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PubMed2004

Anaerobic growth does not support biofilm formation in Escherichia coli K-12.

Association with a surface in a structure known as biofilm is the prevailing microbial lifestyle. Here we show the kinetics of biofilm formation of Escherichia coli W3110 in static cultures growing under aerobic or anaerobic conditions. Aerobically growing cells in LB medium started to produce detectable amounts of biofilm after 4 to 8 h, displaying maximal accumulation of formed biofilm at 24 h, corresponding to the onset of stationary phase. Then an abrupt reduction in the biomass of the biofilm was observed. This decrease was not prevented by external addition of fresh nutrients and coincided with the depletion of oxygen as measured by the enzymatic activity of the AdhE protein. No biofilm formation was detected in cultures grown anaerobically in LB or LB supplemented with nitrate, nitrite, DMSO or fumarate, even after 72 h of incubation, well inside the stationary phase, suggesting that under anaerobic growth conditions E. coli cannot form biofilms.

Anaerobiosis↗
PubMed2005

Biofilm formation is prevalent among field isolates of Actinobacillus pleuropneumoniae.

A total of 77 field isolates and 15 reference strains of the porcine respiratory pathogen Actinobacillus pleuropneumoniae were tested for their ability to form biofilms in a polystyrene microtiter plate assay. More than half of all field isolates, which included strains representing serotypes 1, 5 and 7, but only two reference strains (serotypes 5B and 11) exhibited biofilm formation. Strains that formed biofilms in microtiter plates also formed thick biofilms at the air-liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth. Our findings indicate that biofilm formation is a prevalent phenotype among A. pleuropneumoniae field isolates, and that this phenotype may have been previously overlooked because of its tendency to be lost upon subculturing in broth. Biofilm formation may have relevance to the colonization, pathogenesis and transmission of this bacterium.

Actinobacillus Infections↗
PubMed2006

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates.

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.

Animals↗
PubMed2004

UV disinfection in a model distribution system:; biofilm growth and microbial community.

Two model distribution systems were operated in parallel to investigate the impact of UV disinfection on water distribution system biofilms and microbial community composition. One system received an influent irradiated with UV light, whereas the control received the same influent with no treatment. The biofilm in the UV system, as compared to the control, was more responsive (i.e., had a greater increase in steady-state density of heterotrophic bacteria) to the increased nutrient availability afforded by a decrease in HRT from 12 to 2 h. However, the UV treatment did not have a consistent impact on the biofilm community, indicating the processes controlling HPC density were independent of the specific strains of bacteria forming the biofilm. There was evidence that particle shielding contributed to the survival of UV-susceptible bacteria. This hypothesis was consistent with the presence of UV-susceptible bacteria in the UV system, as well as the high similarity of the biofilm communities in the UV and control systems in one of the experiments. To simulate an intrusion event, opportunistic pathogens were added to each system after the biofilm community reached steady-state. Opportunistic pathogen attachment was not affected by the UV treatment, but was instead correlated to the biofilm density of heterotrophic bacteria.

Bacteria↗
PubMed2005

Effects of temperature and biodegradable organic matter on control of biofilms by free chlorine in a model drinking water distribution system.

This study used annular reactors (AR) to investigate, under controlled laboratory conditions, the effects of temperature and biodegradable organic matter (BOM) on the free chlorine residual needed to control biofilm accumulation, as measured by heterotrophic plate count (HPC) bacteria. Biofilm was grown on PVC coupons, initially in the absence of chlorine, at 6, 12, and 18 degrees C, in the presence and absence of a BOM supplement (250 microg C/L) added as acetate. During the early stages of chlorine addition, when no measurable free chlorine residual was present, a reduction in biofilm HPC numbers was observed. Subsequently, once sufficient chlorine was added to establish a residual, the biofilm HPC numbers expressed as log CFU/cm2 fell exponentially with the increase in free chlorine residual. Temperature appeared to have an important effect on both the chlorine demand of the system and the free chlorine residual required to control the biofilm HPC numbers to the detection limit (3.2 Log CFU/cm2). For the water supplemented with BOM, a strong linear correlation was found between the temperature and the free chlorine residual required to control the biofilm. At 6 degrees C, the presence of a BOM supplement appeared to substantially increase the level of free chlorine residual required to control the biofilm. The results of these laboratory experiments provide qualitative indications of effects that could be expected in full-scale systems, rather than to make quantitative predictions.

