The peptidoglycan-associated lipoprotein OprL helps protect a Pseudomonas aeruginosa mutant devoid of the transactivator OxyR from hydrogen peroxide-mediated killing during planktonic and biofilm culture

OxyR controls H(2)O(2)-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10(7)/ml) organisms are easily killed by micromolar H(2)O(2). The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H(2)O(2). We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H(2)O(2) than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H(2)O(2). Given the sensitivity of oxyR oprL bacteria to planktonic H(2)O(2), we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H(2)O(2)-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H(2)O(2). Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H(2)O(2) in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.     

Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms.

Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria. Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent. This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853). Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates. The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples. Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples. Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction. However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples. Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment. The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.  In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.     

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalis hmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.     

Impact of oleic acid (cis-9-octadecenoic acid) on bacterial viability and biofilm production in Staphylococcus aureus

Staphylococcus aureus is responsible for a broad variety of chronic infections. Most S. aureus clinical isolates show the capacity to adhere to abiotic surfaces and to develop biofilms. Because S. aureus growing in a biofilm is highly refractory to treatment, inhibition of biofilm formation represents a major therapeutic objective. We evaluated the effects of oleic acid on primary adhesion and biofilm production in eight genotypically different S. aureus strains as well as in the biofilm-negative Staphylococcus carnosus strain TM300. Oleic acid inhibited primary adhesion but increased biofilm production in every S. aureus strain tested. Staphylococcus aureus strain UAMS-1 was then selected as a model organism for studying the mechanisms triggered by oleic acid on the formation of a biofilm in vitro. Oleic acid inhibited the primary adhesion of UAMS-1 dose dependently with an IC(50) around 0.016%. The adherent bacterial population decreased proportionally with increasing concentrations of oleic acid whereas an opposite effect was observed on the planktonic population. Overall, the total bacterial counts remained stable. Macroscopic detachments and clumps were visible from the adherent bacterial population. In the presence of oleic acid, the expression of sigB, a gene potentially involved in bacterial survival through an effect on fatty acid composition, was not induced. Our results suggest a natural protective effect of oleic acid against primary adhesion.     

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) biofilm by cationic poly (D,L-lactide-co-glycolide) nanoparticles

Abstract

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7?nm, 26.1?mv) showed time- and concentration-dependent activity against MRSA growth, killing ～ 90% of planktonic bacterial cells in 3?h at 400?μg ml^?1, and completely inhibiting biofilm formation at 1000?μg ml^?1. Moreover, CNPs at 400?μg ml^?1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (～ 25%) and biofilm formation (～ 50%). The CNPs–bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.     

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up‐ or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.     

Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures

Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.     

Assessment of resistance towards biocides following the attachment of micro-organisms to, and growth on, surfaces

AIMS: To develop a rapid method for the assessment of biocidal activity directed towards intact biofilms. METHODS AND RESULTS: Escherichia coli and Staphylococcus epidermidis were cultured for up to 48 h within 96-well microtitre plates. The planktonic phase was removed and the wells rinsed. Residual biofilms were exposed to various concentrations of chloroxylenol, peracetic acid, polyhexamethylene biguanide (PHMB), cetrimide or phenoxyethanol for 1 h. At 15-min intervals, biocide was removed, and the wells washed in neutraliser and filled with volumes of fresh medium. Re-growth of the cultures was monitored during incubation at 35 degrees C in the plate reader. Times taken for the treated wells to re-grow to fixed endpoints were determined and related to numbers of surviving cells. Time--survival curves were constructed and the survival of the attached bacteria, following exposure to the agents for 30 min, interpolated for each biocide concentration. Log--log plots of these survival data and biocide concentration were constructed, and linear regression analysis performed in order to (i) calculate concentration exponents and (ii) compare the effectiveness of the biocides between variously aged biofilm and planktonic cells. From such analyses iso-effective concentrations of biocide (95% kill in 30 min) were calculated and expressed as planktonic : biofilm indices (PBI). CONCLUSION: PBI varied between 1.02 and 0.02, were relatively unaffected by age of the biofilms but differed significantly between organism and biocide. Notably those compounds with the higher activity against planktonic bacteria (PHMB and peracetic acid) were most prone to a biofilm effect but remained the most effective of the agents selected. SIGNIFICANCE AND IMPACT OF THE STUDY: The endpoint method proved robust, enabled the bactericidal effects of the biocides to be assessed against in-situ biofilms, and was suitable for routine screening applications.     

