Ciprofloxacin susceptibility patterns of planktonic and sessile S. aureus, E. coli, and P. aeruginosa – effect of the exposure time

Several aspects of human infections within a clinical arena are related to biofilm development. Various surfaces, like indwelling devices and medical equipments, are prone to biofilm formation, causing subsequent pathogenesis. The application of antimicrobial agents is one of the main strategies to eradicate biofilms. However, the action of antibiotics could be inefficient duo to the high tolerance of biofilmes comparatively to the bacteria in planktonic state. Thus, it is of upmost importance the use of suitable antimicrobials with high efficacies to eradicate biofilms and to control nosocomial infections. Ciprofloxacin (CIP) is a broad spectrum fluoroquinolone antibiotic, often use against both Gram-positive and Gram-negative bacteria, that causes inhibition of bacterial cell division by the inhibition of DNA gyrase and topoisomerase IV. With this study it was aimed to analyze the antimicrobial efficacy of CIP against S. aureus, E. coli K-12, and P. aeruginosa PAO1, in planktonic cultures and in biofilm state, and also to characterize the time–kill kinetics of ciprofloxacin in pre-established 1-day-old biofilms. In planktonic cultures, the CIP susceptibility patterns were achieved by the broth microdilution method and by the determination of the number of viable cells. In sessile bacteria, the anti-biofilm activity of CIP was assessed using a standardized biofilm assay, quantifying the biofilm mass, through crystal violet, the respiratory activity, using the XTT reduction assay, and the number of viable cells. The time-kill kinetics of CIP were attained exposing 24-hour-old biofilms of each bacteria to ciprofloxacin (6 ug/ml) over time (until 24 h), and determining biofilm metabolic activity, biomass and cellular viability at regular time intervals. Ciprofloxacin displayed dose-dependent activity against both bacteria in planktonic and biofilm states. The MIC values ranged between 0.185 ug/ml for S. aureus, 0.5 ug/ml for E. coli and 0.75 ug/ml for P. aeruginosa. These values revealed that it was needed a 4-fold increase in CIP dose to cause growth inhibition of Gram- bacteria comparatively to the Gram+ strain. The presence of CIP during biofilm formation did not kill totally the biofilm-associated cells neither eradicated the biomass adhered. CIP exhibited only a bacteriostatic effect for all strains studied emphasizing that this antibiotic is more effective on suspended than on biofilm cells. The application of a constant dose of 6 ug/ml of CIP against pre-formed biofilms revealed an evident time-dependent effect in the antibiotic action, since a gradual reduction of biofilm activity, biomass and cell number occurred over time. However, the time of CIP exposure needed to reduce the biofilm characteristics varied from each bacterial species. In fact, the antibacterial effect was evident after 4 h of exposure of the antibiotic with the S. aureus, around 8 h of exposure with the E. coli and 6 h of exposure with the P. aeruginosa pre-formed biofilms. Once more, ciprofloxacin only presented bacteriostatical activity against all the bacterial biofilms, even when the exposure time reached 24 h. In the range of experimental concentrations and exposure times tested, CIP didn´t reveal to be effective against bacteria cells neither in the pre-established biofilm nor during the process of biofilm development, in spite of the improved reduction of the metabolic activity, biofilm biomass and in the number of viable cells of S. aureus, E. coli and P. aeruginosa biofilms. Alternative treatments or combination of antibiotic therapies should be studied for the implementation of more effective ways of eradication biofilm-associated infections.IBB-CEB, and the Portuguese Foundation for Science and Technology (Project PTDC/SAU-ESA/64609/2006; PhD Grant SFRH/BD/31065/2006

Impact of nutritional conditions on colony morphology variants isolated from P. aeruginosa and S. aureus biofilms

