Lúcia C. Simões

Species association increases biofilm resistance to chemical and mechanical treatments.

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re(A)). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re(A) were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each others survival to antimicrobials and the biofilm shear stress stability.

Biofilm Interactions between Distinct Bacterial Genera Isolated from Drinking Water

ABSTRACT In the environment, multiple microorganisms coexist as communities, competing for resources and often associated as biofilms. In this study, single- and dual-species biofilm formation by, and specific activities of, six heterotrophic intergeneric bacteria were determined using 96-well polystyrene plates over a 72-h period. These bacteria were isolated from drinking water and identified by partial 16S rRNA gene sequencing. A series of planktonic studies was also performed, assessing the bacterial growth rate, motility, and production of quorum-sensing inhibitors (QSI). This constituted an attempt to identify key attributes allowing bacteria to effectively interact and coexist in a drinking-water environment. We observed that in both pure and dual cultures, all of the isolates formed stable biofilms within 72 h, with specific metabolic activity decreasing, in most cases, with an increase in biofilm mass. The largest single- and dual-biofilm amounts were found for Methylobacterium sp. and the combination of Methylobacterium sp. and Mycobacterium mucogenicum, respectively. Evidences of microbial interactions in dual-biofilm formation, associated with appreciable biomass variation in comparison with single biofilms, were found for the following cases: synergy/cooperation between Sphingomonas capsulata and Burkholderia cepacia, S. capsulata and Staphylococcus sp., and B. cepacia and Acinetobacter calcoaceticus and antagonism between S. capsulata and M. mucogenicum, S. capsulata and A. calcoaceticus, and M. mucogenicum and Staphylococcus sp. A neutral interaction was found for Methylobacterium sp.-M. mucogenicum, S. capsulata-Staphylococcus sp., M. mucogenicum-A. calcoaceticus, and Methylobacterium sp.-A. calcoaceticus biofilms, since the resultant dual biofilms had a mass and specific metabolic activity similar to the average for each single biofilm. B. cepacia had the highest growth rate and motility and produced QSI. Other bacteria producing QSI were Methylobacterium sp., S. capsulata, and Staphylococcus sp. However, only for S. capsulata-M. mucogenicum, S. capsulata-A. calcoaceticus, and M. mucogenicum-Staphylococcus sp., dual-biofilm formation seems to be regulated by the QSI produced by S. capsulata and Staphylococcus sp. and by the increased growth rate of S. capsulata. The parameters assessed by planktonic studies did not allow prediction and generalization of the exact mechanism regulating dual-species biofilm formation between the drinking-water bacteria.

Influence of the Diversity of Bacterial Isolates from Drinking Water on Resistance of Biofilms to Disinfection

ABSTRACT Single- and multispecies biofilms formed by six drinking water-isolated bacterial species were used to assess their susceptibilities to sodium hypochlorite (SHC). In general, multispecies biofilms were more resistant to inactivation and removal than single biofilms. Total biofilm inactivation was achieved only for Acinetobacter calcoaceticus single-species biofilms and for those multispecies biofilms without A. calcoaceticus. Biofilms with all bacteria had the highest resistance to SHC, while those without A. calcoaceticus were the most susceptible. A. calcoaceticus formed single biofilms susceptible to SHC; however, its presence in multispecies biofilms increased their resistance to disinfection.

Potential of the adhesion of bacteria isolated from drinking water to materials

Heterotrophic bacteria (11 genera, 14 species, 25 putative strains) were isolated from drinking water, identified either biochemically or by partial 16s rDNA gene sequencing and their adherence characteristics were determined by two methods: i. thermodynamic prediction of adhesion potential by measuring hydrophobicity (contact angle measurements) and ii. by measuring adherence to eight different substrata (ASI 304 and 316 stainless steel, copper, polyvinyl chloride, polypropylene, polyethylene, silicone and glass). All the test organisms were hydrophilic and inter‐species variation in hydrophobicity occurred only for Comamonas acidovorans. Stainless steel 304 (SS 304), copper, polypropylene (PP), polyethylene (PE) and silicone thermodynamically favoured adhesion for the majority of test strains (>18/25), whilst adhesion was generally less thermodynamically favorable for stainless steel 316 (SS 316), polyvinyl chloride (PVC) and glass. The predictability of thermodynamic adhesion test methods was validated by comparison with 24‐well microtiter plate assays using nine reference strains and three adhesion surfaces (SS 316, PVC and PE). Results for Acinetobacter calcoaceticus, Burkolderia cepacia and Stenotrophomonas maltophilia sp. 2 were congruent between both methods whilst they differed for the other bacteria to at least one material. Only A. calcoaceticus had strongly adherent properties to the three tested surfaces. Strain variation in adhesion ability was detected only for Sphingomonas capsulata. Analysis of adhesion demonstrated that in addition to physicochemical surface properties of bacterium and substratum, biological factors are involved in early adhesion processes, suggesting that reliance on thermodynamic approaches alone may not accurately predict adhesion capacity. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Intergeneric Coaggregation among Drinking Water Bacteria: Evidence of a Role for Acinetobacter calcoaceticus as a Bridging Bacterium

ABSTRACT Intergeneric coaggregation of drinking water bacteria was tested. Acinetobacter calcoaceticus was found not only to autoaggregate but also to coaggregate with four of the five other isolates (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata, and Staphylococcus sp.). In its absence, no coaggregation was found. Interactions were lectin-saccharide mediated. The putative bridging function of A. calcoaceticus was evidenced by multispecies biofilm studies, through a strain exclusion process.

