Biofilm Response Research Articles

Effects of electrolysed water combined with ultrasound on inactivation kinetics and metabolite profiles of Escherichia coli biofilms on food contact surface

This study aimed to better understand the overall metabolic responses of Escherichia coli biofilms to the combined stresses of ultrasound and low concentration acidic electrolysed water (LcAEW, free available chlorine: 4 mg/L). The inactivation kinetics of all E. coli strains (ATCC 25922, ATCC 35150 and ATCC 43895) were simulated well by the modified Weibull model (R2: 0.81–0.97; RMSE: 0.04–0.71). By analysing metabolite profiles, ultrasound mainly disrupted nucleotide metabolism in E. coli cells within biofilms, as most intracellular nucleotide-related compounds (e.g., uridine, ATP, ADP) showed decreased trend especially in ATCC 25922 and ATCC 35150. Increased contents of most amino acids and decreased contents of most carbohydrates were shown in all strains after LcAEW treatment. Under combined treatment, adaptive strategies like glutamate decarboxylase system and mixed acid fermentation were activated to different extents among the three strains. These findings revealed that NMR-based metabolomics technology is promising in identifying strain-specific metabolic responses of biofilms to different antimicrobial treatments, providing guidance for future mechanism studies related to food contact surface sanitisation.

H-NOX proteins in the virulence of pathogenic bacteria.

Nitric oxide (NO) is a toxic gas encountered by bacteria as a product of their own metabolism or as a result of a host immune response. Non-toxic concentrations of NO have been shown to initiate changes in bacterial behaviors such as the transition between planktonic and biofilm-associated lifestyles. The heme nitric oxide/oxygen binding proteins (H-NOX) are a widespread family of bacterial heme-based NO sensors that regulate biofilm formation in response to NO. The presence of H-NOX in several human pathogens combined with the importance of planktonic–biofilm transitions to virulence suggests that H-NOX sensing may be an important virulence factor in these organisms. Here we review the recent data on H-NOX NO signaling pathways with an emphasis on H-NOX homologs from pathogens and commensal organisms. The current state of the field is somewhat ambiguous regarding the role of H-NOX in pathogenesis. However, it is clear that H-NOX regulates biofilm in response to environmental factors and may promote persistence in the environments that serve as reservoirs for these pathogens. Finally, the evidence that large subgroups of H-NOX proteins may sense environmental signals besides NO is discussed within the context of a phylogenetic analysis of this large and diverse family.

Self-targeting of zwitterion-based platforms for nano-antimicrobials and nanocarriers.

Self-targeting antimicrobial platforms have yielded new possibilities for the treatment of infectious biofilms. Self-targeting involves stealth transport through the blood circulation towards an infectious biofilm, where the antimicrobial platform penetrates and accumulates in a biofilm in response to a change in environmental conditions, such as local pH. In a final step, nano-antimicrobials need to be activated or the antimicrobial cargo of nanocarriers released. Zwitterions possess both cationic and anionic groups, allowing full reversal in zeta potential from below to above zero in response to a change in environmental conditions. Electrolyte-based platforms generally do not have the ability to change their zeta potentials from below to above zero. Zwitterions for use in self-targeting platforms are usually hydrophilic and have a negative charge under physiological conditions (pH 7.4) providing low adsorption of proteins and assisting blood circulation. However, near or in the acidic environment of a biofilm, they become positively-charged yielding targeting, penetration and accumulation in the biofilm through electrostatic double-layer attraction to negatively-charged bacteria. Response-times to pH changes vary, depending on the way the zwitterion or electrolyte is built in a platform. Self-targeting zwitterion-based platforms with a short response-time in vitro yield different accumulation kinetics in abdominal biofilms in living mice than platforms with a longer response-time. In vivo experiments in mice also proved that self-targeting, pH-responsive zwitterion-based platforms provide a feasible approach for clinical control of bacterial infections. Clinically however, also other conditions than infection may yield an acidic environment. Therefore, it remains to be seen whether pH is a sufficiently unique recognition sign to direct self-targeting platforms to an infectious biofilm or whether (additional) external targeting through e.g. near-infrared irradiation or magnetic field application is needed.

Electrochemical Response of Electroactive Biofilms from Real Wastewater to Metal Ion Shock

Electrochemical Response of Electroactive Biofilms from Real Wastewater to Metal Ion Shock

Functional Responses of Biofilm and Hydrophyte To Silver Nanoparticles Varied with Exposure Duration in Constructed Wetlands

Functional Responses of Biofilm and Hydrophyte To Silver Nanoparticles Varied with Exposure Duration in Constructed Wetlands

Metabolomics insight into the influence of environmental factors in responses of freshwater biofilms to the model herbicide diuron.

