Aeromonas spp. in Freshwater Bodies: Antimicrobial Resistance and Biofilm Assembly.

Aeromonas spp. are environmental bacteria able to infect animals and humans. Here, we aim to evaluate the role of biofilms in Aeromonas persistence in freshwater. Aeromonas were isolated from water and biofilm samples and identified by Vitek-MS and 16S rRNA sequencing. Antibiotic susceptibility profiles were determined according to EUCAST, and a crystal violet assay was used to assess biofilm assembly. MTT and the enumeration of colony-forming units were used to evaluate biofilm and planktonic Aeromonas susceptibility to chlorination, respectively. Identification at the species level was challenging, suggesting the need to improve the used methodologies. Five different Aeromonas species (A. salmonicida, A. hydrophila, A. media, A. popoffii and A. veronii) were identified from water, and one species was identified from biofilms (A. veronii). A. veronnii and A. salmonicida presented resistance to different antibiotics, whith the highest resistance rate observed for A. salmonicida (multiple antibiotic resistance index of 0.25). Of the 21 isolates, 11 were biofilm producers, and 10 of them were strong biofilm producers (SBPs). The SBPs presented increased tolerance to chlorine disinfection when compared with their planktonic counterparts. In order to elucidate the mechanisms underlying biofilm tolerance to chlorine and support the importance of preventing biofilm assembly in water reservoirs, further research is required.

Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 Gene.

This work presents a comparative analysis of two clinical isolates of C. parapsilosis, isolated from haemoculture (HC) and central venous catheter (CVC). Both strains harboured Y132F and R398I mutations in the gene ERG11 associated with resistance to fluconazole (FLC). Differences between the HC and CVC isolates were addressed in terms of virulence, resistance to FLC, and lipid distribution. Expression of the ERG6 and ERG9 genes, lipid analysis, fatty acid composition, and lipase activity were assessed via qPCR, thin-layer chromatography/high-performance liquid chromatography, gas chromatography, and spectrophotometry, respectively. Regulation of the ERG6 and ERG9 genes did not prove any impact on FLC resistance. Analysis of lipid metabolism showed a higher accumulation of lanosterol in both the isolates regardless of FLC presence. Additionally, a decreased level of triacylglycerols (TAG) with an impact on the composition of total fatty acids (FA) was observed for both isolates. The direct impact of the ERG11 mutations on lipid/FA analysis has not been confirmed. The higher lipase activity observed for C. parapsilosis HC isolate could be correlated with the significantly decreased level of TAG. The very close relatedness between both the isolates suggests that one isolate was derived from another after the initial infection of the host.

Effect of Quorum Sensing Molecule Farnesol on Mixed Biofilms of Candida albicans and Staphylococcus aureus.

The natural bioactive molecule farnesol (FAR) is widely studied mainly for its antibiofilm and antimicrobial properties. In addition, it increases the effectiveness of some antimicrobial substances, which makes it interesting for the development of combined therapy. In the present work, the effect of FAR either alone or in combination with oxacillin (OXA) on mixed biofilms formed by clinically relevant pathogens, Candida albicans and Staphylococcus aureus, was studied. S. aureus isolates used for biofilm formation originated from blood cultures and central venous catheters (CVC) were characterized in terms of antimicrobial resistance. The minimal biofilm inhibitory concentration (MBIC50) for FAR of 48 h mixed biofilms formed by the C. albicans and methicillin-sensitive S. aureus (MSSA) was determined to be 125 µM, and for the mixed biofilms with methicillin-resistant S. aureus (MRSA) was determined to be 250 µM. Treatment of mixed biofilms with OXA (2 mg/mL) showed ≤4% inhibition; however, the combination of OXA (2 mg/mL) and FAR (300 µM) resulted in 80% inhibition of biofilms. In addition, planktonic cells of S. aureus exhibited an increased susceptibility to OXA, cefoxitin and kanamycin in the presence of FAR (150 and 300 µM). Scanning electron microscopy (SEM) micrographs confirmed patchy biofilm and lack of candidal hyphae in the samples treated with FAR and FAR/OXA in comparison to control and mixed biofilms treated only with OXA. Intriguingly, in a pilot experiment using fluorescence in situ hybridization (FISH), considerable differences in activity (as indicated by ribosome content) of staphylococcal cells were detected. While the activity rate of the staphylococci in mixed biofilms treated with FAR was high, no FISH-positive signal for staphylococcal cells was found in the biofilm treated with FAR/OXA.

