Assessment of bismuth thiols and conventional disinfectants on drinking water biofilms

AIMS: Biofilms in water distribution systems represent a far more significant reservoir of micro-organisms than the water phase. Biofilms are (i) resistant to disinfectants, (ii) nuclei for microbial regrowth, (iii) a refuge for pathogens, (iv) accompanied by taste and odour problems, and (v) corrode surfaces. The effects of the current strategies for disinfection of drinking water systems in large buildings (chlorination, copper and silver ionization, and hyper-heating) were compared with a new generation of bismuth thiol (BT) biocides. METHODS AND RESULTS: Multispecies biofilms were treated with 0.8 mg l(-1) of free chlorine, 400 and 40 microg l(-1) of copper and silver ions, respectively, at 55 and 70 degrees C, and bismuth-2,3-dimercaptopropanol (BisBAL). Furthermore, the effect of combined heat and BisBAL on planktonic cell viability was examined in monoculture using Escherichia coli suspensions. Inactivation rates for BisBAL were similar to copper-silver ions, where the effects were slower than for free chlorine or temperature. The BisBAL effect on E. coli monocultures was augmented greatly by increasing temperatures. CONCLUSIONS: Like copper-silver ions, BTs show more persistent residual effects than chlorine and hyper-heating in water systems. BT efficiency increased with temperature. Like copper-silver ions, BT action is relatively slow. SIGNIFICANCE AND IMPACT OF THE STUDY: BT presents a new approach to containing water biofilms. BT action is not as rapid, but is more thorough than chlorine, and less caustic. BTs may also be more efficacious in hot water systems. At sub-minimum inhibition concentration levels, BTs uniquely inhibit bacterial exopolysaccharide, thereby retarding biofilm formation. Thus, the combination of bactericidal and residual effects may prevent slime build-up in hot water systems.     

Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence

AIMS: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system. METHODS AND RESULTS: A pilot-scale domestic water system consisting of seven galvanized steel re-circulation loops and copper dead legs was constructed. Culture techniques, confocal laser scanning microscopy after fluorescent in situ hybridization and viability staining with the BacLight LIVE/DEAD kit were used for planktonic and biofilm flora monitoring. Before starting the treatments, the system was highly contaminated with Legionella pneumophila and biofilm populations mainly consisted of beta-proteobacteria. In the water and the biofilm of the loops, continuous application of chlorine dioxide (0.5 mg l(-1)), or chlorine (2.5 mg l(-1)) were very effective in reducing the microbial flora, including L. pneumophila. Heterotrophic bacteria, although strongly reduced, were still detectable after ozone application (0.5 mg l(-1)), whereas with monochloramine (0.5 mg l(-1)) and copper-silver ionization (0.8/0.02 mg l(-1)), the contamination remained significantly higher. Monochloramine and copper-silver did not remove the biofilm. During copper-silver application, Legionella re-growth was observed. Only chlorine dioxide led to detectable effects in the dead leg. Amoebae could not be eliminated, and after interrupting the treatments, L. pneumophila quickly recovered their initial levels, in all cases. CONCLUSIONS: Chlorine dioxide, applied as a continuous treatment, was identified in this study as the most efficient for controlling L. pneumophila in a domestic water system. Chlorine dioxide showed a longer residual activity, leading to improved performance in the dead leg. Amoebae resisted to all the treatments applied and probably acted as reservoirs for L. pneumophila, allowing a quick re-colonization of the system once the treatments were interrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of microbial contamination requires maintenance of a constant disinfectant residual throughout the water system. Treatment strategies targeting free-living amoebae should lead to improved control of L. pneumophila. Such treatment strategies still have to be investigated.     

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Peracetic acid as an alternative wastewater disinfectant to chlorine dioxide

