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.This work was supported by the Operational Programme for Competitiveness Factors - COMPETE and by FCT - the Portuguese Foundation for Science and Technology through Project Bioresist - PTDC/EBB-EBI/105085/2008 (Manuel Simoes) and the post-doctoral grant SFRH/BPD/81982/2011 (Lucia C. Simoes)

The influence of microbial ecology of drinking water biofilms on their resistance to disinfection

The knowledge of the role of microbial ecology of drinking water (DW) biofilms on disinfection might help to improve our understanding of their resistance mechanisms and allow the development of effective strategies to apply in drinking water distribution systems (DWDS). In this study six opportunistic bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.) isolated from a DWDS were used to form single and multispecies biofilms. Those biofilms were exposed to sodium hypochlorite (SHC) at different oncentrations for 1 h and biofilm control was assessed in terms of mass removal and metabolic activity, cultivability and viability reduction. Biofilm recovery was also assessed 24 h after SHC treatment. The results demonstrate that total biofilm mass removal (single and multispecies biofilms) was not achieved for the SHC concentrations tested. Total biofilm inactivation was only achieved for A. calcoaceticus and Staphylococcus sp. single species biofilms and for multispecies biofilms without A. calcoaceticus, when exposed to high SHC concentrations. From the single species biofilms, Methylobacterium sp. and M. mucogenicum had the highest resistance to SHC, while Staphylocooccus sp. and A. calcoaceticus formed the most susceptible biofilms. Multispecies biofims with all the six bacteria had the highest resistance to SHC, while those without A. calcoaceticus were the most susceptible. However, in general multispecies biofilms were more resistant to inactivation and removal than the single biofilms. The recovery results demonstrated that only biofilms without A. calcoaceticus were not able to recover their biomass from the SHC treatments. Also, those biofilms had a decreased ability to recover their viability. This study highlights the importance of A. calcoaceticus in the resistance and functional resilience of DW biofilms. Despite this bacterium being one of the most susceptible to SHC, its presence in multispecies biofilms increased their resistence to disinfection and their ability to recover from SHC exposure

Biofilms in drinking water

The provision of safe drinking water (DW) is a top priority issue in any civilized society. Safe DW is a basic need to human development, health and well-being. The main challenge to the DW industry is to deliver a product that is microbiologically and chemically safe, aesthetically pleasing and adequate in quantity and delivery pressure. Normally, the water that leaves a treatment station has quality, but its quality decreases along the travel in the drinking water distribution systems (DWDS). Water industries and governments over the world are working together in order to improve DW quality through the effective treatment, monitoring of its physicochemical and microbiological properties, and the design and the operational management of the distribution networks. Although DW is strictly monitored in developed countries, waterborne outbreaks are still being reported due to microbial contamination. Biofilms contribute notoriously to these events, creating a protective and nutritional reservoir for pathogens growth and survival. Nevertheless, the dynamics of microbial growth in DW networks is very complex, as a large number of interacting processes (physicochemical and biological) are involved. DW biofilms constitute one of the major microbial problems in DWDS that most contributes to the deterioration of water quality. Although biofilm elimination from DWDS is almost impossible, several aspects can be manipulated in order to prevent and control their growth. This book chapter provides a contribution to better understand the important biological and ecological mechanisms involved in biofilm formation in DWDS, with intent to control and prevent their formation, in order to improve DW quality that reaches to consumer’s tap

The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.The authors acknowledge the financial support provided by the Portuguese Foundation for Science and Technology (SFRH/BD/31661/2006 - Lucia C. Simoes)

Intergeneric coaggregation among drinking water bacteria: evidence of a role for acinetobacter calcoaceticus as a bridging bacterium

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.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/31661/2006; SFRH/BPD/20582/2004

The effects of emerging environmental contaminants on Stenotrophomonas maltophilia isolated from drinking water in planktonic and sessile states

Concerns on the presence of emerging contaminants (ECs) in water sources have increased in recent years. The lack of efficient technologies to remove ECs from residual waters contributes for their appearance in drinking water distribution systems (DWDS). Therefore, sessile microorganisms on DWDS pipes are continuously exposed to trace concentrations of ECs. However, no data exists on the role of ECs on the resident microbiota. The present work aims to understand the effects of prolonged exposure of a bacterial strain of Stenotrophomonas maltophilia, isolated from a DWDS, in both planktonic and biofilm states, to trace concentrations of selected ECs (antipyrineANTP; diclofenac sodium saltDCF; ibuprofenIBP; galaxolideGAL; tonalideTON; carbamazepineCBZ; clofibric acidCA; tylosinTY) on its tolerance to sodium hypochlorite (NaOCl) and resistance to antibiotics. Pre-established S. maltophilia biofilms were exposed to ECs for 26 d. Subsequently, the planktonic behaviour of the biofilm cells grown in the presence of ECS was characterized in terms of susceptibility to NaOCl and to selected antibiotics (levofloxacin and trimethoprim-sulfamethoxazole). Moreover, S.maltophilia was tested on its biofilm productivity in the presence of ECs (alone and mixed). These biofilms were challenged by NaOCl in order to assess the role of ECs on biofilm susceptibility. The results did not evidence remarkable effects of ECs on planktonic S. maltophilia susceptibility to NaOCl and antibiotics. However, S. maltophilia biofilm production and susceptibility to NaOCl was affected from ECs pre-exposure, particularly by the combination of different ECs (CA+CBZ, CA+IBP, CA+CBZ+IBP). S. maltophilia biofilms became more resistant to removal by NaOCl when developed in the presence of mixtures of CA+CBZ and CA+CBZ+IBP. Also, biofilm production was significantly affected. CA was present in all the combinations that altered biofilm behaviour. The overall results propose that exposure to ECs for 26days had not a huge impact on S. maltophilia planktonic antimicrobial susceptibility. Nevertheless, the prolonged exposure to some ECs altered biofilm production and tolerance to NaOCl, with a potential practical outcome of hindering DWDS disinfection. The simultaneous presence of different ECs in the environment may amplify biofilm resilience.This work was the result of the projects: POCI-01-0145-FEDER-030219; POCI-01-0145-FEDER-006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the European Regional Development Fund (ERDF), through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds, through FCT - Fundação para a Ciência e a Tecnologia. NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Grants attributed by Portuguese Foundation for Science and Technology – FCT – to Inês Gomes (SFRH/BD/103810/2014) and Lúcia Simões (SFRH/BPD/81982/2011).info:eu-repo/semantics/publishedVersio

Adhesion potential of bacteria isolated from tap water to several materials using a modified microtiter-plate test

Autochthonous heterotrophic aerobic bacteria from drinking water were isolated, identified by growth on selective media, biochemical tests and 16S rDNA gene sequence. From 25 different isolated bacteria, 8 representative bacteria were selected in order to test their adhesion ability to four different support materials. The bacteria selected were Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata, Staphylococcus sp., Stenotrophomonas maltophilia and the materials used for adhesion were stainless steel ASI 316 (SS), polyethylene (PE), polyvinyl chloride (PVC) and polystyrene (PS). Strain variation on adherence ability was assessed by using two distinct strains of Sph. capsulata (sp. 1 and sp. 2) and S. maltophilia (sp.1 and sp. 2). The adhesion assays were performed during 2 h using a modified microtiter-plate test. The results obtained revealed that the bacteria adhered in a higher extent to PE (P < 0.05), with the exception of Methylobacterium sp. (higher colonization of SS) and Staphylococcus sp. (higher colonization of PS). Strong and moderate adherence were detected for A. calcoacticus and Staphylococcus sp. - adhered to the four materials used, while only Sph. capsulata sp. 1 was non-adherent to the tested materials. Furthermore, it is expected that some of the strong and moderately (A. calcoaceticus, Staphylococcus sp. and S. maltophilia sp. 1) adherent bacteria will play a determinant role in the primary colonization of the surfaces. The use of distinct S. maltophilia and Sph. capsulata strains showed the existence of varying ability of adherence for the distinct strains, demonstrating that no strain can effectively represent its species. This study provides information about a rapid and reliable methodology for bacteria adherence ability assessment and gives useful clues about the behaviour of drinking water autochthonous bacteria when exposed to potential adhesion surfaces.European Commission Research Project SAFER; Portuguese Foundation for Science and Technology (FCT

Comparison of methods to assess biofilm disinfection and recovery by drinking water-isolated bacteria

Drinking water (DW) distribution systems are known to harbour biofilms even in the presence of disinfectants. DW biofilms are constituted by microbial communities adapted to low nutrient concentrations and high chlorine levels. Biofilm formation and resistance to disinfection have been recognized as important factors that contribute to the survival and persistence of microbial contamination in DW. The purpose of this work was the comparison of diverse methods to assess the disinfection of biofilms formed by six DW-isolated opportunistic bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.) by sodium hypochlorite (SHC). Single and multi-species biofilms (composed of combinations of 6 and 5 bacteria) were developed in 96- wells microtiter plates for 3 days, afterwards, were exposed to several independent SHC concentrations (0.1, 0.5, 1 and 10 mg/L) during 1 h. The potential of biofilms to recover was assessed 24 h after disinfection. The disinfection efficacy and recovery were assessed in terms of variation in: biofilm mass (crystal violet staining); metabolic activity (XTT staining); cultivability (CFUs) and viability (Live/Dead staining). The results indicated that biomass removal increased with increasing SHC concentration, but total biofilm mass removal was not achieved. The effects of SHC on the biofilm activity, cultivability and viability were also concentration dependent. Total biofilm inactivation was achieved only for A. calcoaceticus biofilms and for multi-species biofilms without A. calcoaceticus, when exposed to high SHC concentrations. Almost all multispecies biofilms were more resistant to removal and inactivation than the single biofilms. Methylobacterium sp. and A. calcoaceticus formed the most resistant and the most susceptible biofilms, respectively. On the other hand, biofilm combination with the six DW bacteria was the most resistant to SHC and combination without A. calcoaceticus was the least resistant, for all concentration tested. The several methods used to assess of biofilm activity (metabolic activity, cultivability and viability) provided comparable results. However the viability results provide the worst case scenario in terms of biofilm control analysis (higher number of viable cells for all the SHC concentrations tested). The recovery results demonstrated that only biofilms without A.calcoaceticus were not able to recover their biomass from the SHC treatments. Also, those biofilms had a decreased ability to recover their metabolic activity, cultivability and viability. Conversely, multi-species biofilms without Staphylococcus sp. had the highest ability to recover from disinfection. Biofilm mass and activity recovery were not correlated for all the biofilms tested. However, the data of biofilm recovery in terms of metabolic activity, cultivability and viability also provided comparable results