Biofilms in drinking water: problems and solutions

The main goal of water companies is to deliver to each consumer microbiologically safe drinking water (DW), adequate in quantity and delivery pressure and acceptable in terms of taste, odour and appearance. Drinking water distribution systems (DWDS) are known to harbour biofilms, even in the continuous presence of a disinfectant. These biofilms are a source of planktonic bacteria, which will remain present when the water is delivered through a consumer’s tap. The presence of biofilms in DWDS constitutes one of the currently recognized hazards affecting the microbiological quality of the product and may lead to a number of unwanted effects on the organoleptic quality of the distributed water. Importantly, biofilms constitute a persistent reservoir of pathogenic microorganisms, which are responsible for several waterborne diseases. Antimicrobial products, particularly chlorine, have been the main weapons used to disinfect DW. Although this strategy is widespread, there are not yet standardized disinfection strategies with reliable efficacy in the control of biofilms. This review covers the advances in the knowledge of public health problems caused by the presence of biofilms in DWDS and the current strategies for DW disinfection and associated biofilms.

Antimicrobial Activity of Selected Phytochemicals against Escherichia coli and Staphylococcus aureus and Their Biofilms

Abstract Bacteria can be resistant to multiple antibiotics and we are fast approaching a time when antibiotics will not work on some bacterial infections. New antimicrobial compounds are urgently necessary. Plants are considered the greatest source to obtain new antimicrobials. This study aimed to assess the antimicrobial activity of four phytochemicals—7-hydroxycoumarin (7-HC), indole-3-carbinol (I3C), salicylic acid (SA) and saponin (SP)—against Escherichia coli and Staphylococcus aureus, either as planktonic cells or as biofilms. These bacteria are commonly found in hospital-acquired infections. Some aspects on the phytochemicals mode of action, including surface charge, hydrophobicity, motility and quorum-sensing inhibition (QSI) were investigated. In addition, the phytochemicals were combined with three antibiotics in order to assess any synergistic effect. 7-HC and I3C were the most effective phytochemicals against E. coli and S. aureus. Both phytochemicals affected the motility and quorum-sensing (QS) activity, which means that they can play an important role in the interference of cell-cell interactions and in biofilm formation and control. However, total biofilm removal was not achieved with any of the selected phytochemicals. Dual combinations between tetracycline (TET), erythromycin (ERY) and ciprofloxacin (CIP) and I3C produced synergistic effects against S. aureus resistant strains. The overall results demonstrates the potential of phytochemicals to control the growth of E. coli and S. aureus in both planktonic and biofilm states. In addition, the phytochemicals demonstrated the potential to act synergistically with antibiotics, contributing to the recycling of old antibiotics that were once considered ineffective due to resistance problems.

Drinking water biofilm assessment of total and culturable bacteria under different operating conditions

Abstract Monitoring of biofilms subjected to different operating conditions was performed using a flow cell system. The system was fed by chlorine-free tap water, with and without added nutrients (0.5 mg l−1 carbon, 0.1 mg l−1 nitrogen and 0.01 mg l−1 phosphorus), and biofilms were grown on polyvinyl chloride (PVC) and stainless steel (SS) coupons, both in laminar and turbulent flow. The parameters analysed were culturable cells, using R2A, and total bacteria, which was assessed using the 4,6-diamino-2-phenylindole (DAPI) staining method. The impact of the different operating conditions in the studied parameters was established using Multivariate Analysis of Variance (MANOVA). From the most relevant to the least relevant factor, the total and culturable bacteria in biofilms increased due to the addition of nutrients to water (F = 20.005; p < 0.001); the use of turbulent (Re = 11000) instead of laminar (Re = 2000) hydrodynamic flows (F = 9.173; p < 0.001); and the use of PVC instead of SS as the support material (F = 2.848; p = 0.060). Interactions between these conditions, namely between surface and flow (F = 8.235; p < 0.001) and also flow and nutrients (F = 5.498; p < 0.05) have also proved to significantly influence biofilm formation. This work highlights the need for a deeper understanding of how the large spectrum of conditions interact and affect biofilm formation potential and accumulation with the final purpose of predicting the total and culturable bacteria attached to real drinking water distribution pipes based on the system characteristics.

Antagonism between Bacillus cereus and Pseudomonas fluorescens in planktonic systems and in biofilms

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steady-state biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.

An overview on the reactors to study drinking water biofilms

The development of biofilms in drinking water distribution systems (DWDS) can cause pipe degradation, changes in the water organoleptic properties but the main problem is related to the public health. Biofilms are the main responsible for the microbial presence in drinking water (DW) and can be reservoirs for pathogens. Therefore, the understanding of the mechanisms underlying biofilm formation and behavior is of utmost importance in order to create effective control strategies. As the study of biofilms in real DWDS is difficult, several devices have been developed. These devices allow biofilm formation under controlled conditions of physical (flow velocity, shear stress, temperature, type of pipe material, etc), chemical (type and amount of nutrients, type of disinfectant and residuals, organic and inorganic particles, ions, etc) and biological (composition of microbial community - type of microorganism and characteristics) parameters, ensuring that the operational conditions are similar as possible to the DWDS conditions in order to achieve results that can be applied to the real scenarios. The devices used in DW biofilm studies can be divided essentially in two groups, those usually applied in situ and the bench top laboratorial reactors. The selection of a device should be obviously in accordance with the aim of the study and its advantages and limitations should be evaluated to obtain reproducible results that can be transposed into the reality of the DWDS. The aim of this review is to provide an overview on the main reactors used in DW biofilm studies, describing their characteristics and applications, taking into account their main advantages and limitations.