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.     

Metal resistance in Candida biofilms

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

Pleurotus ostreatus biofilm‐forming ability and ultrastructure are significantly influenced by growth medium and support type

Aims

To investigate the effect of support and growth medium (GM) on Pleurotus ostreatus biofilm production, specific metabolic activity (SMA) and ultrastructure.

Methods and Results

Biofilms were developed on membranes covering a broad range of surface properties and, due to the applicative implications of mixed biofilms, on standard bacterial GM in stationary and shaken culture. Hydrophilic (glass fibre, Duran glass and hydroxyapatite) and mild hydrophobic (polyurethane, stainless steel, polycarbonate, nylon) supports were more adequate for biofilm attachment than the hydrophobic Teflon. Among the GM, sucrose–asparagine (SA) was more conducive to biofilm production than Luria–Bertani and M9. GM was more influential than support type on biofilm ultrastructure, and a high compactness was evident in biofilms developed on SA. Biofilms on Duran glass were more efficient than planktonic cultures in olive‐mill wastewater treatment.

Conclusions

The main effects of support and GM variables and their binary interactions on both biofilm production and SMA were all highly significant (P < 0·001): thus, the magnitude of the effect of each variable strongly depended on the level of the other one.

Significance and Impact of the Study

There is a lack of basic information regarding physiology and ultrastructure of P. ostreatus biofilms. To our knowledge, this is the first attempt to fill this gap, thus representing a basis for future studies.     

Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces

Aim: We compared the MBEC?‐HTP assay plates made of polystyrene with metal discs composed of TMZF^® and CrCo as substrates for biofilm formation. Methods and Results: Staphylococcus aureus was grown on polystyrene and on metal discs made of titanium and chrome–cobalt. Antibiotic susceptibility was assessed by examining the recovery of cells after antibiotic exposure and by measuring the biofilm inhibitory concentration (BIC). The minimal inhibitory concentration (MIC) was assessed with planktonic cells. Bacterial growth was examined by scanning electron microscopy. The antibiotic concentration for biofilm inhibition (BIC) was higher than the MIC for all antibiotics. Microscopic images showed the biofilm structure characterized by groups of cells covered by a film. Conclusions: All models allowed biofilm formation and testing with several antibiotics in vitro. Gentamicin and rifampicin are the most effective inhibitors of Staph. aureus biofilm‐related infections. We recommend MBEC?‐HTP assay for rapid testing of multiple substances and TMZF^® and CrCo discs for low‐throughput testing of antibiotic susceptibility and for microscopic analysis. Significance and Impact of the Study: In vitro assays can improve the understanding of biofilms and help developing methods to eliminate biofilms from implant surfaces. One advantage of the TMZF^® and CrCo discs as biofilm in vitro assay is that these metals are commonly used for orthopaedic implants. These models are usable for future periprosthetic joint infection studies.     

Chl-<Emphasis Type="Italic">a</Emphasis> fluorescence parameters as biomarkers of metal toxicity in fluvial biofilms: an experimental study

A study was carried out to evaluate the sensitivity of different chlorophyll-a (chl-a) fluorescence parameters measured in freshwater biofilms as metal pollution biomarkers of short- and long-term metal exposures at environmentally realistic concentrations. A microcosm experiment was performed using indoor channels. Mature biofilms were exposed from hours to weeks to three different treatments: No-Metal, Zn (400 μg l⁻¹); and Zn plus Cd (400 μg l⁻¹ and 20 μg l⁻¹, respectively). Metal concentration was based on a real case study: the Riou-Mort River (France). Biofilms exposed to Zn bioaccumulated similar Zn contents per dry weight to those exposed to the mixture (Zn plus Cd) causing a similar inhibition of the effective quantum yield (\Upphi_\textM^￠)(\Upphi_{\text{M}}^{\prime}) during the first hours of exposure. A reduction of the algal biomass, a shift in the community composition (a high reduction of diatoms), a reduction of the maximal quantum yield (Φ_M) and a strong reduction of non-photochemical quenching (NPQ) were observed from day 14 until the end of the experiment (35 days). The results indicate that the effects of the metal mixture present in the Riou-Mort on biofilms could be attributed to Zn toxicity. The use of a set of chl-a fluorescence measurements, including photochemical and NPQ parameters, are recommended as a reliable biomarker tool box to evaluate both short- and long-term effects of metals on biofilms containing oxygenic photoautotrophs, suggesting its use in field applications.     

