Biofilm State Research Articles

Synthesis, characterization, and antibacterial activities of a heteroscorpionate derivative platinum complex against methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the species with the greatest clinical importance and greatest impact on public health. In fact, methicillin-resistant S. aureus (MRSA) is considered a pandemic pathogen, being essential to develop effective medicines and combat its rapid spread. This study aimed to foster the translation of clinical research outcomes based on metallodrugs into clinical practice for the treatment of MRSA. Bearing in mind the promising anti-Gram-positive effect of the heteroscorpionate ligand 1,1'-(2-(4-isopropylphenyl)ethane-1,1-diyl)bis(3,5-dimethyl-1H-pyrazole) (2P), we propose the coordination of this compound to platinum as a clinical strategy with the ultimate aim of overcoming resistance in the treatment of MRSA. Therefore, the novel metallodrug 2P-Pt were synthetized, fully characterized and its antibacterial effect against the planktonic and biofilm state of S. aureus evaluated. In this sense, three different strains of S. aureus were studied, one collection strain of S. aureus sensitive to methicillin and two clinical MRSA strains. To appraise the antibacterial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) were determined. Moreover, successful outcomes on the development of biofilm in a wound-like medium were obtained. The mechanism of action for 2P-Pt was proposed by measuring the MIC and MBC with EDTA (cation mediated mechanism) and DMSO (exogenous oxidative stress mechanism). Moreover, to shed light on the plausible antistaphylococcal mechanism of this novel platinum agent, additional experiments using transmission electron microscopy were carried out. 2P-Pt inhibited the growth and eradicated the three strains evaluated in the planktonic state. Another point worth stressing is the inhibition in the growth of MRSA biofilm even in a wounded medium. The results of this work support this novel agent as a promising therapeutic alternative for preventing infections caused by MRSA.

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria package various cargo, including DNA that can be transferred to other bacteria or to host cells. OMV-associated DNA has been implicated in mediating horizontal gene transfer (HGT) between bacteria, which includes the dissemination of antibiotic resistance genes within and between bacterial species. Despite the known ability of OMVs to mediate HGT, the mechanisms of DNA packaging into OMVs remain poorly characterized, as does the effect of bacterial growth conditions on the DNA cargo composition of OMVs and their subsequent abilities to mediate HGT. In this study, we examined the DNA content of OMVs produced by the opportunistic pathogen Pseudomonas aeruginosa grown in either planktonic or biofilm conditions. Analysis of planktonic growth-derived OMVs revealed their ability to package and protect plasmid DNA from DNase degradation and to transfer plasmid-encoded antibiotic resistance genes to recipient, antibiotic-sensitive P. aeruginosa bacteria at a greater efficiency than transformation with plasmid alone. Comparisons of planktonic and biofilm-derived P. aeruginosa OMVs demonstrated that biofilm-derived OMVs were smaller but were associated with more plasmid DNA than planktonic-derived OMVs. Additionally, biofilm-derived P. aeruginosa OMVs were more efficient in the transformation of competent P. aeruginosa bacteria, compared to transformations with an equivalent number of planktonic-derived OMVs. The findings of this study highlight the importance of bacterial growth conditions for the packaging of DNA within P. aeruginosa OMVs and their ability to facilitate HGT, thus contributing to the spread of antibiotic resistance genes between P. aeruginosa bacteria. IMPORTANCE Bacterial membrane vesicles (BMVs) mediate interbacterial communication, and their ability to package DNA specifically contributes to biofilm formation, antibiotic resistance, and HGT between bacteria. However, the ability of P. aeruginosa OMVs to mediate HGT has not yet been demonstrated. Here, we reveal that P. aeruginosa planktonic and biofilm-derived OMVs can deliver plasmid-encoded antibiotic resistance to recipient P. aeruginosa. Additionally, we demonstrated that P. aeruginosa biofilm-derived OMVs were associated with more plasmid DNA compared to planktonic-derived OMVs and were more efficient in the transfer of plasmid DNA to recipient bacteria. Overall, this demonstrated the ability of P. aeruginosa OMVs to facilitate the dissemination of antibiotic resistance genes, thereby enabling the survival of susceptible bacteria during antibiotic treatment. Investigating the roles of biofilm-derived BMVs may contribute to furthering our understanding of the role of BMVs in HGT and the spread of antibiotic resistance in the environment.

