The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria

RESUMO: Sessenta e três derivados de hidantoína foram utilizados para avaliar possíveis efeitos de modulação na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um método fluorimétrico semi-automático. Nenhum dos compostos apresentaram actividade anti-bacteriana até concentrações de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulação de effluxo na presença de glucose. Para testar esta actividade, estirpes de Salmonella resistentes à ciprofloxacina, induzidas a elevados níveis de resistência com sobre-expressão de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes à ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitório /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de iões em eucariotas, foi questionado o papel dos iões na regulação de BE bacterianas, que poderão influenciar a eficácia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido à importância de iões de cálcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulação do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo é independente de energia metabólica; contudo, a pH 8 é absolutamente dependente, sendo que o Ca2+ é indispensável para manter a actividade das ATPases bacterianas. A acumulação/effluxo de EtBr pela E. coli AG100 foi determinada na presença/ausência de Ca2+, clorpromazina (inibidor de ligação de Ca2+ a proteínas), e ácido etilenodiamino tetra-acético (quelante de Ca2+). Acumulação/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importância no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evidência que muitos aspectos bioquímicos e bioenergéticos devem ser tomados em consideração no estudo da resistência bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.

Lack of methicillin-resistant Staphylococcus aureus nasal carriage among patients at a primary-healthcare unit in Porto Alegre, Brazil

INTRODUCTION: Community-associated methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a pathogen in individuals without traditional risk factors. MATERIAL AND METHODS: MRSA nasal carriage was assessed in individuals consulting at a Primary Health Unit in Brazil. RESULTS: A total of 336 individuals were included: 136 were tested only for MRSA and 200 for any S. aureus. No MRSA was found among the 336 individuals and 23 (11.5%) of 200 were colonized by S. aureus. DISCUSSION: Low prevalence rates have been found in non-hospitalized individuals, but MRSA surveillance should be encouraged to monitor clinical and molecular epidemiology of CA- MRSA.

Low Dose Thalidomide for Treatment of Resistant Discoid Lupus Erythematosus. A Case Report

Discoid lupus erythematosus (DLE) is a chronic, indolent, disfiguring disease that is characterized by scaly, erythematous, disk-shaped patches and plaques followed by atrophy, scarring and depigmentation. In a small number of patients, it is refractory to standard therapies. In several studies, thalidomide has been reported to be an effective treatment in those cases. The most fearful side effects are teratogenicity and neuropathy. Adequate counseling and vigilance must be given to the patients. We report a 45-year-old Portuguese woman who presented with a 20-year history of severe facial and scalp DLE confirmed by histopathology. For several years, it failed to respond to several therapies, including topical, intralesional and oral corticosteroids, hydroxychloroquine, methotrexate, azathioprine and topical tacrolimus. Thalidomide was initiated at a dosage of 50mg/day and the skin lesions had improved dramatically after three weeks with complete clinical remission. Two months later, the dose was reduced to 50mg, five days per week without disease rebound. The patient´s concomitant medications during the treatment included sunscreen, hydroxycholoroquine, enoxaparin and aspirin to prevent thromboembolic events. Pregnancy testing, routine laboratory and electrocardiography were performed at regular intervals for safety monitoring and the results were within normal limits. Only minor side effects as nausea, constipation and somnolence were noted, however, they improved with dose reduction. Our data confirm that thalidomide therapy is an alternative or adjunctive treatment for patients with severe, chronic DLE that is refractory to standard therapies. In this patient, low-dose thalidomide was an effective treatment with minimal side effects.

Community-Associated Methicillin-Resistant Staphylococcus Aureus Lacking PVL, as a Cause of Severe Invasive Infection Treated with Linezolid

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging public health problem worldwide. Severe invasive infections have been described, mostly associated with the presence of Panton-Valentine leukocidin (PVL). In Portugal limited information exists regarding CA-MRSA infections. In this study we describe the case of a previously healthy 12-year-old female, sport athlete, who presented to the hospital with acetabulofemoral septic arthritis, myositis, fasciitis, acetabulum osteomyelitis, and pneumonia.The MRSA isolated from blood and synovial fluid was PVL negative and staphylococcal enterotoxin type P (SEP) and type L (SEL) positive, with a vancomycin MIC of 1.0mg/L and resistant to clindamycin and ciprofloxacin. The patient was submitted to multiple surgical drainages and started on vancomycin, rifampicin, and gentamycin. Due to persistence of fever and no microbiological clearance, linezolid was started with improvement. This is one of the few reported cases of severe invasive infection caused by CA-MRSA in Portugal,which was successfully treated with linezolid. In spite of the severity of infection, the MRSA isolate did not produce PVL.