Freshwater biofilms have been increasingly used during the last decade in ecotoxicology due to their ecological relevance to assess the effect(s) of environmental stress at the community level. Despite growing knowledge about the effect of various stressors on the structure and the function of these microbial communities, a strong research effort is still required to better understand their response to chemical stress and the influence of environmental stressors in this response. To tackle this challenge, untargeted metabolomics is an approach of choice because of its capacity to give an integrative picture of the exposure to multiple stress and associated effect as well as identifying the molecular pathways involved in these responses. In this context, the present study aimed to explore the use of an untargeted metabolomics approach to unravel at the molecular/biochemical level the response of the whole biofilm to chemical stress and the influence of various environmental factors in this response. To this end, archived high-resolution mass spectrometry data from previous experiments at our laboratory on the effect of the model photosynthesis inhibitor diuron on freshwater biofilm were investigated by using innovative solutions for OMICs data (e.g., DRomics) and more usual chemometric approaches (multivariate and univariate statistical analyses). The results showed a faster (1 min) and more sensitive response of the metabolome to diuron than usual functional descriptors, including photosynthesis. Also, the metabolomics response to diuron resulted from metabolites following various trends (increasing, decreasing, U/bell shape) along increasing concentration and time. This metabolomics response was influenced by the temperature, photoperiod, and flow. A focus on a plant-specific omega-3 (eicosapentaenoic acid) playing a key role in the trophic chain highlighted the potential relevance of metabolomics approach to establish the link between molecular alteration and ecosystem structure/functioning impairment but also how complex is the response and the influence of all the tested factors on this response at the metabolomics level. Altogether, our results underline that more fundamental researches are needed to decipher the metabolomics response of freshwater biofilm to chemical stress and its link with physiological, structural, and functional responses toward the unraveling of adverse outcome pathways (AOP) for key ecosystem functions (e.g., primary production).

New Frontiers on Adjuvants Drug Strategies and Treatments in Periodontitis

Causes of the progression of periodontitis such as an imbalance between the immune response by the host by the release of inflammatory mediators in the response of the oral pathogenic dysbiotic biofilm have been identified. New insights on specific cell signaling pathways that appear during periodontitis have attracted the attention of researchers in the study of new personalised approaches for the treatment of periodontitis. The gold standard of non-surgical therapy of periodontitis involves the removal of supra and subgingival biofilm through professional scaling and root planing (SRP) and oral hygiene instructions. In order to improve periodontal clinical outcomes and overcome the limitations of traditional SRP, additional adjuvants have been developed in recent decades, including local or systemic antibiotics, antiseptics, probiotics, anti-inflammatory and anti-resorptive drugs and host modulation therapies. This review is aimed to update the current and recent evolution of therapies of management of periodontitis based on the adjunctive and target therapies. Moreover, we discuss the advances in host modulation of periodontitis and the impact of targeting epigenetic mechanisms approaches for a personalised therapeutic success in the management of periodontitis. In conclusion, the future goal in periodontology will be to combine and personalise the periodontal treatments to the colonising microbial profile and to the specific response of the individual patient.

Architecture and physico-chemical properties of Bacillus amyloliquefaciens L-17 pellicle formed at the air–liquid interface

Bacillus amyloliquefaciens is a ubiquitous soil and plant-associated bacterial species which shows structural and adaptative responses to the environment. This present paper explores the ability of the strain L-17 to form subaerial biofilms on a liquid surface. Hydrophobic and non-wetting properties were observed for the rough top biofilm layer in contact with the air, which are quite different to the hydrophilic properties which were observed for the smooth biofilm layer in contact with the liquid. Both pellicle interfaces were visualized by scanning electron microscopy revealing a complex three-dimensional architecture composed of exopolymers organized in stacked fibrous network or sheet-like structures in the vicinity of the subaerial surface. Disruption of the extracellular matrix by combining physical and chemical treatments indicated that both loosely and tightly bound polysaccharides were found as major components of this complex pellicle. Proteins were also involved in the aggregation and cohesion of the matrix as multi extraction steps were needed to recover some tightly bounded proteins. This was confirmed by applying protease treatment which was able to significantly disrupt the pellicle. Overall results underline the ability of B.amyloliquefaciens L-17 to survive on air-liquid interfaces. This feature offers an interesting strategy to escape aquatic environments and develop aerial biofilm in response to environmental changes involving wet-dry cycles.

