Diagnóstico das infecções relacionadas a cateter em recém-nascidos e epidemiologia molecular dos Staphylococcus spp. isolados

Pós-graduação em Biologia Geral e Aplicada - IBB

Frequency of patient-reported infections among sicker adults in high-income countries: an international perspective

The frequency of patient-reported health care-associated infections across several high-income countries was analyzed in representative population samples based on data from "The Commonwealth Fund's 2011 International Survey of Sicker Adults in Eleven countries." Across countries, 8.9% of patients who were hospitalized and/or had surgery reported an infection, but this rate varied considerably from 5.3% in the United States to 11.9% in New Zealand. Patients who reported infection were more likely to rate the quality of medical care received as fair or poor (odds ratio [OR], 2.4; 95% confidence interval [CI]: 1.9-3.1, P < .001). Female sex (OR, 1.2; 95% CI: 1.0-1.5, P = .027), reporting 2 or more chronic conditions (OR, 1.5; 95% CI: 1.1-2.0, P = .004), poor health (OR, 1.6; 95% CI: 1.2-2.1, P < .001), and surgery (OR, 1.8; 95% CI: 1.4-2.3, P < .001) were significant predictors for health care-associated infection across countries. Being above 64 years of age (OR, 0.78; 95% CI: 0.64-0.95, P = .013) and day-surgery (OR, 0.62; 95% CI: 0.48-0.79, P < .001) decreased the likelihood for reporting infection.

Antimicrobial efficacy of chlorhexidine digluconate alone and in combination with eucalyptus oil, tea tree oil and thymol against planktonic and biofilm cultures of Staphylococcus epidermidis

Objectives Effective skin antisepsis and disinfection of medical devices are key factors in preventing many healthcare-acquired infections associated with skin microorganisms, particularly Staphylococcus epidermidis. The aim of this study was to investigate the antimicrobial efficacy of chlorhexidine digluconate (CHG), a widely used antiseptic in clinical practice, alone and in combination with tea tree oil (TTO), eucalyptus oil (EO) and thymol against planktonic and biofilm cultures of S. epidermidis. Methods Antimicrobial susceptibility assays against S. epidermidis in a suspension and in a biofilm mode of growth were performed with broth microdilution and ATP bioluminescence methods, respectively. Synergy of antimicrobial agents was evaluated with the chequerboard method. Results CHG exhibited antimicrobial activity against S. epidermidis in both suspension and biofilm (MIC 2–8 mg/L). Of the essential oils thymol exhibited the greatest antimicrobial efficacy (0.5–4 g/L) against S. epidermidis in suspension and biofilm followed by TTO (2–16 g/L) and EO (4–64 g/L). MICs of CHG and EO were reduced against S. epidermidis biofilm when in combination (MIC of 8 reduced to 0.25–1 mg/L and MIC of 32–64 reduced to 4 g/L for CHG and EO, respectively). Furthermore, the combination of EO with CHG demonstrated synergistic activity against S. epidermidis biofilm with a fractional inhibitory concentration index of <0.5. Conclusions The results from this study suggest that there may be a role for essential oils, in particular EO, for improved skin antisepsis when combined with CHG.

Isolation and characterisation of bacteria associated with flying insects in hospitals, with particualr emphasis on Clostridum difficile

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies.

Endemic Methicillin-Resistant Staphylococcus Aureus: Nurses' Risk Perceptions and Attitudes

Dissemination of methicillin-resistant Staphylococcus aureus (MRSA) remains one of the most difficult challenges for prevention, control, and treatment of health care-associated infections. A survey and interviews were conducted on nurses from a hospital center. We found that most nurses' perceived risk of acquiring MRSA related to themselves (72%), other nurses (88.5%), and patients (97.8%). This perception influences attitudes, leading to compliance with the existing recommendations.