Biofilm form and function: carbon availability affects biofilm architecture, metabolic activity and planktonic cell yield

Aims: To investigate carbon transformation by biofilms and changes in biofilm architecture, metabolic activity and planktonic cell yield in response to fluctuating carbon availability. Methods and Results: Pseudomonas sp. biofilms were cultured under alternating carbon‐replete and carbon‐limited conditions. A shift to medium without added carbon led to a 90% decrease in biofilm respiration rate and a 40% reduction in planktonic cell yield within 1 h. Attached cell division and progeny release were shown to contribute to planktonic cell numbers during carbon limitation. Development of a significantly enlarged biofilm surface area during carbon limitation facilitated a rapid increase in whole‐biofilm metabolic activity, cell yield and biomass upon the re‐introduction of carbon after 8 days of limitation. The cumulative number of planktonic cells (>10¹⁰ CFU) released from the biofilm during the cultivation period contained only 1·0% of the total carbon available to the biofilm, with 6·5% of the carbon retained in the biofilm and 54% mineralized to CO₂. Conclusions: Biofilm‐derived planktonic cell yield is a proliferation mechanism. The rapid response of biofilms to environmental perturbations facilitates the optimal utilization of resources to promote both proliferation and survival. Biofilms function as efficient catalysts for environmental carbon transformation and mineralization. Significance and Impact of the study: A greater understanding of the relationship between biofilm form and function can inform strategies intended to control and/or promote biofilm formation.     

Irradiation sensitivity of planktonic and biofilm-associated Escherichia coli O157:H7 isolates is influenced by culture conditions

Ionizing radiation effectively inactivates Escherichia coli O157:H7, but the efficacy of the process against biofilm cells versus that against free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm cells was determined for three isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894). Biofilms were formed on sterile glass slides incubated at 37 degrees C for either 24 h, 48 h, or 72 h. The biofilm and planktonic cultures were gamma irradiated at doses ranging from 0.0 (control) to 1.5 kGy. The dose of radiation value required to reduce the population by 90% (D10) was calculated for each isolate, culture, and maturity based on viable populations at each radiation dose. For each of the times sampled, the D10 values of isolate 43894 planktonic cells (0.454 to 0.479 kGy) were significantly (P<0.05) higher than those observed for biofilm cells (0.381 to 0.385 kGy), indicating a significantly increased sensitivity to irradiation for cells in the biofilm habitat. At the 24-h sampling time, isolate C9490 showed a similar pattern, in which the D10 values of planktonic cells (0.653 kGy) were significantly higher than those for biofilm cells (0.479 kGy), while isolate 35150 showed the reverse, with D10 values of planktonic cells (0.396 kGy) significantly lower than those for biofilm cells (0.526 kGy). At the 48-h and 72-h sampling times, there were no differences in radiation sensitivities based on biofilm habitat for C9490 or 35150. Biofilm-associated cells, therefore, show a response to irradiation which can differ from that of planktonic counterparts, depending on the isolate and the culture maturity. Culture maturity had a more significant influence on the irradiation efficacy of planktonic cells but not on biofilm-associated cells of E. coli O157:H7.     

Carbon load in aquatic ecosystems affects the diversity and biomass of water biofilm consortia and the persistence of the pathogen Campylobacter jejuni within them

The influence of carbon load on autochthonous water microflora population distribution and diversity, and on the persistence of Campylobacter jejuni, was examined with a two-stage aquatic biofilm model. Serine was chosen since it is a carbon source utilised by C. jejuni and concentrations were chosen to reflect upper limits of amino acid load reported in surface water. The total viable count of the autochthonous biofilm microflora increased with increasing serine concentration (10-fold and 20-fold with 5 nM and 5 μM serine, respectively), as did the counts of the microflora in the planktonic phase. Differences in biofilm species distribution as determined by culture were small with changes in temperature or the addition of serine; but was markedly affected by serine as determined by light microscopy, becoming more luxuriant and dominated by long filamentous cells. The addition of serine to the water significantly and progressively reduced the persistence of C. jejuni, which decreased by 25% and 50% with serine concentrations of 5 nM and 5 μM respectively. We have demonstrated that carbon load affects the species diversity and density of both the planktonic and biofilm phase of aquatic autochthonous microflora. Although the survival of C. jejuni in water in a culturable form was sufficient for this to be an important vehicle for its transmission, carbon load significantly influenced survival; an increase in serine concentration significantly reduced survival.     