AIMS: The aim of this study was to compare the efficiency of peracetic acid with that of chlorine dioxide in the disinfection of wastewater from a sewage treatment plant (serving about 650 000 inhabitants) that has been using peracetic acid as a disinfectant since 1998. METHODS AND RESULTS: A total of 23 samplings were made, each consisting of three samples: from secondary effluent, effluent disinfected with 2 mg l(-1) of peracetic acid and effluent disinfected with 2.2 mg l(-1) of chlorine dioxide (contact time 20 min). For each sample, measurements were made of the heterotrophic plate count at 36 degrees C, total and faecal coliforms, Escherichia coli, enterococci, pH, suspended solids and chemical oxygen demand (COD). During the first phase of the experiment the peracetic acid was seen to be less efficient than chlorine dioxide. To improve the disinfectant action a system of mechanical agitation was added which led to a greater efficiency in the inactivation of bacteria of faecal origin. CONCLUSIONS: Both products were found to be influenced by the level of microbial contamination, the amount of suspended solids and COD but not by the pH of the effluent before disinfection. The immediate mixing of the wastewater and disinfectant caused a greater reduction in enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Since peracetic acid was seen to produce a high abatement of micro-organisms, it can be considered as a valid alternative to chlorine dioxide in the disinfection of wastewaters.     

Chlorine dioxide disinfection of single and dual species biofilms, detached biofilm and planktonic cells

Disinfection efficacy testing is usually done with planktonic cells or more recently, biofilms. While disinfectants are much less effective against biofilms compared to planktonic cells, questions regarding the disinfection tolerance of detached biofilm clusters remain largely unanswered. Burkholderia cepacia and Pseudomonas aeruginosa were grown in chemostats and biofilm tubing reactors, with the tubing reactor serving as a source of detached biofilm clusters. Chlorine dioxide susceptibility was assessed for B. cepacia and P. aeruginosa in these three sample types as monocultures and binary cultures. Similar doses of chlorine dioxide inactivated samples of chemostat and tubing reactor effluent and no statistically significant difference between the log(10) reductions was found. This contrasts with chlorine, shown previously to be generally less effective against detached biofilm particles. Biofilms were more tolerant and required chlorine dioxide doses ten times higher than chemostat and tubing reactor effluent samples. A second species was advantageous in all sample types and resulted in lower log(10) reductions when compared to the single species cultures, suggesting a beneficial interaction of the species.     

Disinfection of feline calicivirus (a surrogate for Norovirus) in wastewaters

AIMS: To compare the inactivation of feline calicivirus (FCV) (a surrogate for Norovirus, NV) with the reduction of a bacterial water quality indicator (Escherichia coli), a human enteric virus (poliovirus) and a viral indicator (MS2, FRNA bacteriophage), following the disinfection of wastewaters. METHODS AND RESULTS: Bench-scale disinfection experiments used wastewater (sterilized by gamma-irradiation) seeded with laboratory-cultured organisms. Seeded primary effluent was treated with different doses of applied free chlorine (8, 16 and 30 mg l(-1)). FCV and E. coli were easily inactivated by >4 log10, within 5 min with a dose of 30 mg l(-1) of applied chlorine. Poliovirus was more resistant and a reduction of 2.85 log10 was seen after 30 min, MS2 was the most resistant organism (1 log10 inactivation). In further experiments seeded secondary effluent was treated with different doses of u.v. irradiation. To achieve a 4-log10 reduction of E. coli, FCV, poliovirus and MS2 doses of 5.32, 19.04, 27.51 and 62.50 mW s cm(-2), respectively, were required. CONCLUSIONS: Feline calicivirus and E. coli seeded in primary wastewater were very susceptible to chlorination compared with poliovirus and MS2. In contrast, FCV seeded in secondary wastewater was more resistant to u.v. irradiation than E. coli but more sensitive than poliovirus and MS2. SIGNIFICANCE AND IMPACT OF THE STUDY: FRNA phage was more resistant to inactivation than all the viruses tested. This suggests FRNA phage would be a useful and conservative indicator of virus inactivation following disinfection of wastewaters with chlorination or u.v. irradiation.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Abstract

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

A pilot study of bacteriological population changes through potable water treatment and distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

Formation of biofilms in drinking water distribution networks,a case study in two cities in Finland and Latvia

The formation of biofilms in drinking water distribution networks is a significant technical, aesthetic and hygienic problem. In this study, the effects of assimilable organic carbon, microbially available phosphorus (MAP), residual chlorine, temperature and corrosion products on the formation of biofilms were studied in two full-scale water supply systems in Finland and Latvia. Biofilm collectors consisting of polyvinyl chloride pipes were installed in several waterworks and distribution networks, which were supplied with chemically precipitated surface waters and groundwater from different sources. During a 1-year study, the biofilm density was measured by heterotrophic plate counts on R2A-agar, acridine orange direct counting and ATP-analyses. A moderate level of residual chorine decreased biofilm density, whereas an increase of MAP in water and accumulated cast iron corrosion products significantly increased biofilm density. This work confirms, in a full-scale distribution system in Finland and Latvia, our earlier in vitro finding that biofilm formation is affected by the availability of phosphorus in drinking water.     