The Preyssler-Type Polyoxotungstate Exhibits Anti-Quorum Sensing, Antibiofilm, and Antiviral Activities.

The increase in bacterial resistance to antibiotics has led researchers to find new compounds or find combinations between different compounds with potential antibacterial action and with the ability to prevent the development of antibiotic resistance. Polyoxotungstates (POTs) are inorganic clusters that may fulfill that need, either individually or in combination with antibiotics. Herein, we report the ability of the polyoxotungstates (POTs) with Wells-Dawson P2W18, P2W17, P2W15, and Preyssler P5W30 type structures to differently affect Gram-negative and Gram-positive microorganisms, either susceptible or resistant to antibiotics. The compound P5W30 showed the highest activity against the majority of the tested bacterial strains in comparison with the other tested POTs (P2W15, P2W17 and P2W18) that did not show inhibition zones for the Gram-negative bacteria, A. baumanii I73775, E. coli DSM 1077, E. coli I73194, K. pneumoniae I7092374, and P. aeruginosa C46281). Generally, the results evidenced that Gram-positive bacteria are more susceptible to the POTs tested. The compound P5W30 was the one most active against S. aureus ATCC 6538 and MRSA16, reaching <0.83 mg·mL−1 (100 µM) and 4.96 mg·mL−1 (600 µM), respectively. Moreover, it was verified by NMR spectroscopy that the most promising POT, P5W30, remains intact under all the experimental conditions, after 24 h at 37 °C. This prompted us to further evaluate the anti-quorum sensing activity of P5W30 using the biosensor Chromobacterium violaceum CV026, as well as its antibiofilm activity both individually and in combination with the antibiotic cefoxitin against the methicillin-resistant Staphylococcus aureus 16 (MRSA16). P5W30 showed a synergistic antibacterial effect with the antibiotic cefoxitin and chloramphenicol against MRSA16. Moreover, the antibiofilm activity of P5W30 was more pronounced when used individually, in comparison with the combination with the antibiotic cefoxitin. Finally, the antiviral activity of P5W30 was tested using the coliphage Qß, showing a dose-dependent response. The maximum inactivation was observed at 750 µM (6.23 mg·mL−1). In sum, P5W30 shows anti-quorum sensing and antibiofilm activities besides being a potent antibacterial agent against S. aureus and to exhibit antiviral activities against enteric viruses.

Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis.

Biofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism's distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.

Occurrence of polycyclic aromatic hydrocarbons, microplastics and biofilms in Alqueva surface water at touristic spots.

Freshwater pollution is a huge concern. A study aiming to evaluate physico-chemical characteristics, microbiota, occurrence of two groups of persistent environmental pollutants with similar chemical properties (polycyclic aromatic hydrocarbons- PAHs and microplastics - MPs) in Alqueva's surface water was performed during 2021. Water samples were collected at three spots related to touristic activities (two beaches and one marina) during the Winter, Spring, Summer and Autumn seasons. In addition, the presence of biofilms on plastic and natural materials (stone, wood/ vegetal materials) were assessed and compared. Water quality based on physicochemical parameters was acceptable with a low eutrophication level. PAHs concentration levels were lower than the standard limits established for surface waters by international organizations. However, carcinogenic compounds were detected in two sampling locations, which can pose a problem for aquatic ecosystems. PAHs profiles showed significant differences when comparing the dry seasons with the rainy seasons, with a higher number of different compounds detected in Spring. Low molecular weigh compounds, usually associated with the atmospheric deposition and petroleum contamination, were more prevalent. MPs were detected in all samples except one during the Winter season. The polymers detected were poly(methyl-2-methylpropenoate), polystyrene, polyethylene terephthalate, polyamide, polypropylene, styrene butadiene, polyvinyl chloride and low /high density polyethylene with the last being the most frequent. Biofilms were more often detected on plastics than on natural materials. In addition, biofilms detected on plastics were more complex with higher microbial diversity (e.g., bacteria, fungi/yeast and phytoplancton organisms) and richer in extrapolymeric material. Based on morphological analysis a good agreement between microbiota and microorganism present in the biofilms was found. Among microbiota were identified microorganisms previously linked to plastic and PAHs detoxification suggesting the need for further studies to evaluate the viability of using biofilms as part of a green bioremediation strategy to mitigate water pollution.