Structural and Functional Characteristics of Microplastic Associated Biofilms in Response to Temporal Dynamics and Polymer Types

The colonization of bacterial communities and biofilm formation on microplastics (MPs) have aroused great concern recently. However, the influence of time and polymer types on the structural and functional characteristics of biofilms remains unclear. In this study, three types of MPs (polyethylene, polypropylene, and polystyrene) were exposed for different time periods (10, 20 and 30days) in seawater using a microcosm experiment. Microscopic spectroscopy and high-throughput gene sequencing techniques were used to reveal the temporal changes of structural and functional characteristics of MPs associated biofilms. The results indicate that the biofilm formation is affected by both the incubation time and the polymer type. In addition, bacterial diversity and community structure in the biofilms show selectivity towards seawater, and tend to shift over time and among different polymer types. Moreover, biofilms are shown to harbor plastic degrading bacteria, leading to the changes of functional groups and surface hydrophobicity, and thereby enhancing the biodegradation of MPs.

Responses of electroactive biofilms to chronic chlorine exposure: Insights from the composition and spatial structure of extracellular polymeric substances

Extensive amounts of chlorine disinfectants have been applied to wastewater system since the outbreak of coronavirus disease 2019 (COVID-19), which inevitably affects the pollutant degradation via interfering with electron transfer mediated by electroactive bacteria. Herein, the response of electroactive biofilm (EAB) to chronic chlorine exposure was investigated. Results showed the EAB formed without exposure (EAB-0) exhibited a 53% and 123% higher current output than that formed with 0.1 mg L−1 (EAB-0.1) and 0.5 mg L−1 (EAB-0.5) chlorine, respectively. The chronic chlorine exposure of EAB boosted the contents of extracellular polymeric substances (EPS) in EAB-0.1 and EAB-0.5 by over secretion of extracellular polysaccharides. The EAB-0.1 and EAB-0.5 also presented lower electron exchange capacities (EECs) of EPS, coincided with reduced relative abundance of Geobacter from 61% in EAB-0 to 52% in EAB-0.5. This study provided new insights into the application of engineered EAB for wastewater treatment in a disinfection environment.

Electrochemical Sensing and Characterization of Aerobic Marine Bacterial Biofilms on Gold Electrode Surfaces.

Reliable and accurate in situ sensors capable of detecting and quantifying troublesome marine biofilms on metallic surfaces are increasingly necessary. A 0.2 mm diameter gold electrochemical sensor was fully characterized using cyclic voltammetry in abiotic and biotic artificial seawater media within a continuous culture flow cell to detect the growth and development of an aerobic Pseudoalteromonas sp. biofilm. Deconvolution of the abiotic and biotic responses enable the constituent extracellular electron transfer and biofilm responses to be resolved. Differentiation of enhanced oxygen reduction kinetics within the aerobic bacterial biofilm is linked to enzyme and redox mediator activities.

Buffer capacity regulates the stratification of anode-respiring biofilm during brewery wastewater treatment

Improving the buffer capacity of the electrolyte can enhance the anode performance in bioelectrochemical systems (BESs). To elucidate the mechanism underlying the facilitated BESs performance, this study used three different anode biofilms cultured with different concentrations of phosphate buffer (5, 50 and 100 mM) to investigate the biofilm response, in terms of the spatial structure of metabolic activity and microbial community, to different buffer capacities. Results showed that the electrochemical activities of the anode biofilms positively correlated with the buffer concentration. The spatial stratification of metabolic activity and microbial community of the anode biofilms were regulated by the buffer capacity, and the spatial microbial heterogeneity of the anode biofilm decreased as the buffer concentration increased. With increasing buffer capacity, Geobacter spp. were enriched in both the inner and outer layers of the biofilm, and the inhibition of methanogens growth improved the COD removal attributed to anode respiration. Additionally, the stimulation of EPS production in biofilms played a role in increasing the electrochemical performance of biofilms by buffer improvement. This study first revealed the regulation of buffer capacity on the stratification of anode biofilm during brewery wastewater treatment, which provided a deep insight into the relation of biofilm structure to its electrochemical properties.