In natural habitats, microorganisms are challenged all the time due to stress conditions imposed by the surrounding environment. To adapt to these environmental changes, bacteria alter their physiological and genetic traits. This adaptive behavior may be achieved by phenotype switching. This process consists in a reversible switch of phenotypes, as a mechanism ON/OFF, which occurs at high frequencies than spontaneous mutations. Colony morphology variation is the macroscopic feature of the phenotypic switching. Colony variation may have serious impact on bacterial virulence and antimicrobial resistance potentiating its ability to cause disease. Some colony variants are strongly associated to antibiotic resistance due to their presence in chronic infections despite antibiotic therapy. In cystic fibrosis, the switch of P. aeruginosa from non-mucoid to mucoid morphotype, which overproduce alginate, is a crucial stage to the establishment of this recalcitrant disease. Small colony variants (SCV) are other well-known resistant morphotype. These variants exhibited small size because its slow growth rate, pigmentation, haemolysis, reduced range of carbohydrate utilization and higher resistance to aminoglycosides antibiotics and cell-wall inhibitors. It has been growing the number of studies related with phenotypic switching and colony morphology characterization. However, normally each study reports the use of different solid growth media which makes the comparison between studies inaccurate. In order to clarify the role of nutritional conditions on bacterial colony morphologies and on its populational diversity, P. aeruginosa and S. aureus planktonic and biofilm-growing cells were spread onto the most common solid laboratory media (TSA, MHA, LB agar, MacConkey agar and Columbia horse blood agar). Additionally, the reproducibility of each medium was also inspected. Data showed that P. aeruginosa and S. aureus colony morphotypes are strongly influenced by the plating medium used. The main differences observed were the size, texture and form of colonies. The largest colonies were detected in TSA, MHA and LB agar. Colonies grown on MHA and LB agar were very similar possibly due to their identical nutritional composition. All the solid media tested showed reproducibility between assays except the Columbia horse blood agar which exhibited some inconsistency probably due to the presence of blood in its composition. Amongst the solid media tested, for planktonic and biofilm cultures, TSA gave rise to higher number of colony variants. Phenotype diversity seems to be more influenced by nutritional factors when bacteria derived from biofilms. This study allows concluding that, in contrast to fungi, bacterial colony appearance is influenced by the nutritional conditions of the solid media used to spread the cells. This evidence should be taking into account when important phenomena as phenotypic switching are going to be studied. The data obtained with this preliminary work may question the classification of colony morphotypes used until now.Fundação para a Ciência e Tecnologia (FCT) - Projecto PTDC/SAUESA/6460912006/FCOMP-01-0124-FEDER-007480, bolsa de Doutoramento SFRH/BD/31065/2006European CommunityFEDER - Program COMPET

Selecting men for bone densitometry: performance of osteoporosis risk assessment tools in Portuguese men

Clinicians need tools to identify patients most likely to benefit from bone mineral density (BMD) testing, for which cost-effectiveness does not allow generalized screening. This study supports the utility of osteoporosis risk assessment tools in selecting men for BMD testing. Different cutoff values may be appropriate for different countries and/or ethnic origins. INTRODUCTION: Our aim was to evaluate the utility of three osteoporosis (OP) risk assessment tools in a large group of Portuguese men aged 50 or more and to determine the best cutoff value to be used for selecting men for bone densitometry. METHODS: We assessed the performance of three simple tools in 202 randomly selected men: body weight criterion (BWC), osteoporosis self-assessment tool for Asians (OSTA), and a modified version of the OSTA equation (OST). Previously published cutoff values (validated in postmenopausal women) and three additional cutoff values were tested. Sensitivity (SE), specificity (SP), predictive values, and area under the receiver operating characteristic (AUROC) curve for correctly selecting men with OP (defined by BMD testing) were determined. RESULTS: Mean age of the cohort was 63.8 years. According to the World Health Organization diagnostic categories, 16.8% had osteoporosis. The best performing cutoffs for correctly selecting men with OP for BMD testing were OST < 3 (SE = 75.5%, SP = 50.0%, AUROC = 0.632), OSTA < 3 (SE = 73.5%, SP = 58.3%, AUROC = 0.659), and BWC < 75 kg (SE = 73.5%, SP = 61.3%, AUROC = 0.674). CONCLUSIONS: OP risk assessment tools seem to be useful in men aged 50 or more. Best cutoff values are different from those recommended for postmenopausal women. Different cutoff values may be appropriate for different countries and/or ethnic origins