Different cellular fatty acid pattern and gene expression of planktonic and biofilm state Listeria monocytogenes under nutritional stress

Listeria monocytogenes is a Gram-positive bacterium frequently involved in food-borne disease outbreaks and is widely distributed in the food-processing environment. This work aims to depict the impact of nutrition deficiency on the survival strategy of L. monocytogenes both in planktonic and biofilm states. In the present study, cell characteristics (autoaggression, hydrophobicity and motility), membrane fatty acid composition of MRL300083 (Lm83) in the forms of planktonic and biofilm-associated cells cultured in TSB-YE and 10-fold dilutions of TSB-YE (DTSB-YE) were investigated. Additionally, the relative expression of related genes were also determined by RT-qPCR. It was observed that cell growth in different bacterial life modes under nutritional stress rendered the cells a distinct phenotype. The higher autoaggression (AAG) and motility of the planktonic cells in DTSB-YE is associated with better biofilm formation. An increased proportion of unsaturated fatty acid/saturated fatty acid (USFA/SFA) indicates more fluidic biophysical properties for cell membranes of L. monocytogenes in planktonic and biofilm cells in DTSB-YE. Biofilm cells produced a higher percentage of USFA and straight fatty acids than the corresponding planktonic cells. An appropriate degree of membrane fluidity is crucial for survival, and alteration of membrane lipids is an essential adaptive response. The adaptation of bacteria to stress is a multifactorial cellular process, the expression of flagella-related genes fliG, fliP, flgE and the two-component chemotactic system cheA/Y genes of planktonic cells in DTSB-YE significantly increased compared to that in TSB-YE (p < 0.05). This study provides new information on the role of the physiological adaptation and gene expression of L. monocytogenes for planktonic and biofilm growth under nutritional stress.

Comparison of antibiofilm activity of low-concentrated hypochlorites vs polyhexanide-containing antiseptic.

Chronic wound infection is highly associated with morbidity and endangers the patient's life. Therefore, wound care products must have a potent antimicrobial and biofilm-eradicating effect. In this work, the antimicrobial/antibiofilm activity of two low-concentrated chlorine-based and releasing solutions was investigated on a total of 78 strains of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, using the cohesive spectrum of in vitro settings, including microtiter plate models, biofilm-oriented antiseptic test, cellulose-based biofilm model, biofilm bioreactors and Bioflux model. The antiseptic containing polyhexamethylene biguanide was used in the character of usability control of performed tests. The results obtained by static biofilm models indicate that low-concentrated chlorine-based and releasing solutions display none to moderate antibiofilm activity, while data obtained by means of the Bioflux model, providing flow conditions, indicate the moderate antibiofilm activity of substances compared with the polyhexanide antiseptic. Considering in vitro data presented in this manuscript, the earlier reported favorable clinical results of low-concentrated hypochlorites should be considered rather an effect of their rinsing activity combined with low cytotoxicity but not the antimicrobial effect per se. For the treatment of heavily biofilm-infected wounds, polyhexanide should be considered the agent of choice because of its higher efficacy against pathogenic biofilms.

Synergistic antibacterial and biofilm eradication activity of quaternary-ammonium compound with copper ion