Response of exoelectrogens centered consortium to nitrate on collaborative metabolism, microbial community, and spatial structure

Exoelectrogens are widespread and participate in building up interspecies electron-sharing network between anaerobic microbes. However, the exoelectrogens-centered electron-sharing networks might be affected by over-released nitrate, which competes with solid minerals or electrodes as the electron acceptor. The knowledge on the mechanism on the response of exoelectrogens-centered community to nitrate disturbance is still limited. This study applied the microbial electrochemical system as a platform and provided insights into the response of electroactive biofilm to nitrate under a constant potential of + 0.2 V. Under a fixed carbon–nitrogen ratio of 6, the biofilm partially processed dissimilatory nitrate reduction to ammonium (DNRA) transitorily and gradually loses electron-output capability. After sufficient succession, the biofilm achieved complete denitrification without ammonia accumulation and started to acquire electrons from the electrode. With nitrate disturbance, the exoelectrogens-centered biofilm dominated by Geobacter sp. preferred to cooperate with highly enriched hydrogenotrophic denitrifiers for mutualistic growth with hydrogen as the electron mediator. Different scan rates of CVs also suggested the mediated electron transfer process. The extracellular polymer substrate (EPS) serving as a network contributed to aggregates formation. The enhanced humic acid and protein secretion in the EPS of nitrate-affected biofilm suggested frequent interactions between cells. Therefore, a mutualistic interaction was established between exoelectrogens and denitrifiers under nitrate stress.

Novel calcium phosphate ion-rechargeable and antibacterial adhesive to inhibit dental caries.

This study aimed to develop an antibacterial and calcium (Ca) and phosphate (P) rechargeable adhesive and investigate the effects of dimethylaminododecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) on dentin bonding, biofilm response, and repeated Ca and P ion recharge and re-release capability for the first time. Pyromellitic glycerol dimethacrylate (PMGDM), ethoxylated bisphenol A dimethacrylate (EBPADMA), 2-hydroxyethyl methacrylate (HEMA), and bisphenol A glycidyl dimethacrylate (BisGMA) formed the adhesive (PEHB). Three groups were tested: (1) Scotchbond (SBMP, 3M) control, (2) PEHB + 30% NACP, and (3) PEHB + 30% NACP + 5% DMAHDM. Specimens were tested for dentin shear bond strength, and Ca and P ion release, recharge, and re-release. Biofilm lactic acid production and colony-forming units (CFU) on resins were analyzed. The four groups had similar dentin shear bond strengths (p > 0.1). Adhesive with DMAHDM showed significant decrease in metabolic activity, lactic acid production, and biofilm CFU (p < 0.05). The adhesives containing NACP released high levels of Ca and P ions initially and after being recharged. This study developed the first Ca and P ion-rechargeable and antibacterial adhesive, achieving strong antibacterial activity and Ca and P ion recharge and re-release for long-term remineralization. Considering the restoration-tooth bonded interface being the weak link and recurrent caries at the margins being the primary reason for restoration failures, this novel calcium phosphate-rechargeable and antibacterial adhesive is promising for a wide range of tooth-restoration applications to inhibit caries.

Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries.

Three-dimensional fluorescence-based imaging of living cells and organisms requires the sample to be exposed to substantial excitation illumination energy, typically causing phototoxicity and photobleaching. Light sheet fluorescence microscopy dramatically reduces phototoxicity, yet most implementations are limited to objective lenses with low numerical aperture and particular sample geometries that are built for specific biological systems. To overcome these limitations, we developed a single-objective light sheet fluorescence system for biological imaging based on axial plane optical microscopy and digital confocal slit detection, using either Bessel or Gaussian beam shapes. Compared to spinning disk confocal microscopy, this system displays similar optical resolution, but a significantly reduced photobleaching at the same signal level. This single-objective light sheet technique is built as an add-on module for standard research microscopes and the technique is compatible with high-numerical aperture oil immersion objectives and standard samples mounted on coverslips. We demonstrate the performance of this technique by imaging three-dimensional dynamic processes, including bacterial biofilm dispersal, the response of biofilms to osmotic shocks, and macrophage phagocytosis of bacterial cells.

Comparative proteomics and variations in extracellular matrix of Candida tropicalis biofilm in response to citral.