Fatores individuais de risco de infeção hospitalar : conhecer para prevenir

RESUMO - Medidas efetivas de prevenção e controlo de infeção, assim como a sua aplicação diária e consistente, devem fazer parte da cultura de segurança dos profissionais de saúde para promover a excelência da prestação de cuidados. Também a identificação dos fatores de riscos individuais de infeção é crucial e indispensável para a adoção de medidas para a gestão desses mesmos riscos. A avaliação do risco pretende determinar a probabilidade que um doente tem de adquirir ou disseminar uma infeção hospitalar (IH) (WIRRAL, 2008) nas unidades de saúde. A avaliação deve ser efetuada na admissão do doente e, de forma periódica durante o internamento, usando uma grelha de avaliação, integrada no processo global de cuidados do doente. Efetuada a avaliação de risco individual, que pressupõe a identificação dos fatores de risco do doente (fatores de risco intrínsecos e extrínsecos) pode ser implementado um plano de cuidados individualizado para os gerir. Pretendeu-se com este estudo identificar os fatores de risco de infeção hospitalar do doente que estão presentes na admissão e/ou que podem surgir durante o seu internamento, para que posteriormente seja possível determinar as medidas de prevenção (gestão do risco) a aplicar individualmente. Foi realizado um estudo de caso-controlo com os doentes internados no Hospital dos Lusíadas em 2011 com o objetivo de, por um lado, determinar os fatores de risco individuais que contribuem para a aquisição da IH e, por outro, caraterizar os fatores de risco para uma futura identificação de possíveis medidas de prevenção e controlo da aquisição e transmissão cruzada da infeção hospitalar. A população em estudo foi constituída pelos doentes que foram internados, entre 1 de Janeiro e 31 de Dezembro de 2011 sendo os casos os doentes em que foi identificada a presença de infeção hospitalar através do programa institucional de vigilância epidemiológica das infeções, tendo os controlos sido selecionados numa razão de 3:1 caso. Foi utilizado um suporte estruturado para a colheita de dados, com a listagem de fatores de risco identificados na revisão bibliográfica e de todos os fatores de risco apresentados pelos doentes em estudo. Os fatores de risco identificados que apresentaram um maior significado estatístico foram: a idade acima dos 50 anos, o género masculino, a administração de antimicrobianos nas três semanas anteriores ao internamento, a colocação de cateter venoso central, a algaliação e, no caso dos doentes cirúrgicos, a cirurgia de urgência e a classificação ASA 3. Após a identificação dos fatores de risco da população estudada neste hospital, é agora possível utilizar a informação obtida e delinear investigações adicionais, objetivando a construção de instrumentos para a identificação de doentes com risco aumentado de infeção.

Controlo e prevenção da infeção associada aos cuidados de saúde numa unidade de cuidados intensivos

Relatório de estágio de mestrado em Enfermagem da Pessoa em Situação Crítica

Determination of the importance of iron acquisition for Staphylococcus epidermidis biofilm survival in human blood

