Impact of hypoxic and anaerobic environments on multidrug-resistance of emerging species found in cystic fibrosis airways

OBJECTIVES: It is well-known the establishment of steep oxygen gradients in cystic fibrosis (CF) airways mucus, giving rise to hypoxic or anaerobic zones in the deeper mucus layers, where traditional and atypical bacteria may accommodate and proliferate to biofilms. This study aimed to compare the influence of low-oxygen atmospheres on biofilm growth and susceptibility profiles of the CF-atypical species Inquilinus limosus and Dolosigranulum pigrum with the traditional Pseudomonas aeruginosa. METHODS: Single biofilms encompassing each species were formed in vitro under aerobic, micraerophilic and anaerobic environments, and further evaluated in terms of biomass and respiratory activity. The antibiotic resistance propensity of planktonic and biofilm-cells was also analyzed by measuring the MICs and MBECs, respectively. RESULTS: Both traditional and unusual species were proficient to develop biofilms under all oxygen environments, with the facultative anaerobe D. pigrum demonstrating the greatest facility to accumulate high amounts of biomass and respiratory activities. Regarding the resistance propensity, planktonic populations of P. aeruginosa and D. pigrum showed antibiotic tolerance under non oxygen-restricted environments, reducing their resistance under micraerophilic and anaerobic conditions. Independently of the oxygen availability, it was noticed a markedly decline of the antibiotic action against the pre-established biofilms, requiring higher doses to eliminate biofilm-encased cells. Although the resistance of P. aeruginosa biofilms has displayed the same tendency as the planktonic populations, the biofilm consortia involving I. limosus and D. pigrum became exceptionally more resistant to antibiotics when compared to those bacteria seeded from the biofilms, persevering this multidrug resistance under hypoxic and anaerobic atmospheres. CONCLUSION: This study highlights a potential prospect on the impact of non-conventional organisms on CF lung infections, showing their great capacity to easily adapt to biofilm mode of life under atmospheres with restricted oxygen, as it is believed to occur in CF airways, which may endanger the efficacy of current antibiotic regimens in CF

Polymicrobial biofilms in cystic fibrosis – the role of atypical bacteria in the consortia and impact in antibiotic treatment

Cystic Fibrosis (CF) is characterized by high rates of morbidity and mortality caused by pulmonary microbial infections. Pseudomonas aeruginosa is typically the prevailing pathogen in the airways of CF patients. However, an emergent and diverse microbial community inhabiting CF lungs has been disclosed, but how it interacts and contributes to the polymicrobial consortia with CF-common pathogens is still to be revealed. The mail goal of this study was to address the behavior of two CF-atypical bacteria, Inquilinus limosus (IL) and Dolosigranulum pigrum (DP), when associated to P. aeruginosa (PA) under oxygen-atmospheres resembling CF airways. For this, those bacteria were grown in dual- and three-species populations with P. aeruginosa in variable oxygen conditions and biofilms were thoroughly characterized for biomass, activity, CFU numbers, antibiotic resistance profiles and relative distributions of bacterial populations. Dual-species consortia were of difficult eradication, with most antibiotics being ineffective in reducing biofilm-bacteria, particularly under low-oxygen atmospheres. Regarding microbial composition, these biofilms presented similar bacterial proportions, whereas P. aeruginosa and D. pigrum dominated the three-species consortia, with I. limosus being the smallest representative population. In general, biofilm compositions changed as a result of antibiotic treatment, with alterations being dependent on the antibiotic, concentration and oxygen condition implemented. P. aeruginosa and I. limosus dual-biofilms exhibited higher antibiotic resistance, with I. limosus persisting and occupying a significant portion together with P. aeruginosa in the overall biofilm after antibiotic treatment. Interestingly, the three-species biofilms displayed higher sensitivity, with D. pigrum and/or P. aeruginosa dominating and I. limosus populations declining in most cases. This suggests that the preponderance of D. pigrum in the biofilm was decisive to decrease I. limosus and lead to an increase in overall sensitivity of the biofilm to a large number of antibiotics. PNA FISH allowed the direct observation of the location and distribution of the three-species species within the biofilms, corroborating the dominance of D. pigrum and P. aeruginosa within the mixed-species consortia and facilitating the understanding of the real complex interactions among the bacterial species. Data highlighted that emergent species are able to establish polymicrobial consortia with common pathogens in the airways of CF patients, modulating different social activities into those communities and impacting the CF therapeutics