A Pilot Study of Bacteriological Population Changes through Potable Water Treatment and Distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

Physiological responses of bacteria in biofilms to disinfection.

In situ enumeration methods using fluorescent probes and a radioisotope labelling technique were applied to evaluate physiological changes of Klebsiella pneumoniae within biofilms after disinfection treatment. Chlorine (0.25 mg of free chlorine per liter [pH 7.2]) and monochloramine (1 mg/liter [pH 9.0]) were employed as disinfectants in the study. Two fluorgenic compounds, 5-cyano-2,3-ditolyl tetrazolium chloride and rhodamine 123, and tritiated uridine incorporation were chosen for assessment of physiological activities. Results obtained by these methods were compared with those from the plate count and direct viable count methods. 5-Cyano-2,3-ditolyl tetrazolium chloride is an indicator of bacterial respiratory activity, rhodamine 123 is incorporated into bacteria in response to transmembrane potential, and the incorporation of uridine represents the global RNA turnover rate. The results acquired by these methods following disinfection exposure showed a range of responses and suggested different physiological reactions in biofilms exposed to chlorine and monochloramine. The direct viable count response and respiratory activity were affected more by disinfection than were the transmembrane potential and RNA turnover rate on the basis of comparable efficiency as evaluated by plate count enumeration. Information revealed by these approaches can provide different physiological insights that may be used in evaluating the efficacy of biofilm disinfection.     

The influence of the chemical composition of drinking water on cuprosolvency by biofilm bacteria

AIMS: This study investigated the influence of water chemistry on copper solvation (cuprosolvency) by pure culture biofilms of heterotrophic bacteria isolated from copper plumbing. METHODS AND RESULTS: Heterotrophic bacteria isolated from copper plumbing biofilms including Acidovorax delafieldii, Flavobacterium sp., Corynebacterium sp., Pseudomonas sp. and Stenotrophomonas maltophilia were used in laboratory coupon experiments to assess their potential for cuprosolvency. Sterile copper coupons were exposed to pure cultures of bacteria to allow biofilm formation and suspended in drinking waters with different chemical compositions. Sterile coupons not exposed to bacteria were used as controls. After 5 days of incubation, copper release and biofilm accumulation was quantified. The results demonstrated that cuprosolvency in the control experiments was influenced by water pH, total organic carbon (TOC) and conductivity. Cuprosolvency in the presence of biofilms correlated with the chemical composition of the water supplies particularly pH, Langeliers Index, chloride, alkalinity, TOC and soluble phosphate concentrations. CONCLUSIONS: The results suggest water quality may influence cuprosolvency by biofilms present within copper plumbing pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for water chemistry to influence cuprosolvency by biofilms may contribute to the sporadic nature of copper corrosion problems in distribution systems.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Colonization and Disinfection of Biofilms Hosting Coliform-Colonized Carbon Fines

The documented release of carbon fines from granular activated carbon filters is a concern for drinking water utilities, since these particles may carry coliform and even pathogenic bacteria through the disinfection barrier. Such a breakthrough could have an impact on distribution system biofilms. Using total cell counts, specific monoclonal antibody staining, and computerized image analysis, we monitored the colonization of introduced Klebsiella pneumoniae associated with carbon fines in mixed-population biofilms. The particles transported the coliforms to the biofilms and allowed successful colonization. Chlorine (0.5 mg/liter) was then applied as a disinfectant. Most K. pneumoniae along with the carbon fines left the biofilm under these conditions. The impact of chlorine was greater on the coliform bacteria and carbon fines than on the general fixed bacterial population. However, 10% of the introduced coliforms and 20% of the fines remained in the biofilm. The possibility that this represents a mechanism for bacteria of public health concern to be involved in regrowth events is discussed.     