Antibiotics overuse and misuse increase the emergence of multidrug-resistant bacterial strains, which often leads to the failure of conventional antibiotic therapies. Even worse, the tendency of bacteria to form biofilms further increases the therapeutic difficulty, because the extracellular matrix prevents the penetration of antibiotics and triggers bacterial tolerance. Therefore, developing novel antibacterial agents or therapeutic strategies with diverse antibacterial mechanisms and destruction of bacteria biofilm is a promising way to combat bacterial infections. In the present study, the combination of quaternary ammonium compound poly(diallyl dimethyl ammonium chloride) (PDDA) with Cu2+ was screened out to fight common pathogenic Staphylococcus aureus (S. aureus) through multi-mechanisms. This combination appeared strong synergistic antibacterial activity, and the fractional inhibitory concentration index was as low as 0.032. The synergistic antibacterial mechanism involved the destruction of the membrane function, generation of intracellular reactive oxygen, and promotion more Cu2+ into the cytoplasm. Further, the combination of PDDA and Cu2+ reduced the extracellular polysaccharide matrix, meanwhile killing the bacteria embedded in the biofilm. The biocompatibility study in vitro revealed this combination exhibited low cytotoxicity and hemolysis ratio even at 8 times of minimum bactericidal concentration. This work provides a novel antibacterial agents combination with higher efficiency to fight planktonic and biofilm conditions of S. aureus.

Susceptibility and transcriptomic response to plasma-activated water of Listeria monocytogenes planktonic and sessile cells

Plasma-Activated Water (PAW) was generated from tap water using a surface dielectric barrier discharge at different discharge power (26 and 36 W) and activation time (5 and 30 min). The inactivation of a three-strain Listeria monocytogenes cocktail in planktonic and biofilm state was evaluated. PAW generated at 36 W-30 min showed the lowest pH and the highest hydrogen peroxide, nitrates, nitrites contents and effectiveness against cells on planktonic state, resulting in 4.6 log reductions after a 15-min treatment. Although the antimicrobial activity in biofilms formed on stainless steel and on polystyrene was lower, increasing the exposure time to 30 min allowed an inactivation >4.5 log cycles. The mechanisms of action of PAW were investigated using chemical solutions that mimic its physico-chemical characteristics and also RNA-seq analysis. The main transcriptomic changes affected carbon metabolism, virulence and general stress response genes, with several overexpressed genes belonging to the cobalamin-dependent gene cluster.

ApoE Mimetic Peptide COG1410 Exhibits Strong Additive Interaction with Antibiotics Against Mycobacterium smegmatis.

Drug-resistant tuberculosis (TB) is an emerging threat to public health worldwide. Antimicrobial peptide (AMP) is a promising solution to solve the antimicrobial resistance crisis. The apolipoprotein E mimetic peptide COG1410 has been confirmed to simultaneously have neuroprotective, anti-inflammatory, and antibacterial activity. However, whether it is effective to inhibit growth of mycobacteria has not been investigated yet. The peptide COG1410 was synthesized with conventional solid-phase peptide synthesis and qualified by HPLC and mass spectrometry. Micro-dilution method was used to determine the minimal inhibitory concentration. A time-kill assay was used to determine the bactericidal dynamics of antimicrobial peptide and relative antibiotics. Static biofilm formation was conducted in 24-well plate and the biofilm was separated from planktonic cells and collected. The mechanism of action of COG1410 was explored by TEM observation and ATP leak assay. The localization of COG1410 was observed by confocal laser scan microscopy. The drug-drug interaction was determined by a checkerboard assay. COG1410 was a potent bactericidal agent against M. smegmatis in vitro and within the macrophages with MIC 16 μg/mL, but invalid against M. abscess and M. tuberculosis. A time-kill assay showed that COG1410 killed M. smegmatis as potent as clarithromycin, but faster than LL-37, another short synthetic cationic peptide. 1× MIC COG1410 almost reduced 90% biofilm formation of M. smegmatis. Additionally, COG1410 was able to penetrate the cell membrane of macrophage and inhibit intracellular M. smegmatis growth. TEM observation and ATP leak assay found that COG1410 disrupted cell membrane and caused release of cell contents. Confocal fluorescence microscopy showed that FITC-COG1410 aggregated around cell membrane instead of entering the cytoplasm. Although COG1410 had relative high cytotoxicity, it exhibited strong additive interaction with regular anti-TB antibiotics, which reduced the working concentration of COG1410 and expanding safety window. After 30 passages, there was no induced drug resistance for COG1410. COG1410 was a novel and potent AMP against M. smegmatis by disrupting the integrity of cell membrane.

Differential Gene Expression of Peroxide Resistant Protein (AhpC) in Planktonic and Biofilm State of Uropathogenic Escherichia coli Cells

Differential Gene Expression of Peroxide Resistant Protein (AhpC) in Planktonic and Biofilm State of Uropathogenic Escherichia coli Cells

Deciphering Target Protein Cascade in Salmonella typhi Biofilm using Genomic Data Mining, and Protein-protein Interaction.

Salmonella typhi biofilm confers a serious public health issue for lengthy periods and the rise in antibiotic resistance and death rate. Biofilm generation has rendered even the most potent antibiotics ineffective in controlling the illness, and the S. typhi outbreak has turned into a fatal disease typhoid. S. typhi infection has also been connected to other deadly illnesses, such as a gall bladder cancer. The virulence of this disease is due to the interaction of numerous genes and proteins of S. typhi. The study aimed to identify a cascade of target proteins in S. typhi biofilm condition with the help of genomic data mining and protein-protein interaction analysis. The goal of this study was to notice some important pharmacological targets in S. typhi. using genomic data mining, and protein-protein interaction approaches were used so that new drugs could be developed to combat the disease. In this study, we identified 15 potential target proteins that are critical for S. typhi biofilm growth and maturation. Three proteins, CsgD, AdrA, and BcsA, were deciphered with their significant role in the synthesis of cellulose, a critical component of biofilm's extracellular matrix. The CsgD protein was also shown to have high interconnectedness and strong interactions with other important target proteins of S. typhi. As a result, it has been concluded that CsgD is involved in a range of activities, including cellulose synthesis, bacterial pathogenicity, quorum sensing, and bacterial virulence. All identified targets in this study possess hydrophobic properties, and their cellular localization offered proof of a potent therapeutic target. Overall results of this study, drug target shortage in S. typhi is also spotlighted, and we believe that obtained result could be useful for the design and development of some potent anti-salmonella agents for typhoid fever in the future.

Feasibility of biofilm production capacity by Levilactobacillus brevis isolated from motal cheese and evaluation of biofilm resistance produced in vitro and in yogurt

Traditional dairy products are a unique source which have been considered for the extraction of indigenous probiotic strains in recent years. In this study, biofilm formation power of Levilactobacillus brevis that isolated from Mutal traditional cheese were investigated. Survival was assessed during 21 days of storage time and at the presence of residues antibiotics as well as gastrointestinal conditions. The results showed after 120 min of treatment in high acidic conditions (pH 2.0), the survival rate decreased only 0.75 log CFU/mL in biofilm formed. The antibiotic susceptibility evaluated of probiotic to enrofloxacin, sulfadimidine, tetracycline, and oxytetracycline showed reducing the bacterial population in the biofilm form only 2.6 log. probiotic strain that isolated from indigenous dairy sources showed excellent resistance in the biofilm state. Therefore, extracting strong probiotic strains from indigenous resources, it can significantly improve functional products and fermentory engineering.

In silico molecular modelling studies and antibiofilm efficacy of shikonin against Candida albicans: mechanistic insight

In the recent past, the occurrence of fungal infections has increased drastically and candidiasis, caused prominently by Candida albicans, is foremost among them which has caused significant mortality and morbidity majorly in immune-compromised patients. Shikonin is a well-known natural naphthazarin derivative with promising antifungal efficacy, but it's mechanism of action is still unclear. Keeping this in view, present workwas designed to get a mechanistic insight of anti-candida efficacy of shikonin via in vitro experiments and in situ molecular modelling studies. The current exploratory study is based on research that uses both qualitative and quantitative techniques, including minimum inhibitory concentration, minimum biofilm inhibitory concentration, time kill assay, cell cycle analysis and apoptotic assays, static biofilm formation assays, microscopic biofilm assessment assays, ergosterol content estimation and molecular docking/simulation studies. The study revealed a notable effect of shikonin against Candida albicans, including retardation of biofilms. Shikonin, with its increasing concentration leads to candidal cell apoptosis and necrosis establishing its dose-dependent effect. Additionally, it exhibited fungicidal activity via a mechanism of action likely related to ergosterol complexation which was further corroborated by molecular docking and simulation studies.

