The effects of methylparaben exposure on biofilm tolerance to chlorine disinfection.

Parabens are emerging contaminants that have been detected in drinking water. Their presence in DW distribution systems (DWDS) can alter bacterial behaviour, characteristics, and structure, which may compromise DW disinfection. This work provides insights into the impact of methylparaben (MP) on the tolerance to chlorine disinfection and antibiotics from dual-species biofilms formed by Acinetobacter calcoaceticus and Stenotrophomonas maltophilia isolated from DW and grown on high-density polyethylene (HDPE) and polypropylene (PPL). Results showed that dual-species biofilms grown on PPL were more tolerant to chlorine disinfection, expressing a decrease of over 50 % in logarithmic reduction values of culturable cells in relation to non-exposed biofilms. However, bacterial tolerance to antibiotics was not affected by MP presence. Although MP-exposed dual-species biofilms grown on HDPE and PPL were metabolically more active than non-exposed counterparts, HDPE seems to be the material with lower impact on DW risk management and disinfection, if MP is present. Overall, results suggest that MP presence in DW may compromise chlorine disinfection, and consequently affect DW quality and stability, raising potential public health issues.

Parabens as environmental contaminants of aquatic systems affecting water quality and microbial dynamics.

Among different pollutants of emerging concern, parabens have gained rising interest due to their widespread detection in water sources worldwide. This occurs because parabens are used in personal care products, pharmaceuticals, and food, in which residues are generated and released into aquatic environments. The regulation of the use of parabens varies across different geographic regions, resulting in diverse concentrations observed globally. Concentrations of parabens exceeding 100 µg/L have been found in wastewater treatment plants and surface waters while drinking water (DW) sources typically exhibit concentrations below 6 µg/L. Despite their low levels, the presence of parabens in DW is a potential exposure route for humans, raising concerns for both human health and environmental microbiota. Although a few studies have reported alterations in the functions and characteristics of microbial communities following exposure to emerging contaminants, the impact of the exposure to parabens by microbial communities, particularly biofilm colonizers, remains largely understudied. This review gathers the most recent information on the occurrence of parabens in water sources, as well as their effects on human health and aquatic organisms. The interactions of parabens with microbial communities are reviewed for the first time, filling the knowledge gaps on the effects of paraben exposure on microbial ecosystems and their impact on disinfection tolerance and antimicrobial resistance, with potential implications for public health.

Realism-based assessment of the efficacy of potassium peroxymonosulphate on Stenotrophomonas maltophilia biofilm control.

The potential of pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE) to control biofilms in drinking water distribution systems (DWDS) was evaluated and compared to chlorine disinfection. Mature biofilms of drinking water (DW)-isolated Stenotrophomonas maltophilia were formed using a simulated DWDS with a rotating cylinder reactor (RCR). After 30 min of exposure, OXONE at 10 × minimum bactericidal concentration (MBC) caused a significant 4 log reduction of biofilm culturability in comparison to the unexposed biofilms and a decrease in the number of non-damaged cells below the detection limit (4.8 log cells/cm2). The effects of free chlorine were restricted to approximately 1 log reduction in both biofilm culturability and non-damaged cells. OXONE in synthetic tap water (STW) at 25 ºC was more stable over 40 days than free chlorine in the same conditions. OXONE solution exhibited a disinfectant decrease of about 10% of the initial concentration during the first 9 days, and after this time the values remained stable. Whereas possible reaction of chlorine with inorganic and organic substances in STW contributed to free chlorine depletion of approximately 48% of the initial concentration. Electron paramagnetic resonance (EPR) spectroscopy studies confirmed the presence of singlet oxygen and other free radicals during S. maltophilia disinfection with OXONE. Overall, OXONE constitutes a relevant alternative to conventional DW disinfection for effective biofilm control in DWDS.

Antimicrobial and antibiofilm potentiation by a triple combination of dual biocides and a phytochemical with complementary activity.