Chlorine dioxide disinfection of single and dual species biofilms,detached biofilm and planktonic cells

Disinfection efficacy testing is usually done with planktonic cells or more recently, biofilms. While disinfectants are much less effective against biofilms compared to planktonic cells, questions regarding the disinfection tolerance of detached biofilm clusters remain largely unanswered. Burkholderia cepacia and Pseudomonas aeruginosa were grown in chemostats and biofilm tubing reactors, with the tubing reactor serving as a source of detached biofilm clusters. Chlorine dioxide susceptibility was assessed for B. cepacia and P. aeruginosa in these three sample types as monocultures and binary cultures. Similar doses of chlorine dioxide inactivated samples of chemostat and tubing reactor effluent and no statistically significant difference between the log₁₀ reductions was found. This contrasts with chlorine, shown previously to be generally less effective against detached biofilm particles. Biofilms were more tolerant and required chlorine dioxide doses ten times higher than chemostat and tubing reactor effluent samples. A second species was advantageous in all sample types and resulted in lower log₁₀ reductions when compared to the single species cultures, suggesting a beneficial interaction of the species.     

Relationship between physiochemical properties, aggregation and u.v. inactivation of isolated indigenous spores in water

AIMS: The objective of the study was to compare ultraviolet (u.v.) inactivation kinetics of indigenous aerobic spores in surface water with their laboratory-cultured spore isolates and to investigate the relationship between physicochemical characteristics and u.v. inactivation kinetics of spore isolates. METHODS AND RESULTS: Lake water samples were analysed for the presence of indigenous aerobic spores. Different bacterial isolates from the heterogeneous indigenous population were genetically characterized, resporulated and examined for hydrophobicity, surface charge, particle size distribution and survival at different u.v. 254 nm fluence levels. Cultured isolated spores exhibited a three-stage inactivation curve consisting of shoulder, first order and tailing regions whereas indigenous spores exhibited only one stage of linear kinetics. Hydrophobicity of the Bacillus spore isolates was inversely related to the extent of u.v. inactivation before tailing occurred. CONCLUSIONS: Tailing in the u.v. inactivation curves results from aggregation of a portion of the spore population because of hydrophobic interactions, supporting the link between aggregation of spores, hydrophobicity and u.v. inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of the link between spore physicochemical parameters and u.v. disinfection performance furthers the understanding of factors that affect inactivation of microbes in natural waters supplied to drinking water treatment plants.     

Measurement of chlorine dioxide penetration in dairy process pipe biofilms during disinfection

Biofilms are considered a significant health risk in the food and dairy industries because they can harbor pathogens, and direct contact with them can lead to food contamination. Biofilm control is often performed using strong oxidizing agents like chlorine and peracetic acid. Although chlorine dioxide (ClO₂) is being used increasingly to control microbiological growth in a number of different industries, not much is known about disinfection in biofilms using chlorine dioxide. In this study, a microelectrode originally made for chlorine detection was modified to measure the profiles of chlorine dioxide in biofilm as a function of depth into the biofilm. In addition, discarded microelectrodes proved useful for in situ direct measurement of biofilm thicknesses. The chlorine dioxide microelectrode had a linear response when calibrated up to a ClO₂ concentration of 0.4 mM. ClO₂ profiles showed depletion of disinfectant at 100 μm in the biofilm depth, indicating that ClO₂ may not reach bacteria in a biofilm thicker than this using a 25 mg/l solution.     

Multi-species biofilms defined from drinking water microorganisms provide increased protection against chlorine disinfection

A model biofilm, formed of multiple species from environmental drinking water, including opportunistic pathogens, was created to explore the tolerance of multi-species biofilms to chlorine levels typical of water-distribution systems. All species, when grown planktonically, were killed by concentrations of chlorine within the World Health Organization guidelines (0.2–5.0?mg?l^?1). Higher concentrations (1.6–40-fold) of chlorine were required to eradicate biofilm populations of these strains, ～70% of biofilms tested were not eradicated by 5.0?mg?l^?1 chlorine. Pathogenic bacteria within the model multi-species biofilms had an even more substantial increase in chlorine tolerance; on average ～700–1100?mg?l^?1 chlorine was required to eliminate pathogens from the biofilm, 50–300-fold higher than for biofilms comprising single species. Confocal laser scanning microscopy of biofilms showed distinct 3D structures and multiple cell morphologies and arrangements. Overall, this study showed a substantial increase in the chlorine tolerance of individual species with co-colonization in a multi-species biofilm that was far beyond that expected as a result of biofilm growth on its own.     

Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.