Biodegradation, Environmental↗
PubMed2005

Kinetics and stoichiometry of sulfide oxidation by sewer biofilms.

Oxidation of sulfide under aerobic conditions by biofilms grown on municipal wastewater in 6 identical pipe reactors was investigated. The biofilms were grown at pH 7.6 and temperatures of 20 and 25 degrees C under aerobic-anaerobic transient conditions with pulse dosing of sulfide in the bulk water. The pulse dosing of sulfide served to simulate conditions in a gravity sewer located downstream of a pressure main. During growth of the biofilms, sulfide was pulse dosed in concentrations of 0, 0.5, 2.0 and 5.0 g Sm(-3) with a frequency of 1h(-1). Based on a series of batch experiments, kinetics and stoichiometry of sulfide oxidation by the sewer biofilms was investigated and a rate equation and a stoichiometric constant proposed. Sulfide oxidation kinetics was significantly faster for biofilms grown at sulfide loadings of 0.5, 2.0 and 5.0 g Sm(-3)h(-1) than for biofilms grown in the absence of sulfide. However, the kinetics of sulfide oxidation was relatively constant for biofilms grown at sulfide loadings above 0.5 g Sm(-3)h(-1). Mass balance calculations of dissolved oxygen and sulfur compounds suggested the oxidation product to be elemental sulfur. Further oxidation of elemental sulfur could not be documented.

Biofilms↗
PubMed2005

Volatile organic compounds in natural biofilm in polyethylene pipes supplied with lake water and treated water from the distribution network.

The objective of this work was investigation of volatile organic compounds (VOC) in natural biofilm inside polyethylene (HDPE) pipelines at continuously flowing water. VOC in biofilm may contribute to off-flavour episodes in drinking water. The pipelines were supplied with raw lake water and treated water from the distribution network. Biofilm was established at test sites located at two different drinking water distribution networks and their raw water sources. A whole range of volatile compounds were identified in the biofilm, including compounds frequently associated with cyanobacteria and algae, such as ectocarpene, dictyopterene A and C', geosmin, beta-ionone and 6-methyl-5-hepten-2-one. In addition, volatile amines, dimethyldisulphide and 2-nonanone, presumably originating from microorganisms growing in the biofilm, were identified. C8-compounds such as 1-octen-3-one and 3-octanone were believed to be products from microfungi in the biofilm. Degradation products from antioxidants such as Irgafos 168, Irganox 1010 and Irganox 1076 used in HDPE pipes, corresponding to 2,4-di-tert-butylphenol and 2,6-di-tert-butylbenzoquinone, were present in the biofilm.

Biofilms↗
PubMed2001

The formation of malodorous dimethyl oligosulphides in treated groundwater: the role of biofilms and potential precursors.