The failure of current sanitation practices requires the development of effective solutions for microbial control. Although combinations using antibiotics have been extensively studied to look for additive/synergistic effects, biocide combinations are still underexplored. This study aims to evaluate the antimicrobial effectiveness of dual biocide and triple biocide/phytochemical combinations, where phytochemicals are used as quorum sensing (QS) inhibitors. The biocides selected were benzalkonium chloride (BAC) and peracetic acid (PAA) - as commonly used biocides, and glycolic acid (GA) and glyoxal (GO) - as alternative and sustainable biocides. Curcumin (CUR) and 10-undecenoic acid (UA) were the phytochemicals selected, based on their QS inhibition properties. A checkerboard assay was used for the screening of chemical interactions based on the cell growth inhibitory effects against Bacilluscereus and Pseudomonasfluorescens. It was observed that dual biocide combinations resulted in indifference, except the PAA + GA combination, which had a potential additive effect. PAA + GA + CUR and PAA + GA + UA combinations also triggered additive effects. The antimicrobial effects of the combinations were further evaluated on the inactivation of planktonic and biofilm cells after 30 min of exposure. These experiments corroborated the checkerboard results, in which PAA + GA was the most effective combination against planktonic cells (additive/synergistic effects). The antimicrobial effects of triple combinations were species- and biocide-specific. While CUR only potentiate the antimicrobial activity of GA against B.cereus, GA + UA and PAA + GA + UA combinations promoted additional antimicrobial effects against both bacteria. Biofilms were found to be highly tolerant, with modest antimicrobial effects being observed for all the combinations tested. However, this study demonstrated that low doses of biocides can be effective in bacterial control when combining biocides with a QS inhibitor, in particular, the combination of the phytochemical UA (as a QS inhibitor) with GA and PAA.

Curcumin and 10-undecenoic acid as natural quorum sensing inhibitors of LuxS/AI-2 of Bacillus subtilis and LasI/LasR of Pseudomonas aeruginosa.

The quorum sensing (QS) system is related to cell-to-cell communication as a function of population density, which regulates several physiological functions including biofilm formation and virulence gene expression. QS inhibitors have emerged as a promising strategy to tackle virulence and biofilm development. Among a wide variety of phytochemicals, many of them have been described as QS inhibitors. Driven by their promising clues, this study aimed to identify active phytochemicals against LuxS/autoinducer-2 (AI-2) (as the universal QS system) from Bacillus subtilis and LasI/LasR (as a specific QS system) of Pseudomonas aeruginosa, through in silico analysis followed by in vitro validation. The optimized virtual screening protocols were applied to screen a phytochemical database containing 3479 drug-like compounds. The most promising phytochemicals were curcumin, pioglitazone hydrochloride, and 10-undecenoic acid. In vitro analysis corroborated the QS inhibitory activity of curcumin and 10-undecenoic acid, however, pioglitazone hydrochloride showed no relevant effect. Inhibitory effects on LuxS/AI-2 QS system triggered reduction of 33-77% by curcumin (at 1.25-5 µg/mL) and 36-64% by 10-undecenoic acid (at 12.5-50 µg/mL). Inhibition of LasI/LasR QS system was 21% by curcumin (at 200 µg/mL) and 10-54% by 10-undecenoic acid (at 15.625-250 µg/mL). In conclusion, in silico analysis allowed the identification of curcumin and, for the first time, 10-undecenoic acid (showing low cost, high availability, and low toxicity) as alternatives to counteract bacterial pathogenicity and virulence, avoiding the imposition of selective pressure usually related to classic industrial disinfection and antibiotics therapy.

Ultraviolet C irradiation: A promising approach for the disinfection of public spaces?

