Microbiological quality assessment of sand from beaches in Portuguese coast: 15 years of experience

Communication presented at the TEMPH 2014 - Trends in Environmental Microbiology for Public Health. Lisbon, 18-21 September 2014

Bacterial biofilms, antibiotic resistance and healthcare-associated infections: a dangerous connection

In 2012, were estimated 6.7 million cases of healthcare-associated infections (HAI) either in long-term care facilities or acute-care hospitals from which result 37,000 deaths configuring a serious public health problem [1]. The etiological agents are diverse and often resistant to antimicrobial agents. One of the mechanisms responsible for the emergence of drug resistance is biofilm assembly. Biofilms are defined as thin layers of microorganisms adhering to the surface of a structure, which may be organic or inorganic, together with the polymers that they secrete [2]. They are dynamic structures which experience different stages of organization with the ageing and are linked to an increase in bacterial resistance to host defense mechanisms, antibiotics, sterilization procedures other than autoclaving, persistence in water distribution systems and other surfaces. The understanding of bacteria organization within the biofilm and the identification of differences between planktonic and sessile forms of bacteria will be a step forward to fight HAIs. Bacterial isolates were grown in adequate medium. Antibiotic susceptibility was evaluated by broth microdilution method and interpreted according to NCCLS guidelines. A similar assay was performed to evaluate biofilm susceptibility to antibiotics. Bacteria ability to assemble biofilms was assayed by the microtiter-plate test [3] being tested in both abiotic (materials present in healthcare units) and biotic (Hella cells) surfaces. The biofilm structure was assessed by scanning electron microscopy (SEM) in either backscattered electron diffraction or secondary electrons mode. The kinetic of biofilm assembly depends on bacteria growth rate, incubation temperature and medium. Furthermore, the SEM analysis of planktonic and sessile forms of the same bacteria allowed the identification of structural differences which may be involved in virulence (Fig. 1). Bacteria ability to assemble biofilms seems to be independent of the abiotic structure (Fig.2). The same is not observed in biotic surfaces. This fact suggests that biofilm assembly in vivo is dependent of bacteria tropism. The minimum inhibitory concentration (MIC) determine for bacteria organized in biofilms is higher than for their planktonic forms. The increase ranges from 2 to 200 folds and is proportional to the ability of bacteria to assemble biofilms. Further studies will be conducted in order to prevent biofilm assembly within healthcare units which will result in a decrease of HAI and emergence of antibiotic resistant bacteria

A gestão da qualidade e a gestão contabilístico-financeira dos centros de novas oportunidades através do modelo de auto-avaliação da Common Assessment Framework