Emergent bacteria in cystic fibrosis: in vitro biofilm formation and resilience under variable oxygen conditions

Concurrently to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF) airways. Nonetheless, little is known about the contribution of these newly microbes for the resilience of CF-associated biofilms, particularly under variable oxygen concentrations that are known to occur in vivo in the mucus of CF patients. Two CF-emergent bacterial species, Inquilinus limosus and Dolosigranulum pigrum, and the major pathogen Pseudomonas aeruginosa were studied in terms of biofilm development and antibiotic susceptibilities under in vitro atmospheres with different oxygen availabilities. All species were able to develop in vitro biofilms under different oxygen-available environments, with D. pigrum accumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics loosing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multi-drug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of non-conventional organisms I. limosus and D. pigrum on CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently-used antibiotic regimens.The authors acknowledge the financial support provided by the Portuguese Foundation for Science and Technology (Grant SFRH/BD/47613/2008 to S. P. Lopes) and by the ANTIPEP Project (PTDC/SAU-SAP/113196/2009). The authors also acknowledge Dr. Michael Surette (University of Calgary, AB, Canada) for kindly providing I. limosus strain

Synergistic interactions within polymicrobial biofilms formed by atypical and conventional species in cystic fibrosis

A complex microbial ecosystem is present in Cystic fibrosis (CF)-airways, with uncommon species co-existing and establishing dynamic interactions with traditional pathogens. This study aimed to examine in vitro biofilm formation and susceptibility patterns of two CF-atypical bacteria, Inquilinus limosus (IL) and Dolosigranulum pigrum (DP), when associated to P. aeruginosa (PA), under oxygen-atmospheres resembling CF airways. The ability of IL and DP to form dual- and three-species biofilms with PA and to resist against antibiotics was evaluated under in vitro oxygen-restricted conditions. The fitness of biofilms was compared with monospecies consortia by individual species. Atypical species were able to develop biofilms with PA, presenting a great extent of adhesion in microtiter plate wells and achieving high cell-densities over 24h of growth. Three-species populations were well-adapted to poor-oxygen environments, showing high growth rates in these conditions. Both dual- and three-species biofilms were significantly resistant to most antibiotics, with minimum biofilm eradication concentrations (MBECs) in most cases not achievable (>1024 mg/L). Comparing with monospecies consortia, the presence of IL and DP did not disturb PA biofilms, resulting in most cases in polymicribial biofilms with increased biomass, activity and antibiotic resistance, which persisted under oxygen-restricted environments. This study evidenced the contribution of some atypical species to develop resilient polymicrobial biofilms with PA in CF-airway oxygen conditions. Hence, CF treatment will only be successful after recognizing CF-infection as polymicrobial and upon exhaustive modulation of ecological processes, which will be useful to predict the effects of new therapeutic interventions

Impact of environments with distinct oxygen availability on biofilm growth and susceptibility patterns of traditional and emerging species in Cystic Fibrosis

One of the main manifestations of cystic fibrosis (CF) is a decreased clearance of mucus with concomitant bacterial lung infections. Infections are caused by a wide variety of organisms, which includes not only the typical “residents” but also other uncommon bacteria. It has been widely demonstrated that steep oxygen gradients exist within the mucus layers of cystic fibrosis, giving rise to hypoxic/anaerobic areas where bacteria may colonize and proliferate. In this study, it was aimed to investigate the responses of conventional and atypical bacterial species related with CF, when exposed to environments with different oxygen availability, in terms of biofilm growth and antibiotic susceptibility patterns. Single biofilms of the traditional pathogen Pseudomonas aeruginosa, and two uncommon pathogenic bacteria, Inquilinus limosus and Dolosigranulum pigrum, were formed in vitro under aerobic, microaerophilic and anaerobic environments in microtiter plates, and their biomass and respiratory activity were further evaluated. The planktonic and biofilm susceptibility patterns were also tested against eight clinically relevant antibiotics under the same conditions, by measuring the minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC), respectively. All organisms showed ability to grow under milieus with distinct oxygen availability, however, D. pigrum developed biofilms with a higher amount of biomass and respiratory activity, particularly those formed under microaerophilic conditions. The susceptibility patterns of planktonic cultures revealed antibiotic tolerance of microorganisms under aerobic environments, decreasing their resistance under environments with oxygen depletion. However, MBEC data were significantly higher than MIC values for most antibiotics, revealing that the bactericidal activity was significantly disturbed once biofilms are established. Moreover, the biofilms formed by the atypical species surprisingly exhibited significant multidrug resistance comparing with P. aeruginosa biofilms, which was independent of oxygen availability in the environment. This study enabled to conclude that restricted-oxygen atmospheres, as occurs in CF airways, may favor the growth and colonization of other microorganisms that are not conventional, making biofilms more resistant to antibiotics. Thus, CF must be regarded as an environmental habitat where the existence of hypoxic or anaerobic regions affects the ability of the bacteria to allocate, proliferate and resist to antibiotics. A more detailed knowledge on this area might hence be crucial for the success of infection treatment