Solvent-Free Microwave Extraction of Thymus mastichina Essential Oil: Influence on Their Chemical Composition and on the Antioxidant and Antimicrobial Activities.

Solvent-free microwave extraction (SFME) is a combination of microwave heating and dry distillation performed at atmospheric pressure without the addition of water or organic solvents that has been proposed as a green method for the extraction of essential oils from aromatic and medicinal herbs. In this work, SFME and the conventional techniques of steam distillation (SD) and hydrodistillation (HD) were compared with respect to the extraction and antioxidant and antimicrobial activities of Thymus mastichina essential oil. The main constituent of essential oils obtained using different methods was 1,8-cineole (eucalyptol). The results showed that the essential oils extracted by means of SFME in 30 min were quantitatively (yield) and qualitatively (aromatic profile) similar to those obtained using conventional HD over 120 min. In addition, SFME generates less waste and less solvent, consumes less energy, and provides a higher yield for a shorter extraction time, which is advantageous for the extraction of the T. mastichina essential oil compared to SD. The antioxidant and antimicrobial activities of the T. mastichina essential oil obtained from either SFME or conventional extraction methods (SD or HD) showed a similar pattern. Large-scale experiments using this SFME procedure showed a potential industrial application.

Opportunist Coinfections by Nontuberculous Mycobacteria and Fungi in Immunocompromised Patients.

Nontuberculous mycobacteria (NTM) and many fungal species (spp.) are commonly associated with opportunistic infections (OPIs) in immunocompromised individuals. Moreover, occurrence of concomitant infection by NTM (mainly spp. of Mycobacterium avium complex and Mycobacterium abscessus complex) and fungal spp. (mainly, Aspergillus fumigatus, Histoplasma capsulatum and Cryptococcus neoformans) is very challenging and is associated with poor patient prognosis. The most frequent clinical symptoms for coinfection and infection by single agents (fungi or NTM) are similar. For this reason, the accurate identification of the aetiological agent(s) is crucial to select the best treatment approach. Despite the significance of this topic it has not been sufficiently addressed in the literature. This review aims at summarizing case reports and studies on NTM and fungi coinfection during the last 20 years. In addition, it briefly characterizes OPIs and coinfection, describes key features of opportunistic pathogens (e.g., NTM and fungi) and human host predisposing conditions to OPIs onset and outcome. The review could interest a wide spectrum of audiences, including medical doctors and scientists, to improve awareness of these infections, leading to early identification in clinical settings and increasing research in the field. Improved diagnosis and availability of therapeutic options might contribute to improve the prognosis of patients' survival.

Biofilm formation by ST17 and ST19 strains of Streptococcus agalactiae.

Bacterial biofilms are an important virulence factor with a vital role in evasion from the host immune system, colonization and infection. The aim of the present study was to evaluate in vitro the effects of three environmental factors (H+, glucose and human plasma) in biofilm formation, by carrier and invasive Streptococcus agalactiae strains of ST17 and ST19 sequence types, including DNase producers and non-producers. Bacteria ability to assemble biofilms was classified based on crystal violet assay. Biofilm formation was also monitored by scanning electron microscopy. Depending on the growth medium used, each bacterial isolate could fit in different biofilm production categories. Our data showed that optimal conditions for S. agalactiae biofilm assembly were reached after 48 h incubation at pH 7.6 in the presence of glucose and inactivated human plasma. In the presence of inactivated human plasma, the biofilm biomass of ST19 strains experienced a higher increase than ST17 strains. The composition of the extracellular polymeric matrix of the three strongest biofilm producers (all from ST17) was accessed by enzymatic digestion of mature biofilms and proteins were shown to be the predominant component. The detailed identification of the extracellular protein components should contribute to the development of new therapeutic strategies to fight S. agalactiae infections.