Adaptation of the medication adherence report scale in a sample of Portuguese type 2 diabetes patients

O presente estudo teve como objectivo adaptar a Escala de Adesão à Medicação (MARS) a uma amostra de diabéticos portugueses tipo 2. Participaram no estudo 387 diabéticos (42% mulheres e 58% homens) diagnosticados com diabetes tipo 2. 57% dos diabéticos tinha sido diagnosticados nos últimos 6 meses e 38% entre 7 e 12 meses no momento da avaliação. O estudo da validade da escala “Medication Adherence Report Scale” revelou uma solução de um factor tal como o instrumento original que explica 52,15% da variância. O alfa de Cronbach foi de .74. Em termos de validade de construto, a adesão à medicação encontrasse relacionada com as representações da doença ao nível das dimensões consequências, controlo pessoal, controlo do tratamento, preocupação e representações emocionais. Finalmente, a relação entre adesão à medicação e as representações da doença foi sensível ao género e idade. O questionário de Adesão à Medicação possui as qualidades psicométricas que permitem o seu uso na população portuguesa com diabetes tipo 2.The goal of the present study was the adaptation of the Medication Adherence Report Scale in a Portuguese sample of type 2 diabetes patients. 387 diabetes patients participated in the study, (42% women and 58% males) diagnosed with type 2 diabetes. 57% of the diabetes patients were diagnosed in the previous six months and 38% between 7 and 12 months prior to the assessment. The validation study revealed a one factor solution like the original version that explained 52,15% of the variance. Cronbach alpha was .74. In terms of construct validity, adherence to medication is related to illness representations on several dimensions: consequences, personal control, treatment control, worries, and emotional representations. Finally, the relationship between adherence to medication and illness representations is sensitive to gender and age. The Medication Adherence Report Scale presents the psychometric properties that allow its use in Portuguese type 2 diabetes patients.Fundação para a Ciência e a Tecnologia (FCT

Effect of benzalkonium chloride residues on the initial adhesion of adapted P. aeruginosa

Bacteria have developed different strategies to sense, respond and adapt to chemical antimicrobial compounds. One of them involves bacterial adhesion to solid surfaces and subsequent biofilm formation. The biofilm lifestyle, that is usually resistant to antimicrobial treatment, is a phenomenon well noticed in almost all industrial and medical arenas. In the latter, the existence of pathogenic bacteria, that easily adhere to a surface and form biofilms, can give rise to equipment contamination and persistent infections. For instance, Pseudomonas aeruginosa has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance. This bacterium, often found in a biofilm, attached to some surface, can frequently cause life-threatening infections under conditions where the host is injured and/or has the immune system compromised. Benzalkonium chloride (BZK) is a quaternary ammonium compound (QAC) used as a general clinical disinfectant and antiseptic in health care facilities and domestic households. QACs are bacteriostatic at low concentrations and bactericidal at high concentrations, thus their indiscriminate and improper use may favor the development of adaptive resistance. Therefore, they have been pointed out as the possible cause for the selection and persistence of bacterial strains with antibiotic and biocidal resistance. With this study it was aimed to characterize the adhesion ability and biofilm formation of P. aeruginosa non-adapted and adapted to BZK on polyethylene (PET) surfaces non-conditioned and pre-conditioned with BZK solution. For that purpose, bacterial hydrophobicity and changes in surface properties after conditioning were evaluated by means of contact angles determination. The initial bacterial adhesion was followed up along time (2, 4 and 6 h) by the quantification of the adhered biomass, through crystal violet (CV) and respiratory activity, using XTT. The adaptation of P. aeruginosa to BZK was attained after 6 cycles of re-inoculation every 48 h selected in TSA also supplemented with increasing doses of BZK. The free energy of adhesion of the bacteria with the PET surfaces, calculated trough the hydrophobicity components, revealed that the precontioning of the substrata increases the potential initial adhesion of P.aeruginosa, being this more significant for the adapted bacteria. Thus, it can be stated that, in the thermodynamic point of view, the initial adhesion of the P. aeruginosa adapted and non-adapted to the PET is favoured by surface preconditioning. Moreover, BZK preconditioning did not prevent or impair P.aeruginosa adhesion. In fact, the initial adhesion data showed that the accumulation of biomass increased with time, being the non-adapted bacterial cells more able to attach and establish a biofilm. The presence of BZK residues in the adhesion surfaces (preconditioning) promotes the attachment of the bacterial cells, since more biomass was determined. Concerning the activity of the adhered cells, preconditioning seem not cause any disturbance of cells, since equal or higher cellular activity was determined. The overall results suggest that preconditioning of PET surfaces with BZK promotes the attachment and activity of the bacterial cells, especially of the adapted bacteria. These evidences suggest that when the bacteria were previously exposed to a sub-lethal concentration of a chemical antimicrobial compound, they acquired some insusceptibility to that compound allowing their growth when facing adverse conditions. This study allowed gaining insights about the impact of unsuitable disinfection and rinsing procedures that might instigate the residues deposition, leading to bacteria adaptation, bacterial adhesion and biofilm development

