Disinfection of bacterial biofilms in pilot-scale cooling tower systems

The impact of continuous chlorination and periodic glutaraldehyde treatment on planktonic and biofilm microbial communities was evaluated in pilot-scale cooling towers operated continuously for 3 months. The system was operated at a flow rate of 10,080 l day^?1. Experiments were performed with a well-defined microbial consortium containing three heterotrophic bacteria: Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. The persistence of each species was monitored in the recirculating cooling water loop and in biofilms on steel and PVC coupons in the cooling tower basin. The observed bacterial colonization in cooling towers did not follow trends in growth rates observed under batch conditions and, instead, reflected differences in the ability of each organism to remain attached and form biofilms under the high-through flow conditions in cooling towers. Flavobacterium was the dominant organism in the community, while P. aeruginosa and K. pneumoniae did not attach well to either PVC or steel coupons in cooling towers and were not able to persist in biofilms. As a result, the much greater ability of Flavobacterium to adhere to surfaces protected it from disinfection, whereas P. aeruginosa and K. pneumoniae were subject to rapid disinfection in the planktonic state.     

Comparison of the efficacy of free residual chlorine and monochloramine against biofilms in model and full scale cooling towers.

The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion. Biofilms accumulate on all submerged industrial and environmental surfaces. The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm. The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower. The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers. A cooling tower and a laboratory model recirculating water system were used as biofilm reactors. Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers. Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal. Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons. Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.     

Comparison of the Efficacy of Free Residual Chlorine and Monochloramine against Biofilms in Model and Full Scale Cooling Towers

The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion. Biofilms accumulate on all submerged industrial and environmental surfaces. The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm. The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower. The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers. A cooling tower and a laboratory model recirculating water system were used as biofilm reactors. Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers. Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal. Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons. Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.     

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.     

Biofilm parameters influencing biocide efficacy

The influence of biofilm areal cell density, species composition, and the presence of abiotic particles on the disinfection and removal of bacterial biofilms by monochloramine was investigated. Mono- and binary population biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown on stainless-steel slides in a continuous flow annular reactor. Biofilms were treated in the reactor with a pulse/step dose of 4 mg/L monochloramine for 2 h. Biofilm samples were disaggregated and assayed for colony formation on R2A agar and for total cell numbers by acridine orange direct counts. These data were used to determine apparent first order rate coefficients for the processes of disinfection and detachment. Disinfection rate coefficients exceeded detachment rate coefficients by as much as an order of magnitude and the two coefficients were poorly correlated (r = 0.272). The overall decay rate coefficient (disinfection plus detachment) depended strongly on the initial biofilm areal cell density. It displayed a parabolic dependence on cell density with a maximum near 10(8) cfu/cm(2). This result points to multiple factors influencing biofilm susceptibility to antimicrobial challenge. Decay rates of K. pneumoniae measured in binary population biofilms were comparable with those measured in monopopulation biofilms (p = 0.61). P. aeruginosa decayed more slowly in biofilsm dominated by K. pneumoniae (p = 0.028), indicating some interaction between species. The presence of kaolin and calcium carbonate particles in the biofilm reduced disinfection efficacy. (c) 1995 John Wiley & Sons, Inc.     

Observations of binary population biofilms

Biofilm research has focused on studies of undefined mixed microbial populations and, more recently, on investigations of monopopulation biofilms. In the first case, the biofilm is considered a homogeneous mass, ignoring the properties of individual species. The second case concentrates on the properties and processes of one microbial species in the biofilm. This article describes biofilm experiments conducted with monopopulations of Klebsiella pneumoniae and Pseudomonas aeruginosa and with binary populations of K. pneumoniae and P. aeruginosa. Process rates and stoichiometric coefficients were determined for the monopopulation and for the binary population biofilms and evaluated in light of the species distribution in the latter. Results indicate that neither the specific cellular product formation rate nor the glucose-oxygen stoichiometric ratio of K. pneumoniae or P. aeruginosa in the binary biofilm is affected by the presence of the other species. Consequently, species interaction was not observed. Although the specific cellular growth rate of K. pneumoniae is five times that of P. aeruginosa, the former species did not dominate the microbial population in the biofilm. Possible reasons for this unexpected behavior are discussed.     

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.     

Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then we investigated cell-surface properties. And finally, we tested in vitro susceptibility to antibiotics. The results first indicated that sessile and detached cells have similar planktonic growth kinetics and cell-surface properties, distinguishable from those of planktonic cells. Interestingly, the three populations exhibited different biofilm-forming capacities, suggesting that there is a transitional phenotype between sessile and planktonic states, at least during the first hours following cell detachment. It is important to consider this observation when developing treatments to optimize disinfection processes. Surprisingly, the three populations showed the same antibiotic susceptibility profile.     

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.     

Resistance of biofilms to the catalase inhibitor 3-amino-1,2, 4-triazole

Consortia of catalase positive bacteria consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Klebsiella pneumoniae, in both the planktonic form and as biofilms, disproportionate hydrogen peroxide into oxygen and water. The biofilm, however, continued to disproportionate the hydrogen peroxide in the presence of the catalase inhibitor, 3-amino-1,2,4-triazole, while the planktonic organisms did not. While the bacterial catalase-peroxidase-dismutase system was probably responsible for the disproportionation of hydrogen peroxide in both cases, biofilms resisted inhibition of this enzyme system.     