Efficacy of Indolicidin,Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

     

Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO(2) (-), SeO(3) (2-), TeO(3) (2-)) and three metal cations (Cd(2+), Ni(2+), Al(3+)) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria-Bertani) medium were thicker (23 mum) than biofilms grown in minimal (SA saccarose-arginine) medium (13 mum). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50-100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO(3) (2-) to elemental Te(0) or SeO(3) (2-) to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO(3) (2-) and SeO(3) (2-) is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.     

Design of a Simple Model of <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms Formed under Conditions of Flow: Development,Architecture, and Drug Resistance

Candida albicans biofilms on most medical devices are exposed to a flow of body fluids that provide water and nutrients to the fungal cells. While C. albicans biofilms grown in vitro under static conditions have been exhaustively studied, the same is not true for biofilms developed under continuous flow of replenishing nutrients. Here, we describe a simple flow biofilm (FB) model that can be built easily with materials commonly available in most microbiological laboratories. We demonstrate that C. albicans biofilms formed using this flow system show increased architectural complexity compared to biofilms grown under static conditions. C. albicans biofilms under continuous medium flow grow rapidly, and by 8 h show characteristics similar to 24 h statically grown biofilms. Biomass measurements and microscopic observations further revealed that after 24 h of incubation, FB was more than twofold thicker than biofilms grown under static conditions. Microscopic analyses revealed that the surface of these biofilms was extremely compact and wrinkled, unlike the open hyphal layer typically seen in 24 h static biofilms. Results of antifungal drug susceptibility tests showed that C. albicans cells in FB exhibited increased resistance to most clinically used antifungal agents.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

<Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> biofilms subjected to phage phiIBB-PF7A

Background  

Pseudomonas fluorescens is an important food spoilage organism, usually found in the form of biofilms. Bacterial biofilms are inherently resistant to a variety of antimicrobial agents, therefore alternative methods to biofilm control, such as bacteriophages (phages) have been suggested. Phage behavior on biofilms is still poorly investigated and needs further understanding. Here we describe the application of phage ϕIBB-PF7, a newly isolated phage, to control P. fluorescens biofilms. The biofilms were formed under static or dynamic conditions and with or without renewal of medium.     

Outer Membrane Vesicles of Helicobacter pylori TK1402 are Involved in Biofilm Formation

Background  

Helicobacter pylori forms biofilms on glass surfaces at the air-liquid interface in in vitro batch cultures; however, biofilms of H. pylori have not been well characterized. In the present study, we analyzed the ability of H. pylori strains to form biofilms and characterized the underlying mechanisms of H. pylori biofilm formation.     

Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

Novel Bioimaging Techniques of Metals by Laser Ablation Inductively Coupled Plasma Mass Spectrometry for Diagnosis Of Fibrotic and Cirrhotic Liver Disorders

Background and Aims

Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult.

Methods

In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers.

Results

Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples.

Conclusions

Biometal imaging via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.     

Differences in metabolism between the biofilm and planktonic response to metal stress

Bacterial biofilms are known to withstand the effects of toxic metals better than planktonic cultures of the same species. This phenomenon has been attributed to many features of the sessile lifestyle not present in free-swimming populations, but the contribution of intracellular metabolism has not been previously examined. Here, we use a combined GC-MS and (1)H NMR metabolomic approach to quantify whole-cell metabolism in biofilm and planktonic cultures of the multimetal resistant bacterium Pseudomonas fluorescens exposed to copper ions. Metabolic changes in response to metal exposure were found to be significantly different in biofilms compared to planktonic cultures. Planktonic metabolism indicated an oxidative stress response that was characterized by changes to the TCA cycle, glycolysis, pyruvate and nicotinate and niacotinamide metabolism. Similar metabolic changes were not observed in biofilms, which were instead dominated by shifts in exopolysaccharide related metabolism suggesting that metal stress in biofilms induces a protective response rather than the reactive changes observed for the planktonic cells. From these results, we conclude that differential metabolic shifts play a role in biofilm-specific multimetal resistance and tolerance. An altered metabolic response to metal toxicity represents a novel addition to a growing list of biofilm-specific mechanisms to resist environmental stress.     

Biofilm Formation and Biocide Susceptibility Testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC™ assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state. Received: 15 February 2002 / Accepted: 28 March 2002     

A quantitative real-time RT-PCR assay for mature C. albicans biofilms

Background  

Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.Download video file.^{(44M, mov)}