Adaptation of trust in physician scale for patients with type 2 diabetes and their partners

Scale numa amostra Portuguesa de diabéticos tipo 2 e seus companheiros. Participaram no estudo 340 diabéticos diagnosticados no ano anterior a avaliação e respectivos companheiros. O estudo da validade revelou dois fatores que explicaram 53,66% da variância total: “Escala de Confiança na Relação” que explica 40,28% da variância e “Escala de Confi ança na Competência” que explica 13,38%, nos diabéticos, e 53,56% da variância total, 41,43% e 12,12% respectivamente. A escala apresenta boa validade convergente estando correlacionada com a adesão à medicação e uma boa validade descriminante em termos de idade e gênero reunindo as condições para ser utilizado em pacientes diabéticos Portugueses e seus companheiros.The present study focused on the psychometric properties of the Portuguese version of Trust in Physician Scale (TPS) in a sample of Portuguese type 2 diabetics and their partners. Three hundred and forty diabetics diagnosed in the year prior to the assessment participated in the study as well as their partners. The validity study revealed two factors that explained 53.66% of the total variance: “Trust in Relationship” explains 40.28% of the variance and “Trust in Competence” explains 13.38% of the variance for the diabetics; and for their partners 53.56% of total variance, 41.43% and 12.12%, respectively. The scale shows good convergent validity being correlated with adherence to medication and a good descriminant validity in terms of age and gender validity gathering the necessary conditions for its use with Portuguese diabetic patients and their partnersFundação para a Ciência e a Tecnologia (FCT

Impact of quality and type of anticoagulant treatment, and In ammatory biomarkers on quality of life and psychological morbidity in patients with atrial fibrillation: An exploratory study