Dissertação de mestrado em Bioengenharia

NEW METHODS FOR DETECTION, SUSCEPTIBILITY TESTING AND BIOFILM ERADICATION OF DIFFICULT ORGANISMS

Aujourd'hui, les problèmes des maladies infectieuses concernent l'émergence d'infections difficiles à traiter, telles que les infections associées aux implants et les infections fongiques invasives chez les patients immunodéprimés. L'objectif de cette thèse était de développer des stratégies pour l'éradication des biofilms bactériens (partie 1), ainsi que d'étudier des méthodes innovantes pour la détection microbienne, pour l'établissement de nouveaux tests de sensibilité (partie 2). Le traitement des infections associées aux implants est difficile car les biofilms bactériens peuvent résister à des niveaux élevés d'antibiotiques. A ce jour, il n'y a pas de traitement optimal défini contre des infections causées par des bactéries de prévalence moindre telles que Enterococcus faecalis ou Propionibacterium acnés. Dans un premier temps, nous avons démontré une excellente activité in vitro de la gentamicine sur une souche de E. faecalis en phase stationnaire de croissance Nous avons ensuite confirmé l'activité de la gentamicine sur un biofilm précoce en modèle expérimental animal à corps étranger avec un taux de guérison de 50%. De plus, les courbes de bactéricidie ainsi que les résultats de calorimétrie ont prouvé que l'ajout de gentamicine améliorait l'activité in vitro de la daptomycine, ainsi que celle de la vancomycine. In vivo, le schéma thérapeutique le plus efficace était l'association daptomycine/gentamicine avec un taux de guérison de 55%. En établissant une nouvelle méthode pour l'évaluation de l'activité des antimicrobiens vis-à-vis de micro-organismes en biofilm, nous avons démontré que le meilleur antibiotique actif sur les biofilms à P. acnés était la rifampicine, suivi par la penicilline G, la daptomycine et la ceftriaxone. Les études conduites en modèle expérimental animal ont confirmé l'activité de la rifampicine seule avec un taux de guérison 36%. Le meilleur schéma thérapeutique était au final l'association rifampicine/daptomycine avec un taux de guérison 63%. Les associations de rifampicine avec la vancomycine ou la levofloxacine présentaient des taux de guérisons respectivement de 46% et 25%. Nous avons ensuite étudié l'émergence in vitro de la résistance à la rifampicine chez P. acnés. Nous avons observé un taux de mutations de 10"9. La caractérisation moléculaire de la résistance chez les mutant-résistants a mis en évidence l'implication de 5 mutations ponctuelles dans les domaines I et II du gène rpoB. Ce type de mutations a déjà été décrit au préalable chez d'autres espèces bactériennes, corroborant ainsi la validité de nos résultats. La deuxième partie de cette thèse décrit une nouvelle méthode d'évaluation de l'efficacité des antifongiques basée sur des mesures de microcalorimétrie isotherme. En utilisant un microcalorimètre, la chaleur produite par la croissance microbienne peut être-mesurée en temps réel, très précisément. Nous avons évalué l'activité de l'amphotéricine B, des triazolés et des échinocandines sur différentes souches de Aspergillus spp. par microcalorimétrie. La présence d'amphotéricine Β ou de triazole retardait la production de chaleur de manière concentration-dépendante. En revanche, pour les échinochandines, seule une diminution le pic de « flux de chaleur » a été observé. La concordance entre la concentration minimale inhibitrice de chaleur (CMIC) et la CMI ou CEM (définie par CLSI M38A), avec une marge de 2 dilutions, était de 90% pour l'amphotéricine B, 100% pour le voriconazole, 90% pour le pozoconazole et 70% pour la caspofongine. La méthode a été utilisée pour définir la sensibilité aux antifongiques pour d'autres types de champignons filamenteux. Par détermination microcalorimétrique, l'amphotéricine B s'est avéré être l'agent le plus actif contre les Mucorales et les Fusarium spp.. et le voriconazole le plus actif contre les Scedosporium spp. Finalement, nous avons évalué l'activité d'associations d'antifongiques vis-à-vis de Aspergillus spp. Une meilleure activité antifongique était retrouvée avec l'amphotéricine B ou le voriconazole lorsque ces derniers étaient associés aux échinocandines vis-à-vis de A. fumigatus. L'association échinocandine/amphotéricine B a démontré une activité antifongique synergique