Hgc1 Independence of Biofilm Hyphae in Candida albicans.

Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

Phage and Antibiotic Combinations Reduce Staphylococcus aureus in Static and Dynamic Biofilms Grown on an Implant Material.

Staphylococcus aureus causes the majority of implant-related infections. These infections present as biofilms, in which bacteria adhere to the surface of foreign materials and form robust communities that are resilient to the human immune system and antibiotic drugs. The heavy use of broad-spectrum antibiotics against these pathogens disturbs the host's microbiome and contributes to the growing problem of antibiotic-resistant infections. The use of bacteriophages as antibacterial agents is a potential alternative therapy. In this study, bioluminescent strains of S. aureus were grown to form 48-h biofilms on polyether ether ketone (PEEK), a material used to manufacture orthopaedic implants, in either static or dynamic growth conditions. Biofilms were treated with vancomycin, staphylococcal phage, or a combination of the two. We showed that vancomycin and staph phages were able to independently reduce the total bacterial load. Most phage-antibiotic combinations produced greater log reductions in surviving bacteria compared to single-agent treatments, suggesting antimicrobial synergism. In addition to demonstrating the efficacy of combining vancomycin and staph phage, our results demonstrate the importance of growth conditions in phage-antibiotic combination studies. Dynamic biofilms were found to have a substantial impact on apparent treatment efficacy, as they were more resilient to combination treatments than static biofilms.

Dual Anticancer and Antibacterial Properties of Silica-Based Theranostic Nanomaterials Functionalized with Coumarin343, Folic Acid and a Cytotoxic Organotin(IV) Metallodrug.

Five different silica nanoparticles functionalized with vitamin B12, a derivative of coumarin found in green plants and a minimum content of an organotin(IV) fragment (1-MSN-Sn, 2-MSN-Sn, 2-SBA-Sn, 2-FSPm-Sn and 2-FSPs-Sn), were identified as excellent anticancer agents against triple negative breast cancer, one of the most diagnosed and aggressive cancerous tumors, with very poor prognosis. Notably, compound 2-MSN-Sn shows selectivity for cancer cells and excellent luminescent properties detectable by imaging techniques once internalized. The same compound is also able to interact with and nearly eradicate biofilms of Staphylococcus aureus, the most common bacteria isolated from chronic wounds and burns, whose treatment is a clinical challenge. 2-MSN-Sn is efficiently internalized by bacteria in a biofilm state and destroys the latter through reactive oxygen species (ROS) generation. Its internalization by bacteria was also efficiently monitored by fluorescence imaging. Since silica nanoparticles are particularly suitable for oral or topical administration, and considering both its anticancer and antibacterial activity, 2-MSN-Sn represents a new dual-condition theranostic agent, based primarily on natural products or their derivatives and with only a minimum amount of a novel metallodrug.

Review on the Role of Biofilm Formation in Bacterial Pathogenesis

Biofilms are defined as a accumulation of microorganisms living in a self-assembled matrix of polymeric substances that adheres to a variety of surfaces. Depending on the interaction between the surface and constituent cells, biofilms can be monolayer or multilayer. Initial attachment, irreversible attachment, micro colony growth, biofilm maturation, and biofilm dispersal are the five steps of the biofilm formation process. The processes that contribute tobiofilm formation are a dynamic and complicated process. Biofilm Formation Causes many bacteria have the ability to transition between planktonic and biofilm forms. The biofilm state appears to be a natural and prevalent condition of bacteria. Biofilm-forming bacteria are important for animal health. The risk of infection in animal species is likely to be higher than in humans. Because of the differences in animal housing and living conditions. Microorganisms capable of generating biofilms include P. aeruginosa, S. epidermidis, E. colispp, S. aureus, E. cloacae, and K. pneumonia. Bacterial pathogenesis of biofilms occurs frequently on medical devices and bits of dead tissue, such as sequestra of dead bone; nevertheless, they canals of or nonliving tissues. Biofilms in bacterial infections can improve the pathogenicity of the organism while also protecting it from external treatment. UV radiation, pH stress, chemical exposure, phagocytosis, dehydration, and antibiotics are all threats to cells that biofilms protect them. The Role of Signaling in Biofilm Formation is the bond between environmental stimuli and the bacteria’ reciprocation of the relevant signaling is required for the formation of a biofilm. Two-component systems and Quorum sensing can both be induced by these sensing systems. Tissue culture plate, cong red agar, and bioluminescent assay are some of the laboratory tests used to diagnose biofilm. The emergence and spread of antibiotic resistance among bacteria is one of the world’s most serious health issues. One of the implications of the bacterial biofilm communities that lead to persistent illnesses is antibiotic resistance.