Ultraviolet irradiation C (UVC) has emerged as an effective strategy for microbial control in indoor public spaces. UVC is commonly applied for air, surface, and water disinfection. Unlike common 254 nm UVC, far-UVC at 222 nm is considered non-harmful to human health, being safe for occupied spaces, and still effective for disinfection purposes. Therefore, and allied to the urgency to mitigate the current pandemic of SARS-CoV-2, an increase in UVC-based technology devices appeared in the market with levels of pathogens reduction higher than 99.9 %. This environmentally friendly technology has the potential to overcome many of the limitations of traditional chemical-based disinfection approaches. The novel UVC-based devices were thought to be used in public indoor spaces such as hospitals, schools, and public transport to minimize the risk of pathogens contamination and propagation, saving costs by reducing manual cleaning and equipment maintenance provided by manpower. However, a lack of information about UVC-based parameters and protocols for disinfection, and controversies regarding health and environmental risks still exist. In this review, fundamentals on UVC disinfection are presented. Furthermore, a deep analysis of UVC-based technologies available in the market for the disinfection of public spaces is addressed, as well as their advantages and limitations. This comprehensive analysis provides valuable inputs and strategies for the development of effective, reliable, and safe UVC disinfection systems.

Drinking-water isolated Delftia acidovorans selectively coaggregates with partner bacteria and facilitates multispecies biofilm development.

Coaggregation plays an important role in the development of multispecies biofilms in different environments, often serving as an active bridge between biofilm members and other organisms that, in their absence, would not integrate the sessile structure. The ability of bacteria to coaggregate has been reported for a limited number of species and strains. In this study, 38 bacterial strains isolated from drinking water (DW) were investigated for their ability to coaggregate, in a total of 115 pairs of combinations. Among these isolates, only Delftia acidovorans (strain 005P) showed coaggregating ability. Coaggregation inhibition studies have shown that the interactions mediating D. acidovorans 005P coaggregation were both polysaccharide-protein and protein-protein, depending on the interacting partner bacteria. Dual-species biofilms of D. acidovorans 005P and other DW bacteria were developed to understand the role of coaggregation on biofilm formation. Biofilm formation by Citrobacter freundii and Pseudomonas putida strains highly benefited from the presence of D. acidovorans 005P, apparently due to the production of extracellular molecules/public goods favouring microbial cooperation. This was the first time that the coaggregation capacity of D. acidovorans was demonstrated, highlighting its role in providing a metabolic opportunity for partner bacteria.

Impact of parabens on drinking water bacteria and their biofilms: The role of exposure time and substrate materials.

Parabens have been detected in drinking water (DW) worldwide, however, their impact on DW microbial communities remains to be explored. Microorganisms can easily adapt to environmental changes. Therefore, their exposure to contaminants of emerging concern, particularly parabens, in DW distribution systems (DWDS) may affect the microbiological quality and safety of the DW reaching the consumers tap. This work provides a pioneer evaluation of the effects of methylparaben (MP), propylparaben (PP), butylparaben (BP), and their combination (MIX), in bacterial biofilms formed on different surfaces, representative of DWDS materials - high-density polyethylene (HDPE), polypropylene (PPL) and polyvinyl chloride (PVC). Acinetobacter calcoaceticus and Stenotrophomonas maltophilia, isolated from DW, were used to form single and dual-species biofilms on the surface materials selected. The exposure to MP for 7 days caused the most significant effects on biofilms, by increasing their cellular culturability, density, and thickness up to 233%, 150%, and 224%, respectively, in comparison to non-exposed biofilms. Overall, more pronounced alterations were detected for single biofilms than for dual-species biofilms when HDPE and PPL, demonstrating that the surface material used affected the action of parabens on biofilms. Swimming motility and the production of virulence factors (protease and gelatinase) by S. maltophilia were increased up to 141%, 41%, and 73%, respectively, when exposed to MP for 7 days. The overall results highlight the potential of parabens to interfere with DW bacteria in planktonic state and biofilms, and compromise the DW microbiological quality and safety.

An Overview of the Impact of Pharmaceuticals on Aquatic Microbial Communities.