In vitro SCREENING ANTIBACTERIAL ACTIVITY OF Bidens pilosa LINNÉ AND Annona crassiflora MART. AGAINST OXACILLIN RESISTANT Staphylococcus aureus (ORSA) FROM THE AERIAL ENVIRONMENT AT THE DENTAL CLINIC

Currently multiresistant Staphylococcus aureus is one common cause of infections with high rates of morbidity and mortality worldwide, which directs scientific endeavors in search for novel antimicrobials. In this study, nine extracts from Bidens pilosa (root, stem, flower and leaves) and Annona crassiflora (rind fruit, stem, leaves, seed and pulp) were obtained with ethanol: water (7:3, v/v) and their in vitro antibacterial activity evaluated through both the agar diffusion and broth microdilution methods against 60 Oxacillin Resistant S. aureus (ORSA) strains and against S. aureus ATCC6538. The extracts from B. pilosa and A. crassiflora inhibited the growth of the ORSA isolates in both methods. Leaves of B. pilosa presented mean of the inhibition zone diameters significantly higher than chlorexidine 0.12% against ORSA, and the extracts were more active against S. aureus ATCC (p < 0.05). Parallel, toxicity testing by using MTT method and phytochemical screening were assessed, and three extracts (B. pilosa, root and leaf, and A. crassiflora, seed) did not evidence toxicity. On the other hand, the cytotoxic concentrations (CC50 and CC90) for other extracts ranged from 2.06 to 10.77 mg/mL. The presence of variable alkaloids, flavonoids, tannins and saponins was observed, even though there was a total absence of anthraquinones. Thus, the extracts from the leaves of B. pilosa revealed good anti-ORSA activity and did not exhibit toxicity.

Aluminum and Sulphate Removal by a Highly Al-Resistant Dissimilatory Sulphate-Reducing Bacteria Community

A highly Al-resistant dissimilatory sulphatereducing bacteria community was isolated from sludge of the wetland of Urgeiriça mine (community W). This community showed excellent sulphate removal at the presence of Al3+. After 27 days of incubation, 73,86 and 81% of sulphate was removed in the presence of 0.48, 0.90 and 1.30 mM of Al3+, respectively. Moreover,Al3+ was simultaneously removed: 55,85 and 78% of metal was removed in the presence of 0.48, 0.90 and 1.30 mM of Al3+, respectively. The dissociation of aluminiumlactate soluble complexes due to lactate consumption by dissimilatory sulphate-reducing bacteria can be responsible for aluminum removal, which probably precipitates as insoluble aluminium hydroxide. Phylogenetic analysis of 16S rRNA gene showed that this community was mainly composed by bacteria closely related to Desulfovibrio desulfuricans. However, bacteria affiliated to Proteus and Ralstonia were also present in the community.

IDENTIFICATION OF Pseudomonas spp. AS AMOEBA-RESISTANT MICROORGANISMS IN ISOLATES OF Acanthamoeba

Acanthamoeba is a “Trojan horse” of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.

RAW TROPICAL OYSTERS AS VEHICLES FOR MULTIDRUG-RESISTANT Vibrio parahaemolyticus

The following study aimed to determine the antimicrobial susceptibility profile of Vibrio parahaemolyticus strains from fresh and frozen oysters Crassostrea rhizophorae sold in Fortaleza-Brazil. An antibiogram was performed on 87 isolates using nine antibiotics: gentamicin (Gen 10 µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 µg), chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline (Tet 30 µg), vancomycin (Van 30 µg) and erythromycin (Ery 15 µg). All strains were resistant to at least one antibiotic, and 85 (97.7%) were multi-resistant, with predominance of the Van+ Pen+Amp resistance profile (n = 46). Plasmid resistance to Pen, Amp and Ery was detected. Thus, the risk that raw oyster consumption poses to the health of consumers is highlighted, due to the fact that these bivalves may host antibacterial-resistant microorganisms.

Investigation and Control of a Large Outbreak of Multi-Drug Resistant Tuberculosis at a Central Lisbon Hospital

An increase in the number of new cases of tuberculosis (TB) combined with poor clinical outcome was identified among HIV-infected injecting drug users attending a large HIV unit in central Lisbon. A retrospective epidemiological and laboratory study was conducted to review all newly diagnosed cases of TB from 1995 to 1996 in the HIV unit. Results showed that from 1995 to 1996, 63% (109/173) of the Mycobacterium tuberculosis isolates from HIV-infected patients were resistant to one or more anti-tuberculosis drugs; 89% (95) of these were multidrug-resistant, i.e., resistant to at least isoniazid and rifampicin. Eighty percent of the multidrug-resistant strains (MDR) available for restriction fragment length polymorphism (RFLP) DNA fingerprinting clustered into one of two large clusters. Epidemiological data support the conclusion that the transmission of MDR-TB occurred among HIV-infected injecting drug users exposed to infectious TB cases on open wards in the HIV unit. Improved infection control measures on the HIV unit and the use of empirical therapy with six drugs once patients were suspected to have TB, reduced the incidence of MDR-TB from 42% of TB cases in 1996 to 11% in 1999.

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.

Development of biocolonization resistant mortars: preliminary results

Restoration of Buildings and Monuments, vol. 13, nº 6 (2007), p.389-400

Evaluation of salt resistant mortars

Restoration of Buildings and Monuments, vol.11, nº 2 (2005), p.105-110

The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus

Dissertation for the Master’s Degree in Structural and Functional Biochemistry