Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine

The role of two sigma factors, AlgT and RpoS, in mediating Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine was investigated. Two knock out mutant strains, SS24 (rpoS-) and PAO6852 (algT-), were compared with a wild type, PAO1, in their susceptibility to monochloramine and hydrogen peroxide. When grown as biofilms on alginate gel beads (mean untreated areal cell density 3.7 +/- 0.27 log cfu cm-2) or on glass slides (mean untreated areal cell density 7.6 +/- 0.9 log cfu cm-2), wild type bacteria exhibited reduced susceptibility to both antimicrobial agents in comparison with suspended cells. On alginate gel beads, all strains were equally resistant to monochloramine. rpoS- and algT- gel bead biofilms of 24-hour-old were more susceptible to hydrogen peroxide disinfection than were biofilms formed by PAO1. Biofilm disinfection rate coefficients for the two mutant strains were statistically indistinguishable from planktonic disinfection rate coefficients, indicating complete loss of biofilm resistance. While 48-hour-old algT- biofilm cells became resistant to hydrogen peroxide, 48-hour-old rpoS- biofilm cells remained highly susceptible. With the thicker biofilms formed on glass coupons, all strains were equally resistant to both hydrogen peroxide and monochloramine. It is concluded that while RpoS and AlgT may play a transient role in protecting thin biofilms from hydrogen peroxide, these sigma factors do not mediate resistance to monochloramine and do not contribute significantly to the hydrogen peroxide resistance of thick biofilms.     

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.     

Physiological state of epiphytic bacteria on submerged stems of the reed Phragmites australis compared with planktonic bacteria in gravel-pit ponds

Suspensions of epiphytic bacteria from submerged stems of Phragmites australis , collected from a gravel-pit pond, were prepared by treatment in a stomacher and by brushing. The two procedures were equally successful in dislodging bacteria. These epiphytic bacteria were compared with planktonic bacteria in water samples from within the reed bed. Colony-forming units (cfu) as a percentage of acridine-orange direct counts (AODCs), percentage of cells capable of intracellular reduction of 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, percentage of cells able to form microcolonies, and cell length were all greater for epiphytic bacteria. Epiphytic bacteria of P. australis from six further gravel-pit ponds were also compared with bacterioplankton; cfu as a percentage of AODCs, and percentage of cells capable of INT reduction were again greater for epiphytic bacteria. Because the epiphytic bacteria in these non-organically-enriched, gravel-pit ponds were physiologically different from the planktonic bacteria it is suggested that there was not continual casual exchange, by largely identical bacterial cells, between the epiphytic and planktonic mode. Instead, epiphytic and planktonic populations were independent of each other and/or if there was exchange then bacteria were more successful while in the attached mode, perhaps because of greater organic-nutrient availability at the stem surface.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

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.     

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.     

Laser impact on marine planktonic diatoms: an experimental study using a flow cytometry system

A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum. Three flow speeds, i.e. 9, 18 and 27 ml min-1 and three laser fluences, i.e. 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study. The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls. Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species). A maximum mortality of 0.77 log10 (about 83%) for C. gracilis and 0.68 log10 (about 78%) for S. costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence. The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C. gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S. costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2. In general, mortality increased with an increase in the laser fluence. The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures. The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water.     

Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case, resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 10–15 doi:10.1038/sj.jim.7000256 Received 29 October 2001/ Accepted in revised form 18 March 2002     

Biofilm formation by Pseudoalteromonas ruthenica and its removal by chlorine

The distribution of a recently described marine bacterium, SBT 033 GenBank Accession No. AY723742), Pseudoalteromonas ruthenica, at the seawater intake point, outfall and mixing point of an atomic power plant is described, and its ability to form biofilm was investigated. The effectiveness of the antifouling biocide chlorine in the inactivation of planktonic as well as biofilm cells of P. ruthenica was studied in the laboratory. The results show that the planktonic cells were more readily inactivated than the cells enclosed in a biofilm matrix. Viable counting showed that P. ruthenica cells in biofilms were up to 10 times more resistant to chlorine than those in liquid suspension. Using confocal laser scanning microscopy it was shown that significant detachment of P. ruthenica biofilm developed on a glass substratum could be accomplished by treatment with a dose of 1 mg l-1 chlorine. Chlorine-induced detachment led to a significant reduction in biofilm thickness (up to 69%) and substratum coverage (up to 61%), after 5-min contact time. The results show that P. ruthenica has a remarkable ability to form biofilms but chlorine, a common biocide, can be used to effectively kill and detach these biofilms.     