vis-à-vis de A. terreus, contrairement à l'association échinocandine/voriconazole qui ne démontrait aucune amélioration significative de l'activité antifongique. - The diagnosis and treatment of infectious diseases are today increasingly challenged by the emergence of difficult-to-manage situations, such as infections associated with medical devices and invasive fungal infections, especially in immunocompromised patients. The aim of this thesis was to address these challenges by developing new strategies for eradication of biofilms of difficult-to-treat microorganisms (treatment, part 1) and investigating innovative methods for microbial detection and antimicrobial susceptibility testing (diagnosis, part 2). The first part of the thesis investigates antimicrobial treatment strategies for infections caused by two less investigated microorganisms, Enterococcus faecalis and Propionibacterium acnes, which are important pathogens causing implant-associated infections. The treatment of implant-associated infections is difficult in general due to reduced susceptibility of bacteria when present in biofilms. We demonstrated an excellent in vitro activity of gentamicin against E. faecalis in stationary growth- phase and were able to confirm the activity against "young" biofilms (3 hours) in an experimental foreign-body infection model (cure rate 50%). The addition of gentamicin improved the activity of daptomycin and vancomycin in vitro, as determined by time-kill curves and microcalorimetry. In vivo, the most efficient combination regimen was daptomycin plus gentamicin (cure rate 55%). Despite a short duration of infection, the cure rates were low, highlighting that enterococcal biofilms remain difficult to treat despite administration of newer antibiotics, such as daptomycin. By establishing a novel in vitro assay for evaluation of anti-biofilm activity (microcalorimetry), we demonstrated that rifampin was the most active antimicrobial against P. acnes biofilms, followed by penicillin G, daptomycin and ceftriaxone. In animal studies we confirmed the anti-biofilm activity of rifampin (cure rate 36% when administered alone), as well as in combination with daptomycin (cure rate 63%), whereas in combination with vancomycin or levofloxacin it showed lower cure rates (46% and 25%, respectively). We further investigated the emergence of rifampin resistance in P. acnes in vitro. Rifampin resistance progressively emerged during exposure to rifampin, if the bacterial concentration was high (108 cfu/ml) with a mutation rate of 10"9. In resistant isolates, five point mutations of the rpoB gene were found in cluster I and II, as previously described for staphylococci and other bacterial species. The second part of the thesis describes a novel real-time method for evaluation of antifungals against molds, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against molds, have several limitations, especially when combinations of antifungals are investigated. We evaluated the activity of amphotericin B, triazoles (voriconazole, posaconazole) and echinocandins (caspofungin and anidulafungin) against Aspergillus spp. by microcalorimetry. The presence of amphotericin Β or a triazole delayed the heat production in a concentration-dependent manner and the minimal heat inhibition concentration (MHIC) was determined as the lowest concentration inhibiting 50% of the heat produced at 48 h. Due to the different mechanism of action echinocandins, the MHIC for this antifungal class was determined as the lowest concentration lowering the heat-flow peak with 50%. Agreement within two 2-fold dilutions between MHIC and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. We further evaluated our assay for antifungal susceptibility testing of non-Aspergillus molds. As determined by microcalorimetry, amphotericin Β was the most active agent against Mucorales and Fusarium spp., whereas voriconazole was the most active agent against Scedosporium spp. Finally, we evaluated the activity of antifungal combinations against Aspergillus spp. Against A. jumigatus, an improved activity of amphotericin Β and voriconazole was observed when combined with an echinocandin. Against A. terreus, an echinocandin showed a synergistic activity with amphotericin B, whereas in combination with voriconazole, no considerable improved activity was observed.