Rotterdam Prostate Cancer Risk Calculator: Development and Usability Testing of the Mobile Phone App

BACKGROUND: The use of prostate cancer screening tools that take into account relevant prebiopsy information (ie, risk calculators) is recommended as a way of determining the risk of cancer and the subsequent need for a prostate biopsy. This has the potential to limit prostate cancer overdiagnosis and subsequent overtreatment. mHealth apps are gaining traction in urological practice and are used by both practitioners and patients for a variety of purposes. OBJECTIVE: The impetus of the study was to design, develop, and assess a smartphone app for prostate cancer screening, based on the Rotterdam Prostate Cancer Risk Calculator (RPCRC). METHODS: The results of the Rotterdam arm of the European Randomized Study of Screening for Prostate Cancer (ERSPC) study were used to elaborate several algorithms that allowed the risk of prostate cancer to be estimated. A step-by-step workflow was established to ensure that depending on the available clinical information the most complete risk model of the RPCRC was used. The user interface was designed and then the app was developed as a native app for iOS. The usability of the app was assessed using the Post-Study System Usability Questionnaire (PSSUQ) developed by IBM, in a group of 92 participants comprising urologists, general practitioners, and medical students. RESULTS: A total of 11 questions were built into the app, and, depending on the answers, one of the different algorithms of the RPCRC could be used to predict the risk of prostate cancer and of clinically significant prostate cancer (Gleason score ≥7 and clinical stage >T2b). The system usefulness, information quality, and interface quality scores were high-92% (27.7/30), 87% (26.2/30), and 89% (13.4/15), respectively. No usability problems were identified. CONCLUSIONS: The RPCRC app is helpful in predicting the risk of prostate cancer and, even more importantly, clinically significant prostate cancer. Its algorithms have been externally validated before and the usability score shows the app's interface is well designed. Further usability testing is required in different populations to verify these results and ensure that it is easy to use, to warrant a broad appeal, and to provide better patient care.info:eu-repo/semantics/publishedVersio

Role of oxygen-restricted environments in biofilm growth and susceptibility profiles of traditional and atypical bacterial species in cystic fibrosis

It has been widely demonstrated that different predisposing “local environmental” conditions exist within the cystic fibrosis (CF) airways. Steep oxygen gradients are frequently detected in CF airways and may give rise to hypoxic or even anaerobic zones, providing nutritionally rich growth environments where bacteria may penetrate and accommodate, and being also responsible for the increase of antimicrobial resistance of most microorganisms. The aim of this study was to investigate the influence of aerobic, microaerophilic and anaerobic environments in biofilm growth and susceptibility patterns of the conventional CF-pathogen P. aeruginosa and other two atypical species related with CF. Single biofilms formed by Pseudomonas aeruginosa, and two uncommon CF-related bacteria, Inquilinus limosus and Dolosigranulum pigrum, were formed in vitro under environments with distinct oxygen availability, and their biomass and respiratory activity were further evaluated. The planktonic and biofilm susceptibility patterns to eight clinically relevant antibiotics were also determined under the same oxygen conditions, by measuring the minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC), respectively. The results obtained showed that both bacterial species, the traditional and unusual, were able to grow under the distinct environments, with D. pigrum demonstrating a great capability to develop biofilms with higher amount of biomass and higher respiratory activity, particularly when formed under microaerophilic atmospheres. Concerning the susceptibility profiles, all the planktonic cultures showed antibiotic tolerance under aerobic environments, decreasing their resistance under oxygen-restricted environments. As expected, biofilms were notoriously more difficult to eradicate than the same bacteria in the planktonic state (MBECs higher than MICs) which was independent of the oxygen availability within the surrounding environment. Furthermore, the biofilm consortia involving the atypical species were particularly more resistant to most antibiotics than the biofilms formed by the traditional pathogen P. aeruginosa, revealing the ineffectiveness of most antibiotics when applied to those atypical biofilms. This study suggests that the environment where bacteria grow is of great importance, demonstrating that the biofilm formation of traditional and other non-conventional bacteria is favored by restricted-oxygen atmospheres, as occurs in CF airways, which also makes biofilms more resistance to antibiotics. The recognition of CF lung as an environmental habitat, comprising niches ranging from oxic to anoxic, that can be occupied and colonized by a set of different microbial species, could lead to a better understanding of the clinical repercussions that these CF-associated infections can origin and may assist improvements in the management of CF and eventually in the treatment of other pathologies

