Clinical and Bacteriological Survey of Diabetic Foot Infections in Lisbon

AIMS: An epidemiological survey of diabetic foot infections (DFIs) in Lisbon, stratifying the bacterial profile based on patient demographical data, diabetic foot characteristics (PEDIS classification), ulcer duration and antibiotic therapy. METHODS: A transversal observational multicenter study, with clinical data collection using a structured questionnaire and microbiological products (aspirates, biopsies or swabs collected using the Levine method) of clinically infected foot ulcers of patients with diabetes mellitus (DM). RESULTS: Forty-nine hospitalized and ambulatory patients were enrolled in this study, and 147 microbial isolates were cultured. Staphylococcus was the main genus identified, and methicillin-resistant Staphylococcus aureus (MRSA) was present in 24.5% of total cases. In the clinical samples collected from patients undergoing antibiotic therapy, 93% of the antibiotic regimens were considered inadequate based on the antibiotic susceptibility test results. The average duration of an ulcer with any isolated multi-drug resistant (MDR) organism was 29 days, and previous treatment with fluoroquinolones was statistically associated with multi-drug resistance. CONCLUSIONS: Staphylococcus aureus was the most common cause of DFIs in our area. Prevalence and precocity of MDR organisms, namely MRSA, were high and were probably related to previous indiscriminate antibiotic use. Clinicians should avoid fluoroquinolones and more frequently consider the use of empirical anti-MRSA therapy.

Clonal types and antimicrobial resistance profiles of methicillin-resistant Staphylococcus aureus isolates from hospitals in south Brazil

In the present study were evaluated the DNA macrorestriction profile and SCCmec types for nine multi-resistant MRSA selected. Also antimicrobial susceptibility testing by disk diffusion method was evaluated for 68 MRSA isolates against 12 antimicrobial agents. The isolates were recovered from blood culture collected from hospitalized patients in three hospitals of Porto Alegre, Brazil. PFGE and PCR for mecA and SCCmec I, II, III, IV types genes were done on selected nine isolates with susceptibility only to vancomycin, teicoplanin and linezolid. Two clone profiles, with five subtypes, were demonstrated among multi-resistant MRSA analyzed. Eight isolates showed harbor SCCmec type III and one isolate was not typeable. The knowledge of SCCmec type, clone and antimicrobial profiles among S. aureus is essential mainly to prevention and control of dissemination of the antimicrobial resistance.

FAS -670A>G genetic polymorphism Is associated with Treatment Resistant Depression

Background Hippocampal neurogenesis has been suggested as a downstream event of antidepressants (AD) mechanism of action and might explain the lag time between AD administration and the therapeutic effect. Despite the widespread use of AD in the context of Major Depressive Disorder (MDD) there are no reliable biomarkers of treatment response phenotypes, and a significant proportion of patients display Treatment Resistant Depression (TRD). Fas/FasL system is one of the best-known death-receptor mediated cell signaling systems and is recognized to regulate cell proliferation and tumor cell growth. Recently this pathway has been described to be involved in neurogenesis and neuroplasticity. Methods Since FAS -670A>G and FASL -844T>C functional polymorphisms never been evaluated in the context of depression and antidepressant therapy, we genotyped FAS -670A>G and FASL -844T>C in a subset of 80 MDD patients to evaluate their role in antidepressant treatment response phenotypes. Results We found that the presence of FAS -670G allele was associated with antidepressant bad prognosis (relapse or TRD: OR=6.200; 95% CI: [1.875–20.499]; p=0.001), and we observed that patients carrying this allele have a higher risk to develop TRD (OR=10.895; 95% CI: [1.362–87.135]; p=0.008).Moreover, multivariate analysis adjusted to potentials confounders showed that patients carrying G allele have higher risk of early relapse (HR=3.827; 95% CI: [1.072–13.659]; p=0.039). FAS mRNA levels were down-regulated among G carriers, whose genotypes were more common in TRD patients. No association was found between FASL-844T>C genetic polymorphism and any treatment phenotypes. Limitations Small sample size. Patients used antidepressants with different mechanisms of action. Conclusion To the best of our knowledge this is the first study to evaluate the role of FAS functional polymorphism in the outcome of antidepressant therapy. This preliminary report associates FAS -670A>G genetic polymorphism with Treatment Resistant Depression and with time to relapse. The current results may possibly be given to the recent recognized role of Fas in neurogenesis and/or neuroplasticity.

Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.

Optimization of the Tartrate-Resistant Acid Phosphatase Detection by Histochemical Method

According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60ºC. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.

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.

A contribuição das bombas de efluxo QacA e Smr para a multirresistência em Staphylococcus aureus

RESUMO: O efluxo de compostos antimicrobianos é um mecanismo importante na multirresistência em bactérias. Bombas de efluxo codificadas em plasmídeos, como a QacA e a Smr, estão implicadas na susceptibilidade reduzida a biocidas, geralmente utilizados na prevenção e controlo de infecções nosocomiais, incluindo as causadas por estirpes de Staphylococcus aureus resistentes à meticilina (MRSA). Neste trabalho pretendeu-se avaliar a relevância de QacA e Smr no perfil de susceptibilidade dos isolados clínicos MRSA SM39 e SM52, que transportam os plasmídeos pSM39 e pSM52 com os determinantes qacA e smr, respectivamente. A actividade de efluxo das estirpes SM39 e SM39 curada (sem pSM39) e das estirpes SM52 e RN4220:pSM52 (estirpe susceptível RN4220 transformada com pSM52) foi caracterizada por: (1) determinação da concentração mínima inibitória (CMI) de biocidas, corantes e antibióticos, na ausência e presença dos inibidores de efluxo tioridazina, clorpromazina, verapamil e reserpina; e (2) fluorometria em tempo-real. A determinação de CMIs demonstrou que a actividade de efluxo mediada por QacA e Smr está envolvida na susceptibilidade reduzida aos biocidas e corantes testados, que incluíram o brometo de hexadeciltrimetilamónio, a cetrimida, o cloreto de benzalcónio, a berberina, o cloreto de dequalínio, a pentamidina e o brometo de etídeo. Os ensaios fluorométricos confirmaram a elevada actividade de efluxo presente nas estirpes com os genes qacA ou smr. A determinação de CMIs para antibióticos β-lactâmicos em conjunto com o teste da nitrocefina revelou a presença simultânea do gene qacA e de uma β-lactamase no plasmídeo pSM39. Este trabalho evidencia a importância das bombas de efluxo QacA e Smr na resistência a biocidas em estirpes MRSA e na sobrevