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

A review of current and emergent biofilm control strategies

Microbial adhesion to surfaces and the consequent biofilm formation has been documented in many different environments. Biofilms constitute a protected mode of growth that allows microorganisms to survival in hostile environments, being their physiology and behavior significantly different from their planktonic counterparts. In dairy industry, biofilms may be a source of recalcitrant contaminations, causing food spoilage and are possible sources of public health problems such as outbreaks of foodborne pathogens. Biofilms are difficult to eradicate due to their resistant phenotype. However, conventional cleaning and disinfection regimens may also contribute to inefficient biofilm control and to the dissemination of resistance. Consequently, new control strategies are constantly emerging with main incidence in the use of biosolutions (enzymes, phages, interspecies interactions and antimicrobial molecules from microbial origin). The present review will focus on describing the mechanisms involved in biofilm formation and behavior, deleterious effects associated with their presence, and some of the current and emergent control strategies, providing new insight of concern for food industry.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/31661/200

Acinetobacter calcoaceticus plays a bridging function in drinking water biofilms

Intergeneric coaggregation of six drinking water autochthonous heterotrophic bacteria isolated from a model laboratory system were tested for their ability to coaggregate by a visual assay and by two microscopic techniques (epifluorescence and scanning electron microscopies). One isolate, identified as Acinetobacter calcoacticus, 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.) to different degrees as assessed by the visual assay, highlighting a possible bridging function in a biofilm consortium. In its absence, no coaggregation was found. Microscopic observations revealed a higher degree of interaction for all the aggregates than did the visual assay. Heat and protease reversed autoaggregation and coaggregation, suggesting that interactions were lectin-saccharide mediated. The increase/decrease in the level of extracellular proteins and polysaccharides produced during intergeneric bacteria association was not correlated with coaggregation occurrence, but probably with coaggregation strength. The bridging function of A. calcoaceticus was evidenced by multispecies biofilm studies through a strain exclusion process.Este trabalho investiga a co-agregação intergenérica de seis bactérias heterotróficas autóctones de água potável isoladas de um sistema laboratorial modelo, testando assim a sua capacidade de co-agregação através do ensaio visual e de duas técnicas microscópicas (microscopia de epifluorescência e microscopia electrónica de varrimento). Para o isolado identificado como Acinetobacter calcoaceticus, foi detectado através do ensaio visual que não só auto-agrega, mas também co-agrega, a diferentes intensidades, com quatro dos outros cinco isolados (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.), realçando a possível função de ligação em biofilmes multi-espécie. Na sua ausência não foi detectada co-agregação. As observações microscópicas revelaram um maior grau de interacção para todos os agregados do que a detectada pelo ensaio visual. O tratamento com calor e protease reverteram a auto-agregação e a co-agregação, sugerindo que as interacções são mediadas por lectinas-açúcares. O aumento/diminuição no nível de proteínas e polissacarídeos extracelulares produzidos durante os fenómenos de co-agregação não estão relacionados com a sua ocorrência, mas provavelmente com a sua força de interacção. A função de ligação da A. calcoaceticus nos consórcios microbianos foi evidenciada pela formação de biofilmes multi-espécie, através de um processo de exclusão bacteriana.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/31661/2006, FRH/BPD/20582/200

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

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

Drinking water biofilm monitoring by Propella™ and flow cell bioreactors under different operating conditions

Monitoring of biofilm subjected to different process conditions was performed using two distinct bioreactors, Propella™ and flow cell system. Biofilms were grown on polyvinyl chloride (PVC) and stainless steel (SS) coupons under laminar (Reynolds number of 2000) and turbulent (Reynolds number of 11000) flow. The parameters analyzed were culturable cells, using R2A, and total bacteria, which were assessed using a DNA-binding stain coupled with epifluorescence microscopy. The impact of the different operating conditions in the studied parameters was established after the biofilms reached the steady-state. It was found that the biofilm steady-state was achieved 3 d after the starting of operating conditions for turbulent flow and for both bioreactors and adhesion surfaces. Under laminar flow it was only achieved 6 d after. The number of total bacteria was invariably higher than the culturable cells. The number of total and culturable bacteria in turbulent flow-generated biofilms were similar in both bioreactors, regardless the adhesion surface tested. Under laminar flow, the Propella™ bioreactor allowed the formation of steady-state biofilms with a higher number of total and culturable bacteria than those from the flow cell system. Comparing the effect of the flow regime on biofilm accumulation, only turbulent flow-generated biofilms formed on the flow cell system had a higher amount of total and culturable bacteria than those formed under laminar flow. In terms of adhesion surface effect on steady-state biofilms, a higher number of total and culturable cells were found on PVC surfaces comparatively to SS when biofilms were formed using the flow cell system. Biofilm formation on PVC and SS was similar in the Propella™ system for both flow regimes.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/31661/200