O presente trabalho de investigação tem por objectivo avaliar a qualidade da gestão dos Centros de Novas Oportunidades (CNO). Mais especificamente, devido às características peculiares de financiamento dos CNO - transferência por conta do Orçamento do Estado e subsídios da União Europeia, sugerimos que a qualidade da gestão no sector financeiro é determinante para um desempenho de sucesso e sustentável. Nesta sequência, o projecto desenvolve-se em dois níveis: no primeiro, é realizada uma avaliação genérica da qualidade da gestão dos CNO através da análise dos critérios de meios da metodologia CAF e para o efeito é aplicado um questionário de diagnóstico aos Directores e/ou Coordenadores e outro aos Colaboradores do CNO. A escolha deste modelo deveu-se ao facto de haver orientações por parte da Agência Nacional para as Qualificações (ANQ) para que os CNO passassem a ter, como prática comum, a auto-avaliação da actividade Novas Oportunidades A avaliação do CNO feita pelo Director/Coordenador e pelos Colaboradores em relação a alguns critérios permitiu-nos averiguar que estes Centros ainda têm um longo caminho a percorrer até atingir a excelência. No entanto, pode-se dizer que o objectivo inicial deste estudo foi atingido, pois os responsáveis e colaboradores têm a mesma percepção da qualidade do serviço que prestam e do CNO que representam, com excepção para alguns itens que são possíveis de melhorar, nomeadamente a divulgação da Missão, da Visão, dos Valores e do Plano Estratégico de Intervenção (PEI) do CNO às outras partes interessadas. No segundo nível, e por se reconhecer que a gestão contabilístico-financeira é crucial e estratégica numa organização, sobretudo no que respeita à sua sustentabilidade, é avaliado o grau de qualidade organizativa contabilístico-financeira dos Centros com base no modelo de auto-avaliação da CAF. A metodologia de investigação adoptada é o estudo de caso. O estudo de caso foi suportado na análise de Instrumentos de Gestão Contabilístico-Financeira, como o Relatório e Contas do ano de 2003 até ao ano de 2009. Foram definidas medidas de qualidade, baseadas nas que a ANQ e o Gestor do Programa utilizam para análise do projecto, acrescentando outras que consideramos pertinentes para este estudo. Deste estudo, conclui-se que o Centro sabe identificar quais as acções de melhoria que precisa implementar e promover uma boa gestão dos instrumentos financeiros na condução dos destinos a que se predispuseram. – ABSTRACT: Quality Management and Accounting and Financial Management in the Centers for New Opportunities under Model of Common Assessment Framework. The research Project that we have engaged aims to evaluate the quality of the management system of the Centres for New Opportunities (CNO). ln addition, due to the peculiar financing system of the CNO - Government transfers and European Union subsidies, we suggest that the quality of the financial system determines the performance and the sustainability of the CNO. Therefore, in the project we have carried out are two folded. The first one is the general evaluation of the management system of the CNO. The empirical research was based on the set of criteria of means of the Common Assessment Framework (CAF) of the EFQM and questionnaires were administered to the managerial staff and collaborators of the CNO. The choice for CAF as our working model was made under the recommendation of the National Agency for Qualifications that all CNO should practice a self-assessment of their qualifying activities. Our findings tell us that the CNO still have a long way to go in order to achieve the excellence. A very positive point is that both managerial staff and co-workers are quite conscious about the quality of service they should provide to their clients and the importance about their role in their own organization. Some points could be easily improved such as providing a clear communication to the organization about their Mission, Vision, Values and Strategy and, furthermore, to the stakeholders in general. The second and most essential part of our work was to evaluate the quality of the accounting and financial system of a CNO. A good accounting and financial management is a guarantee of good performance and long term sustainability. The methodology adopted was case study. The case study was mostly supported by the analysis of deployment of financial instruments and reporting document from 2003 to 2009. Some measures of quality were identified and evaluated and we could conclude the studied CNO was able to select the adequate projects to proceed with and provided a good governance of financial instruments

Exploring dangerous connections between Klebsiella pneumoniae biofilms and healthcare-associated infections

Healthcare-associated infections (HAI) are a huge public health concern,particularly when the etiological agents are multidrug resistant. The ability of bacteria to develop biofilm is a helpful skill, both to persist within hospital units and to increase antibiotic resistance. Although the links between antibiotic resistance, biofilms assembly and HAI are consensual, little is known about biofilms. Here, electron microscopy was adopted as a tool to investigate biofilm structures associated with increased antibiotic resistance. The K. pneumoniae strains investigated are able to assemble biofilms, albeit with different kinetics. The biofilm structure and the relative area fractions of bacteria and extracellular matrix depend on the particular strain, as well as the minimal inhibitory concentration (MIC) for the antibiotics. Increased values were found for bacteria organized in biofilms when compared to the respective planktonic forms, except for isolates Kp45 and Kp2948, the MIC values for which remained unchanged for fosfomycin. Altogether, these results showed that the emergence of antimicrobial resistance among bacteria responsible for HAI is a multifactorial phenomenon dependent on antibiotics and on bacteria/biofilm features

Bacterial biofilms, antibiotic resistance and healthcare-associated infections: a dangerous connection

In 2012, were estimated 6.7 million cases of healthcare-associated infections (HAI) either in long-term care facilities or acute-care hospitals from which result 37,000 deaths configuring a serious public health problem. The etiological agents are diverse and often resistant to antimicrobial drugs. One of the mechanisms responsible for the emergence of drug resistance is biofilm assembly. Biofilms are defined as thin layers of microorganisms adhering to the surface of a structure, which may be organic or inorganic, together with the polymers that they secrete. They are dynamic structures which experience different stages of organization with the ageing and are linked to an increase in bacterial resistance to host defense mechanisms, antibiotics, sterilization procedures other than autoclaving, persistence in water distribution systems and other surfaces. The understanding of bacteria organization within the biofilm and the identification of differences between planktonic and sessile forms of bacteria will be a step forward to fight HAIs

Community- and hospital-acquired Klebsiella pneumoniae urinary tract infections in Portugal : virulence and antibiotic resistance