Water distributed from the Wanneroo Groundwater Treatment Plant intermittently contains dimethyl trisulphide (DMTS). The compound is responsible for a "swampy odour" in the water. DMTS production from potential precursors was insignificant in the absence of biofilms when compared with DMTS production from precursors in the presence of biofilms in a biofilm reactor. Greatest dimethyl disulphide (DMDS) and DMTS production (> 3000 ng L-1 DMTS) occurred in the reactors when supplied with methane thio-containing compounds, such as methionine, S-methyl cysteine and methyl-3-(methylmercapto)-propionate. Abiotic DMTS production from oligosulphides also occurred through the addition of the methylating agents, methyl iodide or methyl-p-toluene sulphonate. Significant DMTS production also occurred with Wanneroo water that contained added omega-thio-containing compounds such as cysteine (1400 ng L-1 DMTS), and 3-mercaptopropionate (210 ng L-1). Biomethylation, a ubiquitous response by microorganisms for the detoxification of toxic compounds, generated DMDS/TS from biofilm oligosulphides. Biofilms exposed to the toxic compounds selenate or 2,4,6-trichlorophenol methylated oligosulphides in addition to the toxins. Sodium sulphide also stimulated DMTS production. Easily Biodegradable Dissolved Organic Carbon (BDOC) probably contributed indirectly to DMTS production by the biofilms, although whether this was a result of its stimulation of greater microbial activity or consumption of oxygen, or both, remains unresolved. Stagnation of water in the biofilm reactors also increased DMTS production, which was concomitant with depletion of oxygen concentrations in the bulk water. Many processes, such as degradation of methane thio-containing compounds, methylation of sulphides and oligosulphides, and changes in contributions of different metabolic pathways upon depletion of oxygen concentrations upon water stagnation, probably contribute simultaneously to "swampy odour" production in the distribution system.

Bacteria↗
PubMed2001

The potential for biofilm growth in water distribution systems.

Biofilms on pipe walls in water distribution systems are composed of bacteria in a polymeric matrix, which can lead to chlorine demand, coliform growth, pipe corrosion and water taste and odour problems. The majority of previous studies have been laboratory or pilot plant based and few results are available for field conditions. In this study, field observations of biofilm were made using biofilm potential monitors. The monitor results were compared with pipe samples taken from the distribution system and with laboratory pipe reactors. An empirical equation quantified the inhibitory effects of free chlorine and decrease of temperature on biofilm growth. With water having total organic carbon concentrations in the range 1.5-3.9mg/1 a free chlorine residual of 0.2 mg/l was needed to reduce biofilm concentration to below 50 pg ATP cm2. Pipe material influenced biofilm activity far less than chlorine with mean biofilm activity being ranked in the order glass (136 pg ATP/cm2) < cement (212 pg ATP/cm2) < MDPE (302 pg ATP/ cm2) < PVC (509 pg ATP/cm2).

Biofilms↗
PubMed2003

Biofilm responses to ageing and to a high phosphate load in a bench-scale drinking water system.

The effects of ageing and of phosphate load on drinking water biofilms developed on a polycarbonate substratum in the pseudo-equilibrium state have been evaluated. Phosphate was added in an amount higher than the stochiometric nutrient requirements of bacteria, at concentrations commonly applied in a drinking water distribution system for corrosion control. Multiple parameters were monitored: heterotrophic plate counts (HPCs), total direct counts (TDCs) and potential exoproteolytic activity (PEPA) in order to characterise changes in bacterial biofilms. The total carbohydrate, amino acid and phosphate contents of biofilms were analysed to characterise and monitor the biochemical composition of the biofilm.The three enumeration methods showed that a pseudo-equilibrium state was reached after 7 weeks of colonisation after which, the bacterial growth rate in the biofilm was 0.1 log per week on average. Bulk phosphate addition doubled the phosphate in the biofilm, but did not affect the other biological, physiological or chemical parameters measured. Polysaccharides increased in the biofilm with ageing and the dynamics of individual carbohydrate synthesis also varied with the age of the biofilm. Once pseudo-equilibrium, it was found that the total proteins were globally constant, whereas the spectra of some individual amino acids of the proteins had significantly changed.

Biofilms↗
PubMed2000

Diffuse lamellar keratitis related to endotoxins released from sterilizer reservoir biofilms.

OBJECTIVE: To investigate the risk factors and control mechanisms used to control the outbreak of diffuse lamellar keratitis (DLK) associated with laser in situ keratomileusis (LASIK) and examine the relationship between DLK and endotoxins released from sterilizer biofilm reservoirs. DESIGN: Clinic-based cohort and laboratory study. PARTICIPANTS: All patients undergoing LASIK at our clinic from October 7, 1998 through August 31, 1999. The case definition was a diffuse infiltrate in the interface developing within the first week after surgery. INTERVENTIONS: Biofilm control in the sterilizer, changes in sterilizer, distilled water, instruments, and irrigating fluids. MAIN OUTCOME MEASURES: The incidence of DLK after LASIK surgery. RESULTS: There were 983 evaluable patients, with three whose DLK status was not recorded. There were 52 cases of DLK. Burkholderia pickettii was isolated from the sterilizer reservoir. Potential risk factors and associations, for which there was no significant difference, included age and sex of the patients, surgeon, operating suite temperature or humidity, drapes used, saline solutions used, time of day the surgery was performed, and microkeratome use. Sterilizers 1 and 2, before biofilm control, were compared with sterilizer 3, after control. The relative risk was 9.4 (confidence limits [CL], 7.5-11.8) for sterilizer 1 versus 3 and 18. 7 (CL, 11-32) for sterilizer 2 versus 3. Three cases occurred after biofilm control, but were sporadic in nature and associated with epithelial defects. CONCLUSIONS: Clusters of DLK may be related to endotoxins released from gram-negative biofilms in sterilizer reservoirs. We experienced an outbreak of DLK affecting 52 patients and isolated B. pickettii from the sterilizer reservoir. Epidemiologic investigation showed that biofilm control in the sterilizer reservoirs was associated with a significant reduction in the development of DLK. We encourage any clinics that experience a cluster of DLK to consider microbiologic and epidemiologic investigation for the effectiveness of sterilizer biofilm control.

Adolescent↗
PubMed2000

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions.

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1-2 ppm) and under anaerobic conditions (</=0.1 ppm O(2)). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth - biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O(2)-grown biofilm.

Anaerobiosis↗
PubMed2003

Micro-cantilever method for measuring the tensile strength of biofilms and microbial flocs.

Cohesive strength is an important factor in determining the structure and function of biofilm systems, and cohesive strength plays a key role in our ability to remove or control biofilms in engineered systems. A micro-mechanical device has been developed to directly measure the tensile strength of biofilms and other microbial aggregates. An important feature of this method is the combination of a direct measurement of force with particle separations that occur at a scale comparable to that observed for biofilm systems. The force required to separate an aggregate is determined directly from the deflection of cantilevered glass micropipettes with a 20-40-microm diameter. Combined with an estimate of the cross-sectional area of the aggregate at the point of separation this measurement indicates the cohesive strength of the aggregate. Samples of return activated sludge (RAS) and a Pseudomonas aeruginosa biofilm were tested using the device. The measured cohesive strengths of the RAS flocs ranged from 419 to 206,400 N/m(2), while many of the flocs exceeded the range of measurement of the device. Fragments of P. aeruginosa biofilm had cohesive strengths ranging from 395 to 15,640 N/m(2), with a median value of 3020 N/m(2). The median equivalent diameters of the particles detached from the aggregates were 32 microm for RAS and 30 microm for the P. aeruginosa biofilms.

Biofilms↗
PubMed2001

Biofilm formation by salmonella spp. on food contact surfaces and their sensitivity to sanitizers.

Biofilm formation by two poultry isolates of Salmonella on three commonly used food contact surfaces viz plastic, cement and stainless steel were studied. Biofilm formation of both the isolates showed a similar trend with the highest density being on plastic followed by cement and steel. Salmonella weltevreden formed biofilm with a cell density of 3.4 x 10(7), 1.57 x 10(6) and 3 x 10(5) cfu/cm2 on plastic, cement and steel respectively while Salmonella FCM 40 biofilm on plastic, cement and steel were of the order of 1.2 x 10(7), 4.96 x 10(6) and 2.23 x 10(5) cfu/cm2 respectively. The sensitivity of the biofilm cells grown on these surfaces to different levels of two sanitizers namely hypochlorite and iodophor for varying exposure times was studied. Biofilm cells offered greater resistance when compared to their planktonic counterparts. Such biofilm cells in a food processing unit are not usually removed by the normal cleaning procedure and therefore could be a source of contamination of foods coming in contact with such surfaces.