Liquid infused silicon based urinary catheters: A simple laboratory model to study bacterial biofilm formation

Biofilms are organized communities of micro-organisms buried firmly on an extra polymeric matrix attached to the living surface. The matrix which is formed surrounding the bacteria makes them tolerant to harsh environmental conditions as well as resistant to antibacterial treatment. The development of biofilms in clinical or hospital settings is the major reason for several infections. They have a negative impact when they are formed on medical devices as it is difficult to eradicate them using disinfectants or antibiotics available. Several in vitro models have been implemented in the last decade to evaluate antimicrobial activity on bacterial biofilms. Although well-established static biofilm methods are available; there is a lack of simple dynamic biofilm models to replicate the clinical settings of biofilm development. In this scenario, considering the importance of this research area, we planned to design laboratory-scale working models to study biofilms. We used Pseudomonas aeruginosa as a model organism. We divided the biofilm models into two categories namely static and dynamic models. The dynamic model of biofilm, that we used for mimicking the clinical setting, was a silicone-based catheter model. This new model will contribute to deeper knowledge about the physiology, structure, formation, and composition of biofilms. This method can be used as an appropriate and up-to-date technique to study biofilms in laboratory settings.

Inactivation of Staphylococcus aureus by Levulinic Acid Plus Sodium Dodecyl Sulfate and their Antibacterial Mechanisms on S. aureus Biofilms by Transcriptomic Analysis

The combination of levulinic acid (LVA) and sodium dodecyl sulfate (SDS) in recent years has shown a considerable potential to use as an antimicrobial intervention. The objectives of this study were to evaluate the antimicrobial efficacy of the combination against Staphylococcus aureus in both planktonic and biofilm states and to investigate the transcriptional changes in S. aureus biofilms coincubated with sublethal concentrations of LVA and/or SDS. The minimum inhibitory concentrations (MICs) of LVA and SDS determined by the microdilution method were 3.125 and 0.039 mg/mL, respectively. An additive bacteriostatic interaction (fractional inhibitory concentration index = 1) between the two compounds was observed by the checkerboard assay, whereas a synergistic bactericidal activity was displayed by the time-kill assay. The biomass and viable cells in the biofilms were reduced by both antimicrobials either alone or in combination in a dose-dependent manner. Transcriptomics indicated that more differentially expressed (DE) genes were observed in the biofilm treated with SDS (103 up- and 205 downregulated DE genes) and LVA + SDS (187 up and 162 down) than that coincubated with LVA (34 up and 32 down). The SDS and LVA + SDS treatments mainly affected the expression of genes responsible for cell surface proteins, virulence factors, adhesins, and capsular polysaccharides. Both the antibiofilm assay and the transcriptomics indicated that SDS, not LVA, was the major chemical contributing to the antibacterial efficacy of the combination. This study reveals the behavioral responses and protective mechanisms of S. aureus to LVA and SDS applied individually or in combination.

Two novel genes identified by large-scale transcriptomic analysis are essential for biofilm and rugose colony development of Vibrio vulnificus.

