtype biofilms (4.16 × 102 CFUs) also exhibited greater resistance to intermediate-resistant concentrations of vancomycin compared with strains deficient in the recA null mutant (8.15 × 101 CFUs) and lexA genes (8.00 × 101 CFUs). While the VISA phenotype would be an unintended consequence of genetic diversity and potentially gene transfer in the biofilm setting, it demonstrates that mutations occurring within biofilms allow for S. aureus to adapt to new environments, including the presence of widely used antibiotics.

Global transcriptional analysis of Geobacter sulfurreducens gsu1771 mutant biofilm grown on two different support structures.

Electroactive biofilms formation by the metal-reducing bacterium Geobacter sulfurreducens is a step crucial for bioelectricity generation and bioremediation. The transcriptional regulator GSU1771 controls the expression of essential genes involved in electron transfer and biofilm formation in G. sulfurreducens, with GSU1771-deficient producing thicker and more electroactive biofilms. Here, RNA-seq analyses were conducted to compare the global gene expression patterns of wild-type and Δgsu1771 mutant biofilms grown on non-conductive (glass) and conductive (graphite electrode) materials. The Δgsu1771 biofilm grown on the glass surface exhibited 467 differentially expressed (DE) genes (167 upregulated and 300 downregulated) versus the wild-type biofilm. In contrast, the Δgsu1771 biofilm grown on the graphite electrode exhibited 119 DE genes (79 upregulated and 40 downregulated) versus the wild-type biofilm. Among these DE genes, 67 were also differentially expressed in the Δgsu1771 biofilm grown on glass (56 with the same regulation and 11 exhibiting counter-regulation). Among the upregulated genes in the Δgsu1771 biofilms, we identified potential target genes involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). RT-qPCR analyses were then conducted to confirm the differential expression of a selection of genes of interest. DNA-protein binding assays demonstrated the direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Furthermore, heme-staining and western blotting revealed an increase in c-type cytochromes including OmcS and OmcZ in Δgsu1771 biofilms. Collectively, our findings demonstrated that GSU1771 is a global regulator that controls extracellular electron transfer and exopolysaccharide synthesis in G. sulfurreducens, which is crucial for electroconductive biofilm development.

Pyocyanin-dependent electrochemical inhibition of Pseudomonas aeruginosa biofilms is synergistic with antibiotic treatment.

Pseudomonas aeruginosa biofilms are common in chronic wound infections and recalcitrant to treatment. Survival of cells within oxygen-limited regions in these biofilms is enabled by extracellular electron transfer (EET), whereby small redox active molecules act as electron shuttles to access distal oxidants. Here, we report that electrochemically controlling the redox state of these electron shuttles, specifically pyocyanin (PYO), can impact cell survival within anaerobic P. aeruginosa biofilms and can act synergistically with antibiotic treatment. Prior results demonstrated that under anoxic conditions, an electrode poised at sufficiently oxidizing potential (+100 mV vs Ag/AgCl) promotes EET within P. aeruginosa biofilms by re-oxidizing PYO for reuse by the cells. Here, when a reducing potential (-400 mV vs Ag/AgCl) was used to disrupt PYO redox cycling by maintaining PYO in the reduced state, we observed a 100-fold decrease in colony forming units within these biofilms compared with those exposed to electrodes poised at +100 mV vs Ag/AgCl. Phenazine-deficient Δphz* biofilms were unaffected by the potential applied to the electrode but were re-sensitized by adding PYO. The effect at -400 mV was exacerbated when biofilms were treated with sub-MICs of a range of antibiotics. Most notably, addition of the aminoglycoside gentamicin in a reductive environment almost completely eradicated wild-type biofilms but had no effect on the survival of Δphz* biofilms in the absence of phenazines. These data suggest that antibiotic treatment combined with the electrochemical disruption of PYO redox cycling, either through the toxicity of accumulated reduced PYO or the disruption of EET, or both, can lead to extensive killing. IMPORTANCE Biofilms provide a protective environment but also present challenges to the cells living within them, such as overcoming nutrient and oxygen diffusion limitations. Pseudomonas aeruginosa overcomes oxygen limitation by secreting soluble redox active phenazines, which act as electron shuttles to distal oxygen. Here, we show that electrochemically blocking the re-oxidation of one of these electron shuttles, pyocyanin, decreases cell survival within biofilms and acts synergistically with gentamicin to kill cells. Our results highlight the importance of the role that the redox cycling of electron shuttles fulfills within P. aeruginosa biofilms.