Pharmaceuticals are present as pollutants in several ecosystems worldwide. Despite the reduced concentrations at which they are detected, their negative impact on natural biota constitutes a global concern. The consequences of pharmaceuticals' presence in water sources and food have been evaluated with a higher detail for human health. However, although most of the pharmaceuticals detected in the environment had not been designed to act against microorganisms, it is of utmost importance to understand their impact on the environmental native microbiota. Microbial communities can suffer serious consequences from the presence of pharmaceuticals as pollutants in the environment, which may directly impact public health and ecosystem equilibrium. Among this class of pollutants, the ones that have been studied in more detail are antibiotics. This work aims to provide an overview of the impacts of different pharmaceuticals on environmental biofilms, more specifically in biofilms from aquatic ecosystems and engineered water systems. The alterations caused in the biofilm function and characteristics, as well as bacteria antimicrobial tolerance and consequently the associated risks for public health, are also reviewed. Despite the information already available on this topic, the need for additional data urges the assessment of emerging pollutants on microbial communities and the potential public health impacts.

The impact of synthetic musk compounds in biofilms from drinking water bacteria.

Musk fragrances have been detected in drinking water (DW) at trace concentrations. However, their impact on the microbial quality of DW has been disregarded. This work provides a pioneer evaluation of the effects of two synthetic musks contaminants, tonalide (AHTN) and galaxolide (HHCB), in microbial biofilms formed on two different surfaces, polyvinyl chloride (PVC) and stainless steel AISI 316 (SS316). Three bacterial species isolated from DW (Acinetobacter calcoaceticus, Burkholderia cepacia and Stenotrophomonas maltophilia), were used to develop 7-day-old single and mixed species biofilms. The impact of musks was assessed directly on biofilms but also on the bacteria motility, biofilm formation ability and biofilm susceptibility to chlorination. AHTN musk caused the most remarkable effects by increasing the cellular density and viability of mixed biofilms, and the extracellular polysaccharides content of biofilms on SS316. Most of the alterations caused by the direct exposure of biofilms to musks were observed when SS316 was used as an adhesion surface. In contrast, the ability to form biofilms and their susceptibility to chlorine were more affected for bacteria from HHCB-exposed biofilms on PVC. The overall results demonstrate that the presence of musks at residual concentrations influences DW bacterial dynamics, with the potential to impact the DW quality and safety. The type of plumbing material may further impact the effects of musks.

Antibiofilm activity of glycolic acid and glyoxal and their diffusion-reaction interactions with biofilm components.

Biofilms on food-contact surfaces act as potential reservoirs of microbial pathogens and can cause operational problems. The search for effective biofilm control agents is a significant research need. In this study, glycolic acid (GA) and glyoxal (GO) were tested in the control of biofilms formed by Bacilluscereus and Pseudomonasfluorescens. Benzalkonium chloride (BAC) and peracetic acid (PAA) were used as reference biocides for industrial surface disinfection. The action of the selected biocides was assessed in bacterial motility, culturability, biofilm removal and inactivation, interference with biofilm components and limitation of biocide penetration through the biofilms (reaction-diffusion interactions). Bacterial motility was not affected by the exposure to sub-inhibitory biocide concentrations. In terms of antibiofilm activity, B.cereus biofilms were tolerant to the action of BAC, GA, and GO, with reductions of circa 2-log CFU/cm2. Even 10000 µg/mL of PAA had modest effects against B. cereus biofilms (5-log CFU/cm2). On the other hand, P.fluorescens biofilms were more susceptible to the biocides, except BAC which was not effective. The minimum concentrations to cause 3-log CFU/cm2 reduction in P. fluorescens biofilms were 10000 µg/mL of PAA and GA, and 20000 µg/mL of GO. GO and BAC were the most actives for biofilm removal, while high biofilm inactivation was caused by GA and PAA. In general, biofilm components affected the antimicrobial activity of all the biocides. The activity of GA and GO was not notably diffusion-reaction limited in contrast to PAA. Overall, this study demonstrated that GA and GO had potential antibiofilm activity, being limited at a low level by physicochemical interactions with biofilm components.

Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens.