Lichen Vitality After a Space Flight on Board the EXPOSE-R2 Facility Outside the International Space Station: Results of the Biology and Mars Experiment.

As part of the Biology and Mars Experiment (BIOMEX; ILSRA 2009-0834), samples of the lichen Circinaria gyrosa were placed on the exposure platform EXPOSE-R2, on the International Space Station (ISS) and exposed to space and to a Mars-simulated environment for 18 months (2014-2016) to study: (1) resistance to space and Mars-like conditions and (2) biomarkers for use in future space missions (Exo-Mars). When the experiment returned (June 2016), initial analysis showed rapid recovery of photosystem II activity in the samples exposed exclusively to space vacuum and a Mars-like atmosphere. Significantly reduced recovery levels were observed in Sun-exposed samples, and electron and fluorescence microscopy (transmission electron microscope and field emission scanning electron microscope) data indicated that this was attributable to the combined effects of space radiation and space vacuum, as unirradiated samples exhibited less marked morphological changes compared with Sun-exposed samples. Polymerase chain reaction analyses confirmed that there was DNA damage in lichen exposed to harsh space and Mars-like environmental conditions, with ultraviolet radiation combined with space vacuum causing the most damage. These findings contribute to the characterization of space- and Mars-resistant organisms that are relevant to Mars habitability.

Impacts of a changing earth on microbial dynamics and human health risks in the continuum between beach water and sand.

Although infectious disease risk from recreational exposure to waterborne pathogens has been an active area of research for decades, beach sand is a relatively unexplored habitat for the persistence of pathogens and fecal indicator bacteria (FIB). Beach sand, biofilms, and water all present unique advantages and challenges to pathogen introduction, growth, and persistence. These dynamics are further complicated by continuous exchange between sand and water habitats. Models of FIB and pathogen fate and transport at beaches can help predict the risk of infectious disease from beach use, but knowledge gaps with respect to decay and growth rates of pathogens in beach habitats impede robust modeling. Climatic variability adds further complexity to predictive modeling because extreme weather events, warming water, and sea level change may increase human exposure to waterborne pathogens and alter relationships between FIB and pathogens. In addition, population growth and urbanization will exacerbate contamination events and increase the potential for human exposure. The cumulative effects of anthropogenic changes will alter microbial population dynamics in beach habitats and the assumptions and relationships used in quantitative microbial risk assessment (QMRA) and process-based models. Here, we review our current understanding of microbial populations and transport dynamics across the sand-water continuum at beaches, how these dynamics can be modeled, and how global change factors (e.g., climate and land use) should be integrated into more accurate beachscape-based models.

Nontuberculous Mycobacteria Persistence in a Cell Model Mimicking Alveolar Macrophages.

Nontuberculous Mycobacteria (NTM) respiratory infections have been gradually increasing. Here, THP-1 cells were used as a model to evaluate intracellular persistence of three NTM species (reference and clinical strains) in human alveolar macrophages. The contribution of phagosome acidification, nitric oxide (NO) production and cell dead on NTM intracellular fate was assessed. In addition, strains were characterized regarding their repertoire of virulence factors by whole-genome sequencing. NTM experienced different intracellular fates: M. smegmatis and M. fortuitum ATCC 6841 were cleared within 24h. In contrast, M. avium strains (reference/clinical) and M. fortuitum clinical strain were able to replicate. Despite this fact, unexpectedly high percentages of acidified phagosomes were found harbouring rab7, but not CD63. All NTM were able to survive in vitro at acidic pHs, with the exception of M. smegmatis. Our data further suggested a minor role for NO in intracellular persistence and that apoptosis mediated by caspase 8 and 3/7, but not necrosis, is triggered during NTM infection. Insights regarding the bacteria genomic backbone corroborated the virulence potential of M. avium and M. fortuitum. In conclusion, the phenotypic traits detected contrast with those described for M. tuberculosis, pointing out that NTM adopt distinct strategies to manipulate the host immune defense and persist intracellularly.