Prevalence, species differentiation, haemolytic activity, and antibiotic susceptibility of aeromonads in untreated well water

The use of untreated water for drinking and other activities have been associated with intestinal and extraintestinal infections in humans due to Aeromonas species. In the present study aeromonads were isolated from 48.7% of 1,000 water samples obtained from wells and other miscellaneous sources. Aeromonas species were detected in 45% of samples tested in spring, 34.5% in summer, 48% in autumn and 60% of samples tested in winter. Speciation of 382 strains resulted in 225 (59%) being A. hydrophila, 103 (27%) A. caviae, 42 (11%) A. sobria and 11 (3%) atypical aeromonads. Of 171 Aeromonas strains tested for their haemolytic activity, 53%, 49%, 40% and 37% were positive in this assay using human, horse, sheep and camel erythrocytes respectively. The results obtained indicate that potentially enteropathogenic Aeromonas species are commonly present in untreated drinking water obtained from wells in Libya (this may also apply to other neighbouring countries) which may pose a health problem to users of such water supplies. In addition, ceftriaxone and ciprofloxacin are suitable drugs that can be used in the treatment of Aeromonas-associated infections, particularly in the immunocompromised, resulting from contact with untreated sources of water.

Comparison of Methods for Evaluation of the Bactericidal Activity of Copper-Sputtered Surfaces against Methicillin-Resistant Staphylococcus aureus.

Bacteria can survive on hospital textiles and surfaces, from which they can be disseminated, representing a source of health care-associated infections (HCAIs). Surfaces containing copper (Cu), which is known for its bactericidal properties, could be an efficient way to lower the burden of potential pathogens. The antimicrobial activity of Cu-sputtered polyester surfaces, obtained by direct-current magnetron sputtering (DCMS), against methicillin-resistant Staphylococcus aureus (MRSA) was tested. The Cu-polyester microstructure was characterized by high-resolution transmission electron microscopy to determine the microstructure of the Cu nanoparticles and by profilometry to assess the thickness of the layers. Sputtering at 300 mA for 160 s led to a Cu film thickness of 20 nm (100 Cu layers) containing 0.209% (wt/wt) polyester. The viability of MRSA strain ATCC 43300 on Cu-sputtered polyester was evaluated by four methods: (i) mechanical detachment, (ii) microcalorimetry, (iii) direct transfer onto plates, and (iv) stereomicroscopy. The low efficacy of mechanical detachment impeded bacterial viability estimations. Microcalorimetry provided only semiquantitative results. Direct transfer onto plates and stereomicroscopy seemed to be the most suitable methods to evaluate the bacterial inactivation potential of Cu-sputtered polyester surfaces, since they presented the least experimental bias. Cu-polyester samples sputtered for 160 s by DCMS were further tested against 10 clinical MRSA isolates and showed a high level of bactericidal activity, with a 4-log(10) reduction in the initial MRSA load (10(6) CFU) within 1 h. Cu-sputtered polyester surfaces might be of use to prevent the transmission of HCAI pathogens.

Deaths in Northern Ireland, 2008

The number of deaths registered in Northern Ireland in 2008 was 14,900, a small increase on the 14,600 deaths registered in 2007. Cancer continues to be the most common cause of death with nearly 4,000 cancer deaths last year.These findings are contained in provisional 2008 mortality figures released by the Northern Ireland Statistics and Research Agency (NISRA).In 2008, over half of all deaths were caused by three main diseases; cancer (4,000 deaths), ischaemic heart disease (2,400 deaths) and stroke (1,300 deaths). Whilst ischaemic heart disease deaths halved from 4,800 in 1978 to 2,400 last year; cancer deaths have risen from 2,900 in 1978 to 4,000 last year.Over the last few years we have seen marked increases in deaths recorded due to Alzheimer's and other dementia related illnesses; conditions largely associated with the elderly. In 2008, there were 290 deaths due to Alzheimer's disease and a further 520 deaths due to other forms of dementia. Deaths related to healthcare-associated infection also increased last year.

Linezolid alone or combined with rifampin against methicillin-resistant Staphylococcus aureus in experimental foreign-body infection.