Background: Owing to increasing worldwide life expectancy, the step rise in the prevalence of atrial fibrillation (AF) represents an urgent public health issue. Indeed, AF can severely affect a patient's quality of life (QoL) since it is associated with serious outcomes, such as stroke, cardiac failure, and cognitive impairment/dementia, resulting in increased morbidity and mortality and a burden for health-care systems. Anticoagulation therapy prevents stroke in individuals with AF, whether with vitamin K antagonists (VKA) or direct oral anticoagulants (DOACs). So far, effective AF management is missing since its etiology is unraveled. Within the research project [Cognitive Decline Risk Profiles and Quality of Life in Atrial Fibrillation: A Longitudinal Study- 2022.072(057-DEFI/058-CE)] a pilot study was conducted to assess a dynamic cohort of AF outpatients who attended the anticoagulation clinic at Santo António University Hospital Center, a Portuguese university public hospital. In this study, QoL, distress and cognitive impairment in patients with AF was assessed considering quality and type of anticoagulant treatment. Methods: With a cross-sectional design, quality of anticoagulation therapy was measured calculating Time in Therapeutic Range (TTR) using Rosendaal method for patients under VKA (TTR>70% as good control); for those patients under DOAC, the presence of plasma therapeutic levels of this drug, taking into account the time of blood sampling, meant an anticoagulant treatment of quality. QoL and distress were assessed by the Atrial Fibrillation Effect on Quality-of-life Questionnaire (AFEQT) and the Depression, Anxiety and Stress Scale (DASS-21), respectively. Cognitive impairment was assessed by the Montreal Cognitive Assessment (MoCA) scale. Data on sociodemographic, clinic [medical history including quality and type of anticoagulation therapy, stroke(CHA2DS2-VASc) and bleeding(HAS-BLED) risk scores, duration of illness) and inflammatory biomarkers were also collected. Results: A total of 62 AF patients were included (mean age 74.4±10.2 years, 59.7% women) with a mean CHA2DS2-VASc of 4.3±1.6 and HAS-BLED of 2.6±1.2. Fifty patients (80.6%) were on VKA and twelve (19.4%) on DOACs (all in apixaban). Those individuals under VKA showed a mean TTR score of 77.1 ± 14.7% (67.4% with a good control) and 11 patients in apixaban presented DOAC therapeutic plasma level. Participants had an illness duration average of 16.02 years±10.62. The mean scores of AFEQT was 2.65 ± .93 showing a good QoL and for MoCA was 21.66 ± 3.90 indicating mild cognitive impairment. Regarding anxiety, depression, and stress (DASS-21) the mean scores were 4.27 ± 4.08, 6.52 ± 4.92 and 7.37 ± 5.37, respectively showing non-clinical psychological morbidity. Patients with higher plasmatic levels of C Reactive Protein (CRP) reported more depression (r= .482; p= .005) and those with a longer illness duration presented a higher level of cognitive impairment, (r= -.398; p= .001). No other associations were found between inflammatory biomarkers and psychological variables. There were significant differences according to sex, age, and illness duration, with women reporting lower QoL (p = .002), higher depression (p = .004), higher stress (p = .005) and older patients (p = .017) as well as patients under VKA and longer disease duration reporting better TTR. (p = .007). No differences were found on patients' QoL (p = .342), anxiety (p = .370), depression (p = .167) and stress (p = .117), cognitive impairment, (p = .084) between good and non-good quality of anticoagulant treatment and between VKA versus DOAC therapy (p = .444). Conclusion: The majority of the patients showed good quality of anticoagulation and QoL, but they revealed mild cognitive impairment. Patients' QoL, distress and cognitive impairment were independent of quality and type of anticoagulation treatment. Older AF patients, particularly those with longer diagnosis, should be assessed for cognitive function on a regular basis, preventing dementia and economic burden. The association of depression with higher levels of CRP in AF patients underlines the nature of these two inflammatory conditions that coexisting associate to worse outcomes. Future studies should assess cognitive function over time to unravel the pathophysiology of cognitive decline and the effect of AF treatments on cognition to optimize and personalize AF therapy

Synthesis of N-alkyl-C α, α-dimethylglycine derivatives

The application of trialkyloxonium tetrafluoroborates for N-alkylation of the nonnatural amino acid Cα, α-dimethylglycine is described. Several methyl esters of dimethylglycine protected with different amine protecting groups were subject to N-ethylation or N-methylation with triethyloxonium tetrafluoroborate or trimethyloxonium tetrafluoroborate, respectively. The corresponding N-akyl-Cα, α-dimethylglycine derivatives were obtained in good to high yields. Removal of the methyl ester rendered amino acid derivatives ready for application in peptide synthesis.Foundation for Science and Technology (FCT) – Portugal and Fundo Europeu de Desenvolvimento Regional (FEDER) for financial support to Chemistry Centre of University of Minho. The NMR spectrometer Bruker Avance II+ 400 is part of the National NMR Network and was purchased in the framework of the National Program for Scientific Re-equipment; contract REDE/1517/RMN/2005, with funds from POCI 2010, FEDER and FCT.info:eu-repo/semantics/publishedVersio