The present study evaluates the antimicrobial susceptibility of persister cells of Bacillus cereus and Pseudomonas fluorescens after their regrowth in suspension and as biofilms. Two conventional (benzalkonium chloride-BAC and peracetic acid-PAA) and two emerging biocides (glycolic acid-GA and glyoxal-GO) were selected for this study. Persister cells resulted from biofilms subjected to a critical treatment using the selected biocides. All biocide treatments developed B. cereus persister cells, except PAA that effectively reduced the levels of vegetative cells and endospores. P. fluorescens persister cells comprise viable and viable but non-culturable cells. Afterwards, persister cells were regrown in suspension and in biofilms and were subjected to a second biocide treatment. In general, planktonic cultures of regrown persister cells in suspension lost their antimicrobial tolerance, for both bacteria. Regrown biofilms of persister cells had antimicrobial susceptibility close to those regrown biofilms of biocide-untreated cells, except for regrown biofilms of persister P. fluorescens after BAC treatment, which demonstrated increased antimicrobial tolerance. The most active biocide against persister cells was PAA, which did not promote changes in susceptibility after their regrowth. In conclusion, persister cells are ubiquitous within biofilms and survive after critical biocide treatment. The descendant planktonic and biofilms populations showed similar properties as the original ones.

The Effects of Chemical and Mechanical Stresses on Bacillus cereus and Pseudomonas fluorescens Single- and Dual-Species Biofilm Removal.

Biofilm control is mainly based on chemical disinfection, without a clear understanding of the role of the biocides and process conditions on biofilm removal. This study aims to understand the effects of a biocide (benzyldimethyldodecyl ammonium chloride-BDMDAC) and mechanical treatment (an increase of shear stress -τw) on single- and dual-species biofilms formed by Bacillus cereus and Pseudomonas fluorescens on high-density polyethene (HDPE). BDMDAC effects were initially assessed on bacterial physicochemical properties and initial adhesion ability. Then, mature biofilms were formed on a rotating cylinder reactor (RCR) for 7 days to assess the effects of chemical and mechanical treatments, and the combination of both on biofilm removal. The results demonstrated that the initial adhesion does not predict the formation of mature biofilms. It was observed that the dual-species biofilms were the most susceptible to BDMDAC exposure. The exposure to increasing τw emphasised the mechanical stability of biofilms, as lower values of τw (1.66 Pa) caused high biofilm erosion and higher τw values (17.7 Pa) seem to compress the remaining biofilm. In general, the combination of BDMDAC and the mechanical treatment was synergic in increasing biofilm removal. However, these were insufficient to cause total biofilm removal (100%; an average standard deviation of 11% for the method accuracy should be considered) from HDPE.

Bacterial coaggregation in aquatic systems.

The establishment of a sessile community is believed to occur in a sequence of steps where genetically distinct bacteria can become attached to partner cells via specific molecules, in a process known as coaggregation. The presence of bacteria with the ability to autoaggregate and coaggregate has been described for diverse aquatic systems, particularly freshwater, drinking water, wastewater, and marine water. In these aquatic systems, coaggregation already demonstrated a role in the development of complex multispecies sessile communities, including biofilms. While specific molecular aspects on coaggregation in aquatic systems remain to be understood, clear evidence exist on the impact of this mechanism in multispecies biofilm resilience and homeostasis. The identification of bridging bacteria among coaggregating consortia has potential to improve the performance of wastewater treatment plants and/or to contribute for the development of strategies to control undesirable biofilms. This study provides a comprehensive analysis on the occurrence and role of bacterial coaggregation in diverse aquatic systems. The potential of this mechanism in water-related biotechnology is further described, with particular emphasis on the role of bridging bacteria.

Copper Surfaces in Biofilm Control.