Efficacy of bromochlorodimethylhydantoin againstLegionella pneumophila in industrial cooling water

Summary Free residual chlorine and bromine can be generated in water from bromochlorodimethylhydantoin (BCDMH). Efficacy of chlorine from inorganic sources has been studied extensively, but there is much less information on the efficacy of bromine againstL. pneumophila; only a few efficacy studies of organically-derived. halogen appear in the literature and the results from different studies conflict or are difficult to interpret. This paper describes the efficacy of halogen from BCDMH against planktonic, pure cultureL. pneumophila in an industrial cooling water. There was no difference in efficacy between halogen derived from organic or inorganic sources in controlled laboratory experiments. Effective doses in laboratory studies cannot be translated directly to field applications because of significant differences in the microbiology. However, the data suggest that disinfection (>99.9% reduction in viability within 10 min) of planktonic, pure cultureL. pneumophila can be achieved with about 1 ppm free residual halogen (expressed as chlorine) from BCDMH in a typical industrial cooling water.     

The role of surface physicochemical properties in determining the distribution of the autochthonous microflora in mineral water bottles

Investigation of the distribution of the viable autochthonous microflora in three brands of 1-2-month-old bottled mineral water showed that 1.8 x 10(4) (S.E.M. 8.9 x 10(3), n = 5) to 1.2 x 10(5) (S.E.M. 1.3 x 10(4), n = 5) cfu ml-1 were planktonic cells while 11 (S.E.M. 4, n = 5)-632 (S.E.M. 176, n = 5) cfu cm-2 were found in the biofilm. The biofilm represented between 0.03 and 1.79% of the total viable microbial population in the 1.5 litre bottles studied. Scanning electron microscopy studies showed that the cells adhering to the polyethylene terephthalate (PET) bottles were predominantly rod-shaped, sparsely distributed over the surface. In contrast, the cells adhering to the high density polyethylene (HDPE) caps were found to be mainly clumps of coccoid cells, suggesting that the bottle may provide different microhabitats for different microfloras. Large-scale roughness, such as that observed as lettering inside the cap (average height (z) = 93 microns) was associated with a 46-fold increase in cell numbers. Increased small-scale roughness, as measured by atomic force microscopy on PET and HDPE surfaces (average roughness (Ra) = 5-551 (nm), showed no correlation with adhesion. Investigations of surface hydrophobicity by the sessile drop technique showed that contact angles (theta) were greater on the HDPE caps (theta = 89-96 degrees) than on the PET surfaces (theta = 69-80 degrees). However, no correlation was found between contact angle and attached cell numbers. Measurements of surface electrostatic charge by streaming potential showed that the PET carried an overall negative charge, measuring -15.9 to -16.6 mV in mineral water. No significant change in charge occurred when the monomer composition of the PET was altered. It was concluded that surface roughness, in particular the scale of surface topographical features, is the most important physicochemical surface characteristic determining the distribution of the autochthonous microflora in mineral water bottles.     

Biofilm susceptibility to metal toxicity

This study compared bacterial biofilm and planktonic cell susceptibility to metal toxicity by evaluating the minimum inhibitory concentration (MIC), the planktonic minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) using the MBEC device. In total, 17 metal cations and oxyanions, chosen to represent groups VIB to VIA of the periodic table, were each tested on biofilm and planktonic cultures of Escherichia coli JM109, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. In contrast to control antibiotic assays, where biofilm cultures were 2 to 64 times less susceptible to killing than logarithmically growing planktonic bacteria, metal compounds killed planktonic and biofilm cultures at the same concentration in the vast majority of combinations. Our data indicate that, under the conditions reported, growth in a biofilm does not provide resistance to bacteria against killing by metal cations or oxyanions.     

A note on the physiological state of epiphytic bacteria compared with planktonic bacteria in two organically-enriched watercourses

Epiphytic bacteria were compared with planktonic bacteria in two watercourses enriched with sewage works effluent. Colony-forming units as a percentage of acridine orange direct counts were higher for epiphytic bacteria in one watercourse. The percentage of cells capable of intracellular reduction of 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan was higher for epiphytic bacteria in both watercourses. The greater viability of epiphytic over planktonic bacteria was less evident, however, than in previously studied non-enriched gravel pit ponds. This suggests that the greater availability of dissolved organic nutrients in the enriched watercourses reduced stress on planktonic bacteria.     