The ribonuclease PNPase is a key regulator of biofilm formation in Listeria monocytogenes and affects invasion of host cells

Biofilms provide an environment that protects microorganisms from external stresses such as nutrient deprivation, antibiotic treatments, and immune defences, thereby creating favorable conditions for bacterial survival and pathogenesis. Here we show that the RNA-binding protein and ribonuclease polynucleotide phosphorylase (PNPase) is a positive regulator of biofilm formation in the human pathogen Listeria monocytogenes, a major responsible for food contamination in food-processing environments. The PNPase mutant strain produces less biofilm biomass and exhibits an altered biofilm morphology that is more susceptible to antibiotic treatment. Through biochemical assays and microscopical analysis, we demonstrate that PNPase is a previously unrecognized regulator of the composition of the biofilm extracellular matrix, greatly affecting the levels of proteins, extracellular DNA, and sugars. Noteworthy, we have adapted the use of the fluorescent complex ruthenium red-phenanthroline for the detection of polysaccharides in Listeria biofilms. Transcriptomic analysis of wild-type and PNPase mutant biofilms reveals that PNPase impacts many regulatory pathways associated with biofilm formation, particularly by affecting the expression of genes involved in the metabolism of carbohydrates (e.g., lmo0096 and lmo0783, encoding PTS components), of amino acids (e.g., lmo1984 and lmo2006, encoding biosynthetic enzymes) and in the Agr quorum sensing-like system (lmo0048-49). Moreover, we show that PNPase affects mRNA levels of the master regulator of virulence PrfA and PrfA-regulated genes, and these results could help to explain the reduced bacterial internalization in human cells of the ΔpnpA mutant. Overall, this work demonstrates that PNPase is an important post-transcriptional regulator for virulence and adaptation to the biofilm lifestyle of Gram-positive bacteria and highlights the expanding role of ribonucleases as critical players in pathogenicity.

Bayesian estimation of Pseudomonas aeruginosa viscoelastic properties based on creep responses of wild type, rugose, and mucoid variant biofilms

Pseudomonas aeruginosa biofilms are relevant for a variety of disease settings, including pulmonary infections in people with cystic fibrosis. Biofilms are initiated by individual bacteria that undergo a phenotypic switch and produce an extracellular polymeric slime (EPS). However, the viscoelastic characteristics of biofilms at different stages of formation and the contributions of different EPS constituents have not been fully explored. For this purpose, we develop and parameterize a mathematical model to study the rheological behavior of three biofilms — P. aeruginosa wild type PAO1, isogenic rugose small colony variant (RSCV), and mucoid variant biofilms against a range of experimental data. Using Bayesian inference to estimate these viscoelastic properties, we quantify the rheological characteristics of the biofilm EPS. We employ a Monte Carlo Markov Chain algorithm to estimate these properties of P. aeruginosa variant biofilms in comparison to those of wild type. This information helps us understand the rheological behavior of biofilms at different stages of their development. The mechanical properties of wild type biofilms change significantly over time and are more sensitive to small changes in their composition than the other two mutants.

