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The development of biofilms in drinking water distribution systems (DWDS) can cause pipe 24 degradation, changes in the water organoleptic properties but the main problem is related to 25 the public health. Biofilms are the main responsible for the microbial presence in drinking 26 water (DW) and can be reservoirs for pathogens. Therefore, the understanding of the 27 mechanisms underlying biofilm formation and behavior is of utmost importance in order to 28 create effective control strategies. As the study of biofilms in real DWDS is difficult, several

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 (antipyrine-ANTP; diclofenac sodium salt-DCF; ibuprofen-IBP; galaxolide-GAL; tonalide-TON; carbamazepine-CBZ; clofibric acid-CA; tylosin-TY) 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 26 days 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.

A. calcoaceticus was more susceptible to the action of NaOCl than S. maltophilia. • Biofilm removal and killing are distinct phenomena. • High biofilm killing rates were achieved with NaOCl at residual concentrations. • High concentrations of NaOCl cause reduced biofilm removal. • Complementary methods to NaOCl are required for drinking water disinfection.

The aim of this work was to assess the effectiveness of the biocide benzyldimethyldodecyl ammonium chloride (BDMDAC) on the removal of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens formed in a rotating cylinder reactor (RCR), using AISI316 stainless steel (SS) and polymethyl methacrylate (PMMA) as adhesion surfaces. Additional tests were performed to understand the adhesion of B. cereus and P. fluorescens to the selected surfaces.Predictions of the adhesion potential according to the thermodynamic theory showed more favourable adhesion on SS than on PMMA, for both species.Thermodynamically, adhesion was more favourable for B. cereus. After BDMDAC treatment, thermodynamic adhesion ability was favoured for P. flurescens and decreased for B. cereus, mainly on PMMA. Both bacteria had negative surface charge and the exposure to BDMDAC increased the charge to less negative values. In vitro adhesion results were, for most cases, contradictory to those predicted by the thermodynamic theory. Single and dual species biofilms were formed in the RCR for 7 days. Afterwards, the biofilms were exposed to the chemical (use of BDMDAC) and to hydrodynamic stresses (use of increasing Reynolds number of agitation), alone and combined. The applications of BDMDAC or hydrodynamic stress, when applied alone, were insufficient to remove the biofilms from the surfaces. The combined effects of BDMDAC with a series of increasing Reynolds number of agitation promoted additional biofilm removal. This effect was dependent on the surface used. For PMMA, the hydrodynamic stress was more effective on the removal of BDMDAC-treated dual species biofilms. For SS, the synergy of the chemical and hydrodynamic stresses removed more B. cereus and dual species biofilms. The overall results 2 demonstrate that the species association was not advantageous in biofilm resistance to removal when compared with the single species biofilms, particularly those of P. fluorescens. In general, removal by hydrodynamic stress, alone and preceded by the BDMDAC treatment, was higher for biofilms formed on SS. However, even the combined action of BDMDAC and the exposure to a series of increasing Reynolds number of agitation were not effective to obtain biofilm-free surfaces.

The presence of emerging contaminants (ECs) in the environment has been consistently recognized as a worldwide concern. ECs may be defined as chemicals or materials found in the environment at trace concentrations with potential, perceived, or real risk to the “One Health” trilogy (environment, human, and animal health). The main concern regarding pharmaceuticals and in particular antibiotics is the widespread dissemination of antimicrobial resistance. Nevertheless, non-antimicrobials also interact with microorganisms in both bulk phase and in biofilms. In fact, drugs not developed for antimicrobial chemotherapy can exert an antimicrobial action and, therefore, a selective pressure on microorganisms. This review aims to provide answers to questions typically ignored in epidemiological and environmental monitoring studies with a focus on water systems, particularly drinking water (DW): Do ECs exposure changes the behavior of environmental microorganisms? May non-antibiotic ECs affect tolerance to antimicrobials? Do ECs interfere with biofilm function? Are ECs-induced changes in microbial behavior of public health concern? Nowadays, the answers to these questions are still very limited. However, this study demonstrates that some ECs have significant effects in microbial behavior. The most studied ECs are pharmaceuticals, particularly antibiotics, carbamazepine and diclofenac. The pressure caused by antibiotic and other antimicrobial agents on the acquisition and spread of antibiotic resistance seems to be unquestionable. However, regarding the effects of ECs on the development and behavior of biofilms, the conclusions of different studies are still controversial. The dissimilar findings propose that standardized tests are needed for an accurate assessment on the effects of ECs in the microbiome of water systems. The variability of experimental conditions, combined with the presence of mixtures of ECs as well as the lack of information about the effects of non-pharmaceutical ECs constitute the main challenge to be overcome in order to improve ECs prioritization.

The presence of biofilms in drinking water distribution systems (DWDS) is a global public health concern as they can harbor pathogenic microorganisms. Sodium hypochlorite (NaOCl) is the most commonly used disinfectant for microbial growth control in DWDS. However, its effect on biofilm removal is still unclear. This work aims to evaluate the effects of the combination of chemical (NaOCl) and mechanical stresses on the removal of single and dual species biofilms of two bacteria isolated from DWDS and considered opportunistic, Acinectobacter calcoaceticus and Stenotrophomonas maltophilia. A rotating cylinder reactor was successfully used for the first time in drinking water biofilm studies with polyvinyl chloride as substratum. The single and dual species biofilms presented different characteristics in terms of metabolic activity, mass, density, thickness and content of proteins and polysaccharides. Their complete removal was not achieved even when a high NaOCl concentrations and an increasing series of shear stresses (from 2 to 23Pa) were applied. In general, NaOCl pre-treatment did not improve the impact of mechanical stress on biofilm removal. Dual species biofilms were colonized mostly by S. maltophilia and were more susceptible to chemical and mechanical stresses than these single species. The most efficient treatment (93% biofilm removal) was the combination of NaOCl at 175mg·l with mechanical stress against dual species biofilms. Of concern was the high tolerance of S. maltophilia to chemical and mechanical stresses in both single and dual species biofilms. The overall results demonstrate the inefficacy of NaOCl on biofilm removal even when combined with high shear stresses.

Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.