蓝光对浮游状态和生物膜内铜绿假单胞菌的抗菌作用

目的：探讨450 nm-470 nm可见光（蓝光）是否具有杀灭浮游状态和生物膜内铜绿假单胞菌的作用。方法：分别采用不同能量密度的蓝光照射浮游状态铜绿假单胞菌,与红光对照组、空白对照组相比,将照射后细菌采用平板涂板法评价蓝光杀菌效果;制作铜绿假单胞菌生物膜模型,16 J/cm2能量密度蓝光照射后通过激光共聚焦显微镜和扫描电子显微镜观察生物膜内细菌存活情况以及生物膜结构变化。结果：与空白对照组相比,2 J/cm2及以上能量密度组蓝光照射后,细菌数目明显减少,杀菌率明显增加（P〈0.05）,并呈剂量效应关系;16 J/cm2能量密度光照后生物膜内细菌死亡数较空白对照组明显增加且生物膜结构变稀疏。结论：450 nm-470 nm可见光（蓝光）具有高效杀灭浮游状态和生物膜内铜绿假单胞菌的作用。     

The relationship between pipe material and biofilm formation in a laboratory model system

The aim of this study was to compare biofilm accumulation and heterotrophic bacterial diversity on three pipe materials-cast iron, medium density polyethylene (MDPE), and unplasticised polyvinyl chloride (uPVC) - using a laboratory model system run over a short period (21 d) and a longer period (7 months). Newly Modified Robbins Devices (nMRD) were run in parallel, each containing 25 discs of one material with cold tap water flowing through the devices at 3 ml min(-1) (Reynolds Number 9.05) for 21 d. The numbers of bacteria on each material increased exponentially between 0 and 11 d when the biofilm viable count remained constant. The mean doubling times of the heterotrophic population on the materials during the exponential phase was 13.2 h for cast iron and 15.6 h for MDPE and uPVC. The same experiment was repeated under different environmental conditions with a lower temperature, higher free chlorine and lower number of organisms ml(-1) of incoming water. The exponential phase lengthened to 16 d but the steady state count remained the same. The mean viable count after 21 d and after 7 months was on average 97% higher on cast iron than on the other materials. Very few different colony types were isolated from each material with the largest number (nine) recovered from cast iron. The numbers of planktonic bacteria in the effluent water leaving each of the nMRDs directly correlated with the numbers in the biofilm phase on each of the materials. In addition the distribution and thickness of the biofilms on the MDPE and uPVC were observed using confocal scanning laser microscopy. In conclusion, MDPE and uPVC support the lowest numbers of bacteria in a steady state biofilm in the short term (21 d) and over a longer term (7 months). The diversity of heterotrophic bacteria was greatest on cast iron.     

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.     

The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm

A constant-depth film fermenter (CDFF) was used to culture a steady-state multispecies biofilm consisting of one strain each of Listeria monocytogenes, Pseudomonas fragi and Staphylococcus xylosus. These bacteria were initially grown together in a conventional chemostat to achieve a steady state before being inoculated into the CDFF over an 18-h period. A dilute tryptone soya broth (TSB) medium was supplied to the CDFF and the biofilm allowed to develop over a 28-d period. This mature biofilm was then subjected to increasing levels of sodium hypochlorite solution to measure any antimicrobial effect. The three organisms were seen to reach a steady state after 6 d in the chemostat before being transferred to the CDFF where the mature multispecies biofilm reached steady state at 17 d. Listeria monocytogenes in both planktonic and biofilm growth stabilized at 1. 8 and 1.5%, respectively, of the total plate counts, while Ps. fragi and Staph. xylosus were the predominant organisms in the biofilm at 59% and 39.5%, respectively, of the total microbial population. Steady-state biofilms in the CDFF were exposed to increasing strengths of sodium hypochlorite; 200, 500 and 1000 p.p.m. free chlorine, but a substantial two-log cycle drop in bacterial numbers was only achieved at 1000 p.p.m. free chlorine. In planktonic culture all three organisms were completely eliminated when exposed to 10 p.p.m. free chlorine for a 30-s period.     

Distribution of bacteria within operating laboratory water purification systems.

Experiments were conducted to measure communities of bacteria within operating ultrapure water treatment systems intended for laboratory use. Samples from various locations within Milli-Q Plus and Milli-Q UV Plus systems were analyzed for populations of planktonic bacteria at weekly intervals over 3 months of operation. Relatively high initial densities of planktonic bacteria (10(2) to 10(3) bacteria per ml) were seen within both units when they were challenged with source water of poor quality, although the product water continued to be acceptable with regard to bacterial numbers, resistivity, and endotoxin concentration. Under more normal operating conditions, significant differences were seen in planktonic populations throughout the systems with excellent product water quality. A great deal of variability was observed in biofilm populations analyzed from various system surfaces after 3 months of operation. The concentrations of planktonic bacteria and biofilm densities were much lower in the unit containing a UV lamp. These findings suggest that a range of microenvironmental conditions exist within purified water systems, leading to variable populations of bacteria. However, product water of excellent quality was obtained despite the bacterial communities.     

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.