Mussel settlement mediated by bacterial VgrG proteins via extracellular outer membrane vesicles

T6SS plays an important role in biofilm formation, but how bacterial T6SS regulates biofilm formation and its effect on marine invertebrate settlement is still unclear. Here, the role of T6SS spike protein VgrG and VgrG protein synthesis gene on biofilm formation and Mytilus coruscus settlement was examined. Deletion of the core gene vgrG of T6SS resulted in a decrease in the growth capability of Leisingera aquaemixtae and inhibition of the ability of biofilm formation. The OMV content and inducing activity of ΔvgrG biofilms were decreased respectively by 20.72% and 59.70% compared with the wild-type biofilms. OMVs induced the settlement of M. coruscus, while VgrG proteins did not induce mussel settlement directly. Addition of VgrG proteins resulted in the upregulation of OMVs in the ΔvgrG biofilms. Adding OMVs caused no increasing inducing activity of ΔvgrG biofilms. Interestingly, the inducing activity of ΔvgrG biofilms both by adding VgrG proteins alone, and by adding VgrG proteins and OMVs in combination increased significantly, and restored to the inducing capability of wild-type biofilms. Thus, bacterial T6SS spike protein VgrG could mediate mussel settlement by increasing the OMV content of biofilms. The present findings shed new light on the interaction between T6SS and marine invertebrate settlement.

Comparative global gene expression analysis of biofilm forms of Salmonella Typhimurium ATCC 14028 and its seqA mutant

In this study, comparative transcriptomic analyzes (mRNA and miRNA) were performed on the biofilm forms of S. Typhimurium ATCC 14028 wild-type strain and its seqA gene mutant in order to determine the regulation characteristics of the seqA gene in detail. The results of global gene expression analyses showed an increase in the expression level of 54 genes and a decrease in the expression level of 155 genes (p < 0.05) in the seqA mutant compared to the wild-type strain. 10 of the 48 miRNAs identified on behalf of sequence analysis are new miRNA records for Salmonella. Transcripts of 14 miRNAs differed between wild-type strain and seqA mutant (p < 0.05), of which eight were up-regulated and six were down-regulated. Bioinformatic analyzes showed that differentially expressed genes in the wild-type strain and its seqA gene mutant play a role in different metabolic processes as well as biofilm formation, pathogenicity and virulence. When the transcriptomic data were interpreted together with the findings obtained from phenotypic tests such as motility, attachment to host cells and biofilm morphotyping, it was determined that the seqA gene has a critical function especially for the adhesion and colonization stages of biofilm formation, as well as for biofilm stability. Transcriptomic data pointing out that the seqA gene is also a general positive regulator of T3SS effector proteins active in cell invasion in S. Typhimurium wild-type biofilm, proves that this gene is involved in Salmonella host cell invasion.

Synergistic Interaction of Caspofungin Combined with Posaconazole against FKS Wild-Type and Mutant Candida auris Planktonic Cells and Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to cause biofilm-associated outbreaks, where frequently indwelling devices are the source of infections. The number of effective therapies is limited; thus, new, even-combination-based strategies are needed. Therefore, the in vitro efficacy of caspofungin with posaconazole against FKS wild-type and mutant Candida auris isolates was determined. The interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss model, and a LIVE/DEAD assay. Planktonic minimum inhibitory concentrations (pMICs) for the caspofungin-posaconazole combination showed a 4- to 256-fold and a 2- to 512-fold decrease compared to caspofungin and posaconazole alone, respectively. Sessile minimum inhibitory concentrations (sMICs) for caspofungin and posaconazole in combination showed an 8- to 128-fold and a 4- to 512-fold decrease, respectively. The combination showed synergy, especially against biofilms (FICIs were 0.033-0.375 and 0.091-0.5, and Bliss cumulative synergy volumes were 6.96 and 32.39 for echinocandin-susceptible and -resistant isolates, respectively). The caspofungin-exposed (4 mg/L) C. auris biofilms exhibited increased cell death in the presence of posaconazole (0.03 mg/L) compared to untreated, caspofungin-exposed and posaconazole-treated biofilms. Despite the favorable effect of caspofungin with posaconazole, in vivo studies are needed to confirm the therapeutic potential of this combination in C. auris-associated infections.