We investigated the activity of linezolid, alone and in combination with rifampin (rifampicin), against a methicillin-resistant Staphylococcus aureus (MRSA) strain in vitro and in a guinea pig model of foreign-body infection. The MIC, minimal bactericidal concentration (MBC) in logarithmic phase, and MBC in stationary growth phase were 2.5, >20, and >20 microg/ml, respectively, for linezolid; 0.01, 0.08, and 2.5 microg/ml, respectively, for rifampin; and 0.16, 0.63, >20 microg/ml, respectively, for levofloxacin. In time-kill studies, bacterial regrowth and the development of rifampin resistance were observed after 24 h with rifampin alone at 1x or 4x the MIC and were prevented by the addition of linezolid. After the administration of single intraperitoneal doses of 25, 50, and 75 mg/kg of body weight, linezolid peak concentrations of 6.8, 12.7, and 18.1 microg/ml, respectively, were achieved in sterile cage fluid at approximately 3 h. The linezolid concentration remained above the MIC of the test organism for 12 h with all doses. Antimicrobial treatments of animals with cage implant infections were given twice daily for 4 days. Linezolid alone at 25, 50, and 75 mg/kg reduced the planktonic bacteria in cage fluid during treatment by 1.2 to 1.7 log(10) CFU/ml; only linezolid at 75 mg/kg prevented bacterial regrowth 5 days after the end of treatment. Linezolid used in combination with rifampin (12.5 mg/kg) was more effective than linezolid used as monotherapy, reducing the planktonic bacteria by >or=3 log(10) CFU (P < 0.05). Efficacy in the eradication of cage-associated infection was achieved only when linezolid was combined with rifampin, with cure rates being between 50% and 60%, whereas the levofloxacin-rifampin combination demonstrated the highest cure rate (91%) against the strain tested. The linezolid-rifampin combination is a treatment option for implant-associated infections caused by quinolone-resistant MRSA.

Gentamicin Improves the Activities of Daptomycin and Vancomycin against Enterococcus faecalis In Vitro and in an Experimental Foreign-Body Infection Model.

For enterococcal implant-associated infections, the optimal treatment regimen has not been defined. We investigated the activity of daptomycin, vancomycin, and gentamicin (and their combinations) against Enterococcus faecalis in vitro and in a foreign-body infection model. Antimicrobial activity was investigated by time-kill and growth-related heat production studies (microcalorimetry) as well as with a guinea pig model using subcutaneously implanted cages. Infection was established by percutaneous injection of E. faecalis in the cage. Antibiotic treatment for 4 days was started 3 h after infection. Cages were removed 5 days after end of treatment to determine the cure rate. The MIC, the minimal bactericidal concentration (MBC) in the logarithmic phase, and the MBC in the stationary phase were 1.25, 5, and >20 μg/ml for daptomycin, 1, >64, and >64 μg/ml for vancomycin, and 16, 32, and 4 μg/ml for gentamicin, respectively. In vitro, gentamicin at subinhibitory concentrations improved the activity against E. faecalis when combined with daptomycin or vancomycin in the logarithmic and stationary phases. In the animal model, daptomycin cured 25%, vancomycin 17%, and gentamicin 50% of infected cages. In combination with gentamicin, the cure rate for daptomycin increased to 55% and that of vancomycin increased to 33%. In conclusion, daptomycin was more active than vancomycin against adherent E. faecalis, and its activity was further improved by the addition of gentamicin. Despite a short duration of infection (3 h), the cure rates did not exceed 55%, highlighting the difficulty of eradicating E. faecalis from implants already in the early stage of implant-associated infection.

Disinfection with sodium hypochlorite in hospital environmental surfaces in the reduction of contamination and infection prevention: a systematic review

OBJECTIVETo search for evidence of the efficiency of sodium hypochlorite on environmental surfaces in reducing contamination and prevention of healthcare-associated infection HAIs.METHODSystematic review in accordance with the Cochrane Collaboration.RESULTSWe analyzed 14 studies, all controlled trials, published between 1989-2013. Most studies resulted in inhibition of microorganism growth. Some decreased infection, microorganism resistance and colonization, loss of efficiency in the presence of dirty and surface-dried viruses.CONCLUSIONThe hypochlorite is an effective disinfectant, however, the issue of the direct relation with the reduction of HAIs remains. The absence of control for confounding variables in the analyzed studies made the meta-analysis performance inadequate. The evaluation of internal validity using CONSORT and TREND was not possible because its contents were not appropriate to laboratory and microbiological studies. As a result, there is an urgent need for developing specific protocol for evaluating such studies.