Role of the extracellular polymer matrix in resistance of bacterial biofilms to extreme environmental factors

Biofilms of a number of gram-positive and gram-negative bacteria (both environmental strains from the stratal waters of oil fields and collection strains) were found to exhibit higher resistance to extreme physicochemical factors (unfavorable temperature, pH, and salt concentration) than planktonic cultures. The extracellular polymers forming the structure of the biofilm matrix were shown to contribute significantly to this resistance, since suppression of matrix formation by subbacteriostatic concentrations of azithromycin (for Pseudomonas acephalitica) or mutation in the cvil gene encoding N-hexanoyl homoserine lactone synthetase (for Chromobacterium violaceum CV026) resulted in the resistance of biofilms being decreased almost to the level of planktonic cultures. The role of the biofilm matrix for bacterial survival under extreme conditions is discussed.     

Use of heterotrophic CO2 assimilation as a measure of metabolic activity in planktonic and sessile bacteria

We have examined whether assimilation of CO2 can be used as a measure of metabolic activity in planktonic and sessile heterotrophic bacteria. CO2 assimilation by environmental samples and pure cultures of heterotrophic bacteria was studied using 14CO2 and 13CO2 as tracers. Heterotrophic growth on complex organic substrates resulted in assimilation of CO2 into cell biomass by activated sludge, drinking water biofilm, and pure cultures of Escherichia coli ATCC 25922, Es. coli ATCC 13706, Rhodococcus ruber, Burkholderia sp., Bacillus circulans, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas aeruginosa. Analysis of 13C-labelled phospholipid fatty acids (PLFAs) confirmed that heterotrophic bacteria may assimilate 13CO2 into cell macromolecules such as membrane lipids. All major PLFAs extracted from activated sludge and drinking water biofilm samples were enriched in 13C after incubation with CO2. Between 1.4% and 6.5% of the biomass produced by cultures of P. putida and a drinking water biofilm during growth in complex media was apparently derived from assimilation of CO2. Resting cells assimilated less CO2 compared to actively growing cells, and CO2 assimilation activity correlated with the amount of biomass produced during heterotrophic growth. The 14CO2 assimilation assay was evaluated as a tool to examine inhibitory effects of biocides on planktonic and sessile heterotrophs (biofilms). On the basis of 14CO2 assimilation activity, the minimum inhibitory concentration (MIC) of benzalkonium chloride was estimated to 21.1 and 127.2 mg l(-1) for planktonic and biofilm samples, respectively. The results indicate that assimilation of isotopically labelled CO2 can be used as a relatively simple measure of metabolic activity in heterotrophic bacteria. CO2 assimilation assays may be used to study the effects of antimicrobial agents on growth and survival of planktonic and sessile heterotrophic organisms.     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

Poly(3-hydroxybutyrate) influences biofilm formation and motility in the novel Antarctic species <Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis> under cold conditions

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. It has previously shown that polyhydroxybutyrate (PHB) accumulation is essential during the growth under cold conditions. In this work, the relationship between PHB accumulation and biofilm development at low temperature was investigated. P. extremaustralis, an Antarctic strain able to accumulate PHB, and its phaC mutant, impaired in the synthesis of this polymer, were used to analyze microaerobic growth, biofilm development, EPS content and motility. PHB accumulation increased motility and survival of planktonic cells in the biofilms developed by P. extremaustralis under cold conditions. Microaerobic conditions rescued the cold growth defect of the mutant strain. The PHB accumulation capability could constitute an adaptative advantage for the colonization of new ecological niches in stressful environments.