Quantifying biofilm propagation on chemically modified surfaces

Conditions affecting biofilm formation differ among bacterial species and this presents a challenge to studying biofilms in the lab. This work leverages functionalized silanes to control surface chemistry in the study of early biofilm propagation, quantified with a semi-automated image processing algorithm. These methods support the study of Pantoea sp. YR343, a gram-negative bacterium isolated from the poplar rhizosphere. We found that Pantoea sp. YR343 does not readily attach to hydrophilic surfaces but will form biofilms with a “honeycomb” morphology on hydrophobic surfaces. Our image processing algorithm described here quantified the evolution of the honeycomb morphology over time, and found the propagation to display a logarithmic behavior. This methodology was repeated with a flagella-deficient fliR mutant of Pantoea sp. YR343 which resulted in reduced surface attachment. Quantifiable differences between Pantoea WT and ΔfliR biofilm morphologies were captured by the image processing algorithm, further demonstrating the insight gained from these methods.

Impaired amino acid uptake leads to global metabolic imbalance of Candida albicans biofilms

Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development.

Streptococcus mutans PrsA mediates AtlA secretion contributing to extracellular DNA release and biofilm formation in the pathogenesis of infective endocarditis

ABSTRACT The role of secretion chaperone-regulated virulence proteins in the pathogenesis of infective endocarditis (IE) induced by viridans streptococci such as Streptococcus mutans is unclear. In this study, we investigated the contribution of the foldase protein PrsA, a putative parvulin-type peptidyl-prolyl isomerase, to the pathogenesis of S. mutans-induced IE. We found that a prsA-deficient strain had reduced virulence in terms of formation of vegetation on damaged heart valves, as well as reduced autolysis activity, eDNA release and biofilm formation capacity. The secretion and surface exposure of AtlA in vitro was reduced in the prsA-deficient mutant strain, and complementation of recombinant AtlA in the culture medium restored a wild type biofilm phenotype of the prsA-deficient mutant strain. This result suggests that secretion and surface localization of AtlA is regulated by PrsA during biofilm formation. Together, these results demonstrate that S. mutans PrsA could regulate AtlA-mediated eDNA release to contribute to biofilm formation in the pathogenesis of IE.

Direct microbial electron uptake as a mechanism for stainless steel corrosion in aerobic environments

Shewanella oneidensis MR-1 is an attractive model microbe for elucidating the biofilm-metal interactions that contribute to the billions of dollars in corrosion damage to industrial applications each year. Multiple mechanisms for S. oneidensis-enhanced corrosion have been proposed, but none of these mechanisms have previously been rigorously investigated with methods that rule out alternative routes for electron transfer. We found that S. oneidensis grown under aerobic conditions formed thick biofilms (∼50 µm) on stainless steel coupons, accelerating corrosion over sterile controls. H2 and flavins were ruled out as intermediary electron carriers because stainless steel did not reduce riboflavin and previous studies have demonstrated stainless does not generate H2. Strain ∆mtrCBA, in which the genes for the most abundant porin-cytochrome conduit in S. oneidensis were deleted, corroded stainless steel substantially less than wild-type in aerobic cultures. Wild-type biofilms readily reduced nitrate with stainless steel as the sole electron donor under anaerobic conditions, but strain ∆mtrCBA did not. These results demonstrate that S. oneidensis can directly consume electrons from iron-containing metals and illustrate how direct metal-to-microbe electron transfer can be an important route for corrosion, even in aerobic environments.

Staphylococcus aureus flavohaemoglobin contributes to early stage biofilm development under nitrosative stress.

Staphylococcus aureus is a Gram-positive bacterium with capacity to form biofilms, which constitute an important resistance mechanism and virulence factor. Flavohaemoglobin (Hmp) is a major nitric oxide (NO) detoxifier of several bacteria, including S. aureus. Although Hmp has a well-known physiological role linked to response of planktonic cells to nitrosative stress, its contribution to biofilm formation remains unaddressed. Hence, in this work, we investigated the role of Hmp in biofilm development of a methicillin-resistant S. aureus strain. For this purpose, we exposed the hmp mutant to nitrosative stress and examined its behaviour along biofilm development. We observed that cells inactivated in hmp and grown under nitrosative stress conditions have significantly impaired capacity to develop early stage biofilms. Furthermore, the wild-type biofilm phenotype was fully restored by trans-complementation of hmp in the hmp mutant. Coculture studies of NO-producing macrophages with S. aureus revealed that the hmp mutant has significantly lower capacity to develop biofilm biomass when compared with the wild type. Thus, we concluded that the pathogen S. aureus relies on Hmp to establish viable biofilms in the presence of cells of the host innate immune system.