Prostate Cancer Early Detection 2.0: Prediction models and eHealth

Early detection of prostate cancer should be tailored to well informed men using a personalized strategy, supported by multivariable individual risk-stratification. Risk calculators are superior to PSA-based strategies, and their use is advised in guidelines. In this thesis, a pragmatic screening strategy is proposed, starting in the primary care setting, using the readily available and easy to use ERSPC RPCRC smartphone app. Prediction models are expected to improve through research in big-data, biomarkers, and imaging studies. This new paradigm of personalized risk assessment should stimulate individualized management and follow-up, promoting early detection of clinically significant prostate cancer, while reducing overdiagnosis and overtreatment. eHealth impact on Urology practice is expected to grow, supported by software and hardware improvements, innovative services, and original applications. In the case of prostate cancer screening, this can be materialized via decision support tools, empowering patients and assisting healthcare professionals, promoting true informed shared decisions

Detection and discrimination of microorganisms using Locked Nucleic Acid - Fluorescence In Situ Hybridization (LNA-FISH)

The impact of multispecies biofilms on catheter-associated urinary tract infections outcome is still unclear due to the lack of adequate methodologies to discriminate the populations in situ. Employing fluorescence in situ hybridization (FISH) to discriminate the populations in a biofilm, can contribute to the understanding of microorganisms interactions in this structures. Consequently, this information might help to develop efficient strategies to prevent this disease. This work presents the first study that apply the FISH methodology using a set of LNA and/2’-O-Methyl RNA oligonucleotide probes, for the in situ detection of microorganisms in biofilms formed under conditions similar to the catheter-associated urinary tract infections

Dyslipidaemia, and mean blood cholesterol and triglycerides levels in the portuguese population: A systematic review

Introduction: Dyslipidaemia is a major risk factor for cardiovascular disease, the leading cause of death in Portugal. We aimed to critically summarize the evidence from studies that quantified the distribution of total cholesterol, cholesterol fractions and triglycerides, in order to estimate time trends in the Portuguese adult population. Methods: A systematic review was performed through Pubmed search up to January 2011. References' screening and data extraction were performed independently by two researchers and 28 eligible studies identified. Ecologic estimates of mean total cholesterol, LDL, HDL, and triglycerides were computed by linear regression, adjusting for participants' mean age, year of data collection and geographical coverage. Results: In 2005, the mean total cholesterol at 50 years of age was 215 mg/dL [95% confidence interval (95%CI): 210 to 219] among women and 219 mg/dL (95%CI: 206 to 232) among men. Between 1985 and 2005, the mean adjusted variation in total cholesterol per calendar year was 0.4 mg/dL (95%CI: -0.3 to 1.2) among women and -0.1 mg/dL (95%CI: -0.6 to 0.4) among men. Data on LDL, HDL and triglycerides covered a much narrower period, precluding analysis of time trends. In 2001 the adjusted mean levels of LDL, HDL and triglycerides were 132 mg/dL, 59 mg/dL and 111 mg/dL, respectively, among women, and 132 mg/dL, 49 mg/ dL and 150 mg/dL, respectively, among men. Conclusions: Estimated mean cholesterol, respective fractions, and prevalence of dyslipidaemia suggest a high proportion of high-risk subjects in the Portuguese population. Between 1985 and 2005, mean total cholesterol did not vary significantly