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Klebsiella pneumoniae is a clinically relevant pathogen and a frequent cause of hospital-acquired (HA) and community-acquired (CA) urinary tract infections (UTI). The increased resistance of this pathogen is leading to limited therapeutic options. To investigate the epidemiology, virulence, and antibiotic resistance profile of K. pneumoniae in urinary tract infections, we conducted a multicenter retrospective study for a total of 81 isolates (50 CA-UTI and 31 HA-UTI) in Portugal. The detection and characterization of resistance and virulence determinants were performed by molecular methods (PCR, PCR-based replicon typing, and multilocus sequence typing (MLST)). Out of 50 CA-UTI isolates, six (12.0%) carried β-lactamase enzymes, namely blaTEM-156 (n = 2), blaTEM-24 (n = 1), blaSHV-11 (n = 1), blaSHV-33 (n = 1), and blaCTX-M-15 (n = 1). All HA-UTI were extended-spectrum β-lactamase (ESBL) producers and had a multidrug resistant profile as compared to the CA-UTI isolates, which were mainly resistant to ciprofloxacin, levofloxacin, tigecycline, and fosfomycin. In conclusion, in contrast to community-acquired isolates, there is an overlap between virulence and multidrug resistance for hospital-acquired UTI K. pneumoniae pathogens. The study is the first to report different virulence characteristics for hospital and community K. pneumoniae pathogens, despite the production of β-lactamase and even with the presence of CTX-M-15 ESBL, a successful international ST15 clone, which were identified in both settings. This highlights that a focus on genomic surveillance should remain a priority in the hospital environment.This research was funded by the Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (ULisboa).info:eu-repo/semantics/publishedVersio

Exploitation of a link between antibacterial agent-resistance and biofilm-formation by classical and emergent pathogens

Objectives: In recent years nosocomial infections have gained growing importance. Among their etiological agents are “classical” pathogens such as S.aureus and also emergent pathogens, previously neglected, such as nontuberculous mycobacteria (MTM). The ability to resist to antibacterial agents, such as antibiotics and disinfectants, is shared by all of them. Here we aim to establish a link between bacterial virulence, antibacterial agents’ resistance and biofilm formation. Methods: Bacterial reference strains and clinical isolates were grown in adequate medium. Among the “classical” pathogens used are E.coli, K. pneumoniae, S. aureus and P. aeroginosa. The group of emergent pathogens includes M.fortuitum, M.abcessus, M.chelonae, M.avium etc. NTM susceptibility test to antibiotics was evaluated by broth based microdilution method and interpreted according to NCCLS guidelines. The desinfectant (oxygen peroxide, ammonium quaternary salts [AQS] and glutaraldehyde [GA) efficacy was performed according to the approved guidelines. The susceptibility was performed by two different methods: broth microdilution and diffusion in solid medium. In order to evaluate the effect of these agents in bacteria a scanning electron microscopy (SEM) study was performed. Biofilm forming ability was evaluated by the microtiter-plate test. The assay was performed at 25ºC and 37ºC in optimal growth media, phosphate saline buffer pH 7.4 and water during for different periods of time. Fast growing bacteria were assayed for 3 days while slow growers were assayed for 15 days.Results: The results of the antibiotic susceptibility test showed, with no surprise, that the resistant strains are the most prevalent. The resistance spectrum ranged from 1 to 5 antibiotics currently used in therapeutic schemes. Oxygen peroxide was the most effective disinfectant followed by QAS and GA. Nevertheless, among NTM we identified one isolate resistant to all disinfectants tested. The SEM analysis showed that different disinfectants caused different effects on bacteria suggesting different action mechanisms. The ability to form biofilms was time, medium and temperature dependent.Conclusion: Bacteria resistance to antibiotics and disinfectants vary in the same manner. The mechanisms involved in the resistance are not fully elucidated and more studies are needed to provide effective conclusions. Biofilm formation can be part of the mechanism involved both in resistance development and propagation of infections by these agents

Next generation sequencing: The key to understand Klebsiella pneumoniae biofilms?