Exopolysaccharides matrix affects the process of extracellular electron transfer in electroactive biofilm

The applications of bioelectrochemical systems (BESs) in the field of environment and energy are achieved through the bioelectrocatalytic process of electroactive biofilms. As a primary component of biofilm, the role of exopolysaccharides in electroactive biofilm in BESs is poorly understood. This study constructed an exopolysaccharides-deficient Geobacter sulfurreducens-based BES to explore the role of exopolysaccharides in electroactive biofilm. Compared with the wild type, the mutant biofilm expressing less exopolysaccharides decreased the capacity of current generation. In the mutant biofilm, the content of exopolysaccharides decreased significantly, resulting in a thinner biofilm and lower cell viability compared with the wild-type biofilm. However, the mutant with overexpressed pili developed a mature biofilm with extended time, which indicating the importance of exopolysaccharides for early biofilm formation and the compensatory role of pili in biofilm formation. The mutant biofilm had less content of c-type cytochromes (c-Cyts) and lower electrochemical activity of extracellular polymeric substances than the wild-type biofilm, suggesting a function of exopolysaccharides anchoring extracellular c-Cyts that essential to extracellular electron transfer (EET) in electroactive biofilms. Our findings demonstrated the essential role of exopolysaccharides in the process of EET in electroactive biofilm, which contributed to a better understanding and optimization of the performance of BESs.

Candida biofilm matrix as a resistance mechanism against photodynamic therapy

BackgroundAntimicrobial photodynamic therapy (aPDT) efficiency on Candida albicans is recognized in free-floating cultures. Though, the lack of aPDT effectiveness against C. albicans organized in biofilms is still unclear. This study aimed to explore the role of the extracellular matrix (ECM) in the protection against aPDT in C. albicans biofilms. MethodsC. albicans SN 425 wild-type and two mutant strains CNJ 2302; Δ/Δefg1 and CJN 2330; Δ/Δtec1 (ECM deficient) were used. Biofilms were grown on 24-well plates and exposed twice-daily to aPDT with 44 μM toluidine blue-O (TBO) for 5 min followed by red light (635 nm) for 1 min (87.6 J/cm²) or 2 min (175.2 J/cm2). Application of just TBO, light, 0.12% chlorhexidine, and ultrapure water were used as controls. After 48 h, biofilms were assessed for dry-weight (DW), colony forming units (CFU), extracellular DNA (eDNA), soluble and insoluble protein (SP/IP), water-insoluble (alkali-soluble) polysaccharide (ASP), water-soluble polysaccharides (WSP), and confocal scanning laser microscopy. ResultsThe strains with ECM deficient were affected by aPDT. For the mutant strain Δ/Δefg1, aPDT significantly reduced CFU, ASP, DW, eDNA, WSP and IP when compared to NC (p<0.001) and for the Δ/Δtec1, aPDT significantly reduced CFU, eDNA, IP and SP. Whereas CFU, DW, ASP of the wild-type strain biofilms were not reduced (p>0.05). ConclusionsC. albicans strains with reduced ECM compounds were more sensitive to aPDT suggesting that the ECM may have a significant protection role from aPDT in C. albicans biofilms.

The Effect of tonB Gene on the Virulence of Pseudomonas plecoglossicida and the Immune Response of Epinephelus coioides.

Pseudomonas plecoglossicida is the causative agent of “visceral white spot disease” in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides. To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB-RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB-RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida. Meanwhile, the E. coioides infected with the tonB-RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB-RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB-RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.