Biofilms are structures comprising microorganisms associated to surfaces and enclosed by an extracellular polymeric matrix produced by the colonizer cells. These structures protect microorganisms from adverse environmental conditions. Biofilms are typically associated with several negative impacts for health and industries and no effective strategy for their complete control/eradication has been identified so far. The antimicrobial properties of copper are well recognized among the scientific community, which increased their interest for the use of these materials in different applications. In this review the use of different copper materials (copper, copper alloys, nanoparticles and copper-based coatings) in medical settings, industrial equipment and plumbing systems will be discussed considering their potential to prevent and control biofilm formation. Particular attention is given to the mode of action of copper materials. The putative impact of copper materials in the health and/or products quality is reviewed taking into account their main use and the possible effects on the spread of antimicrobial resistance.

Antimicrobial activity of glycolic acid and glyoxal against Bacillus cereus and Pseudomonas fluorescens.

The development of effective disinfection strategies, including the search for new active biocides, is a priority research for the food industry. Glycolic acid (GA) and glyoxal (GO) are two glycolysis by-products approved as biocides for surface disinfection, whose antimicrobial action remains to be understood. This study focused on the understanding of the antimicrobial activity of GA and GO against two foodborne pathogens, Bacillus cereus and Pseudomonas fluorescens. Benzalkonium chloride (BAC) and peracetic acid (PAA) were used as reference biocides for comparison. The influence of biocide concentration and exposure time on the antimicrobial activity of the selected biocides was evaluated based on the European Standard EN 1276. The mode of action of each biocide was characterized based on their effects on the cell envelope and cell replication. According to the Chick-Watson model, all biocides interacted chemically with cell targets of both bacteria, except GO that only stablished physicochemical interactions with P. fluorescens. The survival curves revealed that high concentrations of biocide induced readily effects on cell culturability. The susceptibility profile of P. fluorescens was constant over time. In general, B. cereus increased tolerance to BAC and became more susceptible to cumulative damages of GA. Overall, this study demonstrates that the biocidal activity was species-, dose- and time-dependent. GA, similarly to BAC, was a membrane-active and oxidant agent. GO had no effect on the bacterial surface as well as PAA. GO was mainly categorized as a cell replication inhibitor. For the first time, the antimicrobial activity of GA and GO were characterized revealing their potential for rational combination with other biocides commonly used in the food industry.

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 (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.

Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications.

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.

Long-term results of percutaneous balloon valvuloplasty in neonatal critical pulmonary valve stenosis: a 20-year, single-centre experience.

Introduction Percutaneous balloon valvuloplasty is the primary treatment for critical pulmonary valve stenosis in neonates. Thus far, a few studies have reported long-term results of this technique in neonatal critical pulmonary valve stenosis. METHODS: We carried out a retrospective study of all consecutive newborns with critical pulmonary valve stenosis subjected to percutaneous balloon valvuloplasty at a single centre, between 1994 and 2014, to assess its immediate and long-term safety and efficacy. RESULTS: A total of 24 neonates presented with critical pulmonary valve stenosis. The mean diameter of the pulmonary annulus was 7 mm (±1.19); 33.3% had a dysplastic pulmonary valve, and 92% were started on prostaglandin E1 treatment. Percutaneous balloon valvuloplasty was performed at a mean age of 4.0±4.3 days using, on average, a balloon-to-pulmonary annulus ratio of 1.18 mm (with a range from 0.9 to 1.43). Immediate success was achieved in 22/24 patients (92%) with a reduction in the pulmonary transvalvular peak gradient (p<0.05) and in the right ventricle/systemic pressure ratio (p<0.05). There was one death (4%) 6 days after the procedure, and 29.2% of them had transient rhythm complications. For a mean follow-up time of 8.4 years, the re-intervention rate was 42.9%. In total, 14 re-interventions were performed in nine neonates, including surgery in six. Freedom from re-intervention was 50% at 8 years and 43% at 10 and 15 years. CONCLUSION: This series, to the best of our knowledge, has had the longest follow-up of neonates with critical pulmonary valve stenosis. Percutaneous balloon valvuloplasty is a safe and effective treatment, and in our study 75% of the patients were exclusively treated using this technique.