Many pathogenic bacteria form biofilms to survive under environmental stresses and host immune defenses. Differential expression (DE) analysis of the genes in biofilm and planktonic cells under a single condition, however, has limitations to identify the genes essential for biofilm formation. Independent component analysis (ICA), a machine learning algorithm, was adopted to comprehensively identify the biofilm genes of Vibrio vulnificus, a fulminating human pathogen, in this study. ICA analyzed the large-scale transcriptome data of V. vulnificus cells under various biofilm and planktonic conditions and then identified a total of 72 sets of independently co-regulated genes, iModulons. Among the three iModulons specifically activated in biofilm cells, BrpT-iModulon mainly consisted of known genes of the regulon of BrpT, a transcriptional regulator controlling biofilm formation of V. vulnificus. Interestingly, the BrpT-iModulon additionally contained two novel genes, VV1_3061 and VV2_1694, designated as cabH and brpN, respectively. cabH and brpN were shared in other Vibrio species and not yet identified by DE analyses. Genetic and biochemical analyses revealed that cabH and brpN are directly up-regulated by BrpT. The deletion of cabH and brpN impaired the robust biofilm and rugose colony formation. CabH, structurally similar to the previously known calcium-binding matrix protein CabA, was essential for attachment to the surface. BrpN, carrying an acyltransferase-3 domain as observed in BrpL, played an important role in exopolysaccharide production. Altogether, ICA identified two novel genes, cabH and brpN, which are regulated by BrpT and essential for the development of robust biofilms and rugose colonies of V. vulnificus.

A thiamine transporter is required for biofilm formation by Rhizobium sp. IRBG74.

Rhizobium sp. IRBG74 is a nitrogen fixing symbiont of Sesbania cannabina and a growth promoting endophyte of rice thus making it a good model to compare rhizobial interactions with legumes and cereals. In this report, we show that Rhizobium sp. IRBG74 forms biofilms on the roots of S. cannabina and rice. A mutant defective in biofilm formation was identified by screening a transposon mutant library. The transposon insertion was in thiQ, part of the thiBPQ operon that encodes the components of a thiamine/thiamine pyrophosphate ABC transporter. Complementation with thiBPQ partially restored biofilm formation. Addition of thiamine in growth media led to repression of thiC expression in the wild type strain but not in the thiQ mutant. These results suggest that thiBPQ is involved in thiamine/TPP transport in Rhizobium sp. IRBG74. Using a GUS reporter, we show that the expression of thiC is significantly higher in biofilm as compared to cells in planktonic growth. Based on these results we propose that Rhizobium sp. IRBG74 is thiamine limited and requires thiamine transport for efficient biofilm formation and plant colonization. Thiamine synthesis in aerobic bacteria such as Rhizobium requires O2 and thus could be inhibited in the microaerobic/anaerobic conditions in biofilms.

The impact of osmotic stresses on the biofilm formation, immunodetection, and morphology of Aeromonas hydrophila

Aeromonas hydrophila (Ah) is a zoonotic pathogen of great importance to aquaculture and human health. This study systematically evaluated the impact of salinity, sugar, ammonia nitrogen, and nitric nitrogen levels on the fitness of Ah by using Luria-Bertani (LB) broth supplemented with different concentrations of NaCl, sucrose, NH4Cl, urea, NaNO2 or NaNO3. Results showed that the static biofilm formation of Ah was higher at 28 °C compared to 37 °C (P < 0.05). At 28 °C, as the NaCl (>1 %) and sucrose levels increased, the Ah biofilm formation and the binding between Ah cells and monoclonal antibodies (mAbs, for immunodetection) decreased. Elevated ammonia nitrogen and nitric nitrogen levels generated no significant impact on Ah biofilm formation or immunodetection (P > 0.05). The expression of mAbs-targeted Omp remained unchanged under high NaCl or sucrose conditions. Further analysis showed that high sucrose conditions led to the over-expression of the extracellular polysaccharides (PS) and promoted the formation of capsule-like structures. These over-expressed PS and capsule structures might be one reason explaining the inhibited immunodetection efficacy. Results generated from this study provide crucial insights for the design of recovery and detection protocols for Ah present in food or environmental samples.