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

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

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Identification and characterization of a high-affinity zinc uptake system in Neisseria gonorrhoeae

Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic culture. RpoS levels in biofilms, indicated by immunoblot analysis, were similar to levels in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.     

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.     

Direct measurement of chlorine penetration into biofilms during disinfection.

Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.     

Biofilms in drinking water systems: a possible reservoir for Helicobacter pylori

A laboratory model system was utilised to investigate the persistence of Helicobacter pylori in mixed-species heterotrophic biofilms. A single-stage continuous culture vessel was linked to a modified-Robbins device (mRD) incorporating removable stainless steel coupons. The system was innoculated with H. pylori (NCTC 11637) and the fate of the organism monitored by polymerase chain reaction (PCR) analysis. Helicobacter pylori was detected in biofilm material for a period of up to 192 h. Theoretical washout would have occurred at around 48 h thus detection of H. pylori for a prolonged period after theoretical washout suggested that the organism possessed the ability to persist in the mixed-species heterotrophic biofilm. Preliminary studies using heat-inactivated H. pylori showed that the organism was not detected in biofilm material at any time post-challenge suggesting that the persistence of H. pylori in such material was a phenomenon requiring the organism to be in a viable state. Further investigations to assess the biological basis for the association of H. pylori with drinking water biofilms and the risk that this may pose to public health are being undertaken.     

Effect of phosphate availability on biofilm formation in cooling towers

Abstract

Phosphate limitation has been suggested as a preventive method against biofilms. P-limited feed water was studied as a preventive strategy against biofouling in cooling towers (CTs). Three pilot-scale open recirculating CTs were operated in parallel for five weeks. RO permeate was fed to the CTs (1) without supplementation (reference), (2) with supplementation by biodegradable carbon (P-limited) and (3) with supplementation of all nutrients (non-P-limited). The P-limited water contained ≤10?µg PO₄ l^?1. Investigating the CT-basins and coupons showed that P-limited water (1) did not prevent biofilm formation and (2) resulted in a higher volume of organic matter per unit of active biomass compared with the other CTs. Exposure to external conditions and cycle of concentration were likely factors that allowed a P concentration sufficient to cause extensive biofouling despite being the limiting compound. In conclusion, phosphate limitation in cooling water is not a suitable strategy for CT biofouling control.     

Differential biofilm formation and chemical disinfection resistance of sessile cells of listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions

This study aimed to investigate the possible influence of bacterial intra- and interspecies interactions on the ability of Listeria monocytogenes and Salmonella enterica to develop mixed-culture biofilms on an abiotic substratum, as well as on the subsequent resistance of sessile cells to chemical disinfection. Initially, three strains from each species were selected and left to attach and form biofilms on stainless steel (SS) coupons incubated at 15°C for 144 h, in periodically renewable tryptone soy broth (TSB), under either monoculture or mixed-culture (mono-/dual-species) conditions. Following biofilm formation, mixed-culture sessile communities were subjected to 6-min disinfection treatments with (i) benzalkonium chloride (50 ppm), (ii) sodium hypochlorite (10 ppm), (iii) peracetic acid (10 ppm), and (iv) a mixture of hydrogen peroxide (5 ppm) and peracetic acid (5 ppm). Results revealed that both species reached similar biofilm counts (ca. 10(5) CFU cm(-2)) and that, in general, interspecies interactions did not have any significant effect either on the biofilm-forming ability (as this was assessed by agar plating enumeration of the mechanically detached biofilm bacteria) or on the antimicrobial resistance of each individual species. Interestingly, pulsed-field gel electrophoresis (PFGE) analysis clearly showed that the three L. monocytogenes strains did not contribute at the same level either to the formation of mixed-culture sessile communities (mono-/dual species) or to their antimicrobial recalcitrance. Additionally, the simultaneous existence inside the biofilm structure of S. enterica cells seemed to influence the occurrence and resistance pattern of L. monocytogenes strains. In sum, this study highlights the impact of microbial interactions taking place inside a mixed-culture sessile community on both its population dynamics and disinfection resistance.     

Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multi-species biofilm has reduced susceptibility to disinfectants

Legionella pneumophila will infect biofilm-associated protozoa, and in this way might be protected from disinfectants in potable water systems. A base biofilm containing Pseudomonas aeruginosa, Klebsiella pneumoniae, and Flavobacterium spp. was grown on steel coupons in potable water prior to the addition of L. pneumophila and the protozoan H. vermiformis. After 7 d, coupons were removed and treated with 0.5 mgl(-1) free residual chlorine (FRC) or 0.5 mgl(-1) monochloramine (MCA) for 15, 60, or 180 min or 24 h. In a second experiment, only L. pneumophila and the base biofilm organisms were present but with an identical treatment protocol. Treatment of L. pneumophila for 180 min in a system without H. vermiformis resulted in log reductions of 2.07 and 2.11 for FRC and MCA, respectively. When H. vermiformis was present, however, the treatment resulted in log reductions of 0.67 and 0.81 for FRC and MCA, respectively. A similar pattern was observed for 15 and 60 min contact times. These results indicate that L. pneumophila was less susceptible to MCA or FRC when associated with biofilm-associated H. vermiformis in a model potable water biofilm.