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow paced and often does not pass clinical trials. Citral, a monoterpene aldehyde, has shown effective antimicrobial activities against various microorganisms. However, only few studies have elaborated the action of citral against the biofilm of C. tropicalis. In the present work, the aim was to study the fungicidal effect, differential expression of proteome and changes in extracellular matrix in response to the sub-lethal concentration (16µg/mL) of citral. The administration of citral on C. tropicalis biofilm leads to a fungicidal effect. Furthermore, the differential expression of proteome has revealed twenty-five proteins in C. tropicalis biofilm, which were differentially expressed in the presence of citral. Among these, amino acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p) and biofilm-specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative stress-related proteins indicates the response of biofilm cell to combating oxidative stress during citral treatment. Moreover, the upregulation of Adh1p is of particular interest because it subsidizes the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1p/Erg19p signifies the effect of citral on ergosterol biosynthesis. The presence of citral has also shown an increment in hexosamine and ergosterol component in extracellular matrix of C. tropicalis biofilm. Hence, it is indicated that the cellular response towards citral acts through multifactorial processes. This study will further help in the interpretation of the effect of citral on C. tropicalis biofilm and development of novel antifungal agents against these potential protein targets.

Understanding the role of the master regulator, PecS, in Agrobacterium fabrum

Bacterial plant pathogens have devised means of surviving in a hostile environment created by their hosts during the pathogenic phase of their life cycle. Critical to this survival mechanism are a family of transcriptional regulators known as Multiple antibiotic resistance Regulators (MarR), known to regulate the expression of genes involved in virulence, stress response, multidrug resistance etc. Recent studies have associated PecS, a member of the MarR family, as a master regulator of virulence gene expressions in the Enterobacterium Dickeya dadantii, a soft rot-causing phytopathogen of a range of plant species. D. dadantii PecS controls both the expression of pectinase genes and biosynthesis of a blue pigment known as indigoidine (an antioxidant). PecS has also been characterized in other plant pathogens such as, Pectobacterium atrosepticum, and it has been identified in the human pathogens, Klebsiella pneumoniae and Vibrio vulnificus. However, the role ofPecS in the crown gall-causing Agrobacterium fabrum remains largely unknown. To understand the role of A. fabrum PecS in the physiology of the bacterium, plant association, and promotion of survival in response to host defenses, we created a pecS knockout and determined growth, motility, biofilm and stress response. Our result shows a higher growth rate in pecS knockoutthan the Wild Type over a 14 hour period, while the Wild Type was more motile after 48 hours of incubation in a 0.3% (wt/vol) agar. Biofilm formation was measured at 24, 48 and 72 hours, using crystal violet staining, and the data shows that Wild Type forms more biofilm than the knockout at all the time points. Stress response assays show the Wild Type was more sensitive to 5 mM of both cumene hydroperoxide and hydrogen peroxide, 10 mM tert-butyl hydroperoxide and 25 ug ampicillin compared to the knockout strain, while the knockout was more sensitive to 25 ug and 200 ug trimethoprim and streptomycin respectively. The study expands our knowledge about the master regulatory role of A. fabrum PecS and proves that A. fabrum PecSis implicated in bacterial physiology, plant association and survival.

5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice.

Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.

Interactions of the Streptococcus pneumoniae Toxin-Antitoxin RelBE Proteins with Their Target DNA

Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen Streptococcus pneumoniae is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal relBE operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the −10 sequences of the single promoter that directs the synthesis of the relBE mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.

High fluoride resistance and virulence profile of environmental Pseudomonas isolated from water sources.

In our previous study, all Pseudomonas strains THP6, THP41, and OHP5 were identified as fluoride-resistant bacteria isolated from Dindigul district, Tamilnadu, India. The selected strains exhibiting a high level of fluoride resistance was determined in Luria broth (LB) medium and LB agar plates. In a further effort, fluoride-resistant organisms were tested for hemolytic activity and showed β-hemolysis on blood agar plates. The virulence factors such as gyrB, toxA, algD and lasB, plcH, rhlC and biofilm response genes (pslA, pelA, ppyR) were detected by PCR analysis. The putative genus-specific and species-specific PCR also confirmed that the selected fluoride-resistant strains were belonging to Pseudomonas aeruginosa species. Fluoride-resistance gene crcB was amplified by gene-specific primers. The crcB gene was cloned in TA vector and transformed into E. coli DH5α. Comparative and blast analysis of THP6, THP41, and OHP5 strains crcB gene sequences were high homology with P. aeruginosa fluoride efflux transporter crcB and P. aeruginosa putative fluoride ion transporter crcB. The recombinants were efficiently growing in the NaF containing LB agar plates. The fluoride tolerance of these strains was also associated with resistance to multiple antibiotics. These results can lead to the use of the fluoride resistance gene of P. aeruginosa for the development of a biosensor for fluoride detection.