Animals↗
PubMed1998

Significance of microbial biofilms in food industry: a review.

Biofilms have been of considerable interest in the context of food hygiene. Of special significance is the ability of microorganisms to attach and grow on food and food-contact surfaces under favourable conditions. Biofilm formation is a dynamic process and different mechanisms are involved in their attachment and growth. Extracellular polymeric substances play an important role in the attachment and colonization of microorganisms to food-contact surfaces. Various techniques have been adopted for the proper study and understanding of biofilm attachment and control. If the microorganisms from food-contact surfaces are not completely removed, they may lead to biofilm formation and also increase the biotransfer potential. Therefore, various preventive and control strategies like hygienic plant lay-out and design of equipment, choice of materials, correct use and selection of detergents and disinfectants coupled with physical methods can be suitably applied for controlling biofilm formation on food-contact surfaces. In addition, bacteriocins and enzymes are gaining importance and have an unique potential in the food industry for the effective biocontrol and removal of biofilms. These newer biocontrol strategies are considered important for the maintenance of biofilm-free systems, for quality and safety of foods.

Bacterial Adhesion↗
PubMed2003

Acid tolerance response of biofilm cells of Streptococcus mutans.

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.

Adaptation, Physiological↗
PubMed2003

Effect of fosfomycin alone and in combination with N-acetylcysteine on E. coli biofilms.

Four slime-producing uropathogenic Escherichia coli strains were used to investigate the activity of fosfomycin and N-acetylcysteine (NAC) against biofilms developed on 96-well polystyrene tissue culture plates. Biofilms aged, respectively, 5 (initial) and 48 h (mature) and two fosfomycin concentrations (128 and 2000 mg/l) were used. The effect of various levels (0.007-8 mg/ml) of NAC alone and in combination with fosfomycin on the formation or disruption of biofilms was assessed. Following exposure to the drugs, the percentage of residual slime relative to the control, ranged from 62.5-100 to 26.2-64.1% in the presence of 0.007 and 8 mg/ml of NAC. After treatment of pre-formed biofilms with NAC at the highest concentrations used, the remaining exopolysaccharide matrix was reduced to 25-68% of the amount found with the untreated control. Exposure to fosfomycin at 2000 mg/l reduced biofilms 40-57 and 41-49% for the initial and mature forms, respectively. Fosfomycin was more active at 2000 mg/l combined with NAC 2 mg/ml. Under these conditions initial and mature biofilms were reduced 66-80 and 60-73%, respectively. NAC, when used in combination, enhanced fosfomycin bactericidal activity producing a 99-99.9% reduction in viable cells. Fosfomycin and NAC at concentrations achievable in urine displayed a synergistic effect promoting both the formation of biofilms and reduction of sessile cell viability.

Acetylcysteine↗
PubMed1999

An in vitro study of the use of photodynamic therapy for the treatment of natural oral plaque biofilms formed in vivo.

Seven-day oral plaque biofilms have been formed on natural enamel surfaces in vivo using a previously reported in situ device. The devices are then incubated with a cationic Zn(II) phthalocyanine photosensitizer and irradiated with white light. Confocal scanning laser microscopy (CSLM) of the biofilms shows that the photosensitizer is taken up into the biomass of the biofilm and that significant cell death is caused by photodynamic therapy (PDT). In addition, the treated biofilms are much thinner than the control samples and demonstrate a different structure from the control samples, with little evidence of channels and a less dense biomass. Transmission electron microscopy (TEM) of the in vivo-formed plaque biofilms reveals considerable damage to bacteria in the biofilm, vacuolation of the cytoplasm and membrane damage being clearly visible after PDT. These results clearly demonstrate the potential value of PDT in the management of oral biofilms.

Biofilms↗