Evaluating Efficacy of Antimicrobial and Antifouling Materials for Urinary Tract Medical Devices: Challenges and Recommendations.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

Clonal expansion across the seas as seen through CPLP-TB database: A joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countries.

Tuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analysed the largest CPLP M. tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space. To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends.

Cross-sectional study of adenoidal biofilms in a paediatric population and its clinical implications

INTRODUCTION: Adenoids are nasopharyngeal lymphoid tissue with a relevant role in host defence against infection of upper respiratory tract. Nevertheless, adenoids are also a reservoir of microorganisms that can cause infections of upper respiratory tract and otitis particularly in children. OBJECTIVE: Evaluate and compare the association between biofilm assembly on adenoids and the incidence of recurrent infections in a paediatric population submitted to adenoidectomy by either infectious or non-infectious indication. METHODS: Scanning electron microscopy was used to assess biofilms on adenoid surface; biofilm assembly in vitro was monitored by crystal violet assay; antibiotic susceptibility was assessed following EUCAST guidelines; Hinfluenzae capsular typing was performed by PCR. RESULTS: Biofilms were present in 27.4% of adenoid samples and no statistical difference was found between infectious and non-infectious groups. In vitro, the most clinically relevant bacteria, H.influenzae, S.aureus, S.pyogenes, S.pneumoniae and M.catarrhalis, were mostly moderate biofilm assemblers (71.7%). 55.3% of these bacteria were intermediate/resistant to at least one of the tested antibiotics. No association was found between the ability to assemble biofilms in vitro and the presence of biofilms on adenoids nor antibiotic resistance. All H.influenzae were characterized as non-typeable. CONCLUSION: The presence of biofilms on adenoid surface was independent from clinical sample background. Bacterial ability to assemble biofilms in vitro cannot be used to predict biofilm assembly in vivo. The lack of correlation between biofilm formation and infectious respiratory diseases found contributes to question the relevance of biofilms on the pathogenesis of infectious diseases.

Levofloxacin-loaded bone cement delivery system: Highly effective against intracellular bacteria and Staphylococcus aureus biofilms.

Staphylococcus aureus is a major pathogen in bone associated infections due to its ability to adhere and form biofilms on bone and/or implants. Moreover, recrudescent and chronic infections have been associated with S. aureus capacity to invade and persist within osteoblast cells. With the growing need of novel therapeutic tools, this research aimed to evaluate some important key biological properties of a novel carrier system composed of acrylic bone cement (polymethylmethacrylate - PMMA), loaded with a release modulator (lactose) and an antibiotic (levofloxacin). Levofloxacin-loaded bone cement (BC) exhibited antimicrobial effects against planktonic and biofilm forms of S. aureus (evaluated by a flow chamber system). Moreover, novel BC formulation showed high anti-bacterial intraosteoblast activity. This fact led to the conclusion that levofloxacin released from BC matrices could penetrate the cell membrane of osteoblasts and be active against S. aureus strains in the intracellular environment. Furthermore, levofloxacin-BC formulations showed no significant in vitro cytotoxicity and no allergic potential (measured by the in vivo chorioallantoic membrane assay). Our results indicate that levofloxacin-loaded BC has potential as a local antibiotic delivery system for treating S. aureus associated bone infections.

Integration of cellular and molecular endpoints to assess the toxicity of polycyclic aromatic hydrocarbons in HepG2 cell line.

Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants present in the environment with known mutagenic and carcinogenic properties. In the present study the effects of exposure to single or multiple doses of benzo[a]anthracene (BaA), pyrene (Pyr), and 3 halogenated derivatives of these compounds (1-chloropyrene, 1-bromopyrene [1-BrPyr], and 7-chlorobenzo[a]anthracene [7-ClBaA]) were evaluated in a liver-derived human cell line (HepG2). Cytotoxicity as assessed by the classic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red assays showed a mild toxic effect in response to single or multiple dose exposure for up to 72 h, except for multiple dose exposure to BaA and 7-ClBaA (1 µM/d for 4 d) and single exposure to 10 µM BaA. Furthermore, selective mitochondrial and lysosomal toxicity was observed for Pyr and BaA series, respectively. To understand the underlying molecular mechanisms responsible for this effect, reactive oxygen species production, mitochondrial membrane depolarization, lysosomal pH, DNA fragmentation, and early and late apoptosis mediators were evaluated after exposure to single doses of the compounds. All compounds were able to trigger oxidative stress after 24 h as measured by catalase activity, and a good correlation was found between mitochondrial membrane depolarization, lysosomal pH increase, and MTT and neutral red assays. Evaluation of cell death mediators showed that caspase-3/7, but not annexin-V, pathways were involved in toxicity triggered by the studied compounds. The integration of all results showed that 1-BrPyr and BaA have a higher toxicity potential. Environ Toxicol Chem 2017;36:3404-3414. © 2017 SETAC.

Insights on Klebsiella pneumoniae Biofilms Assembled on Different Surfaces Using Phenotypic and Genotypic Approaches.

Klebsiella pneumoniae is a prominent etiological agent of healthcare associated infections (HAIs). In this context, multidrug-resistant and biofilm-producing bacteria are of special public health concern due to the difficulties associated with treatment of human infections and eradication from hospital environments. Here, in order to study the impact of medical devices-associated materials on the biofilm dynamics, we performed biofilm phenotypic analyses through a classic and a new scanning electron microscopy (SEM) technique for three multidrug-resistant K. pneumoniae isolates growing on polystyrene and silicone. We also applied whole-genome sequencing (WGS) to search for genetic clues underlying biofilm phenotypic differences. We found major differences in the extracellular polymeric substances (EPS) content among the three strains, which were further corroborated by in-depth EPS composition analysis. WGS analysis revealed a high nucleotide similarity within the core-genome, but relevant differences in the accessory genome that may account for the detected biofilm phenotypic dissimilarities, such as genes already associated with biofilm formation in other pathogenic bacteria (e.g., genes coding haemogglutinins and haemolysins). These data reinforce that the research efforts to defeat bacterial biofilms should take into account that their dynamics may be contingent on the medical devices-associated materials.

Adenoid bacterial colonization in a paediatric population.

Adenoids play a key role in both respiratory and ear infection in children. It has also been shown that adenoidectomy improves these symptoms in this population. The main goal of the present study was to evaluate adenoid bacterial colonization and document a possible relation with infectious respiratory disease. A prospective observational study was designed to evaluate the proposed hypothesis in a paediatric population submitted to adenoidectomy by either infectious or non-infectious indications and compare these two cohorts. A total of 62 patients with ages ranging from 1 to 12 years old were enrolled in the study. Adenoid surface, adenoid core and middle meatus microbiota were compared. A close association between adenoid colonization and nasal infection was found, supporting that adenoids may function as bacterial reservoir for upper airway infection. The obtained results also contribute to explain the success of adenoidectomy in patients with infectious indications.

Pathogens in Ornamental Waters: A Pilot Study.

In parks, ornamental waters of easy access and populated with animals are quite attractive to children and yet might hide threats to human health. The present work focuses on the microbiota of the ornamental waters of a Lisboa park, characterized during 2015. The results show a dynamic microbiota integrating human pathogens such as Klebsiella pneumoniae, Aeromonas spp. and Enterobacter spp., and also antibiotic resistant bacteria. K. pneumoniae and Aeromonas spp. were present as planktonic and biofilm organized bacteria. In vitro K. pneumoniae and Aeromonas spp. showed an enhanced ability to assemble biofilm at 25 °C than at 37 °C. Bacteria recovered from biofilm samples showed an increased antibiotic resistance compared to the respective planktonic counterparts.