Background: The incidence of healthcare-associated infections (HAI) is determined by underlying disease conditions and exposure to high risk medical interventions. In Portugal since 1980s K. pneumoniae is a recognized etiological agent of epidemic and endemic infections in healthcare units. An increasing rate of K. pneumoniae strains resistant either to extended cephalosporins or carbapenems has been observed and one of the mechanisms responsible for the emergence of drug resistance could be the biofilm assembly. The capacity of K. pneumoniae to form biofilm was first described in the 1980s for abiotic surfaces and ten years later on biotic surfaces. The antibiotic failure to penetrate through the biofilm layers, the emergence of mutations which might be easily transferred horizontally, and quorum sensing have been pointed as responsible for the increased antibiotic resistance of bacteria within biofilms. The main objective was to study the biofilm structure and the kinetic assembly associated to antibiotic resistance profile in K. pneumoniae strains capsulate or not, and identify the genes involved in biofilm assembly on full genome sequencing of studied strains. Material and Methods: Twoo K. pneumoniae isolates collected in 1980 (Kp45, Kp703) and one (Kp2948) in 2011 were studied. Kp703 was encapsulated and the remaining had capsular type K:2. The bacterial ability to assemble biofilms on cell culture plates was evaluated. For SEM analysis, biofilms were allowed to form on six wells cell culture plates (Nunc) for 12h at 37ºC. DNA was extracted using QiAamp DNA mini kit following the manufactures instructions. Full genome sequence was performed using next-generation sequencing platform MiSeq (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. The RAST platform was used for annotation and MAUVE platform for multiple alignments. Results: The three isolates were able to assemble biofilms although following different kinetics. K. pneumoniae strains Kp703 and Kp45 followed similar kinetics with identical biomass increase nevertheless these bacteria differ in capsule expression. Full-sequencing and annotation of genomes of isolates was performed in order to explain the differences found in biofilm assembly. Preliminary data already revealed that the K. pneumoniae strains displaying enhanced biofilm-forming ability is genetically different from the others, and, in particular, present some specific features enrolling genomic regions believed to be biofilm-related in other bacteria (an intact prophage, genes coding for a filamentous haemoagglutinin, a haemolysin expression modulating protein and an YdeA protein). Conclusion: K. pneumoniae lacking capsule, regarded as less virulent, have a better performance as biofilm assembler and exhibited the highest increase in antibiotic resistance when organized within biofilms. The analysis of the full genome sequence will allow reachingon K.pneumoniaebiofilms and provide novel opportunities to exploit the overall fitness ofK. pneumoniaeunder antibiotic stress

Antioxidant and antimycotic activities of two native lavandula species from portugal

The antioxidant and antimycotic activities of the essential oils and extracts of two native Portuguese Lavandula species, L. stoechas subsp. luisieri and L. pedunculata, were evaluated by in vitro assays. The total phenolics and flavonoids content were also determined. The antioxidant potential was assessed through DPPH radical scavenging, inhibition of lipid peroxidation (ILP), and DNA protection assays. All samples displayed a high DPPH scavenging activity, some of them showing concentration dependence. The majority of the samples were also able to inhibit lipid peroxidation. A strong correlation was observed between the results of DPPH and ILP assays and the flavonoids content of the samples. In the DNA protection assay, all the extracts were able to preserve DNA integrity. The antimycotic activity was performed against twelve fungi belonging to Basidiomycota and Ascomycota Divisions. L. stoechas subsp. luisieri exhibited the broadest activity spectra. L. pedunculata extracts were active against five fungi. Cryptococcus neoformans was the most sensitive, being inhibited by all the extracts. Our results led to the conclusion that L. stoechas subsp. luisieri and L. pedunculata can be useful as new sources of natural antioxidants and antimycotic agents, providing a possible valorization of the existing biodiversity and resources of Portuguese flora

Virulence factors in carbapenem-resistant hypervirulent Klebsiella pneumoniae

Copyright © 2023 Mendes, Santos, Ramalho, Duarte and Caneiras. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Hypervirulence and carbapenem-resistant have emerged as two distinct evolutionary pathotypes of Klebsiella pneumoniae, with both reaching their epidemic success and posing a great threat to public health. However, as the boundaries separating these two pathotypes fade, we assist a worrisome convergence in certain high-risk clones, causing hospital outbreaks and challenging every therapeutic option available. To better understand the basic biology of these pathogens, this review aimed to describe the virulence factors and their distribution worldwide among carbapenem-resistant highly virulent or hypervirulent K. pneumoniae strains, as well as to understand the interplay of these virulence strains with the carbapenemase produced and the sequence type of such strains. As we witness a shift in healthcare settings where carbapenemresistant highly virulent or hypervirulent K. pneumoniae are beginning to emerge and replace classical K. pneumoniae strains, a better understanding of these strains is urgently needed for immediate and appropriate response.This research was partially funded by Fundação para a Ciência e a Tecnologia (FCT), under grant numbers UIDB/04295/2020 and UIDP/04295/2020. Moreover, CC acknowledges the funding provided by the “Research Award in Healthcare Associated Infections” granted by Escola Superior de Saúde Norte da Cruz Vermelha Portuguesa (2019) and by “BInov award,” an innovation award granted by the Southern Regional Section and Autonomous Regions of the Portuguese Pharmaceutical Society (2021). GM was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal, through a Ph.D. Research Studentship Contract (Contrato de Bolsa de Investigação para Doutoramento 2020.07736.BD).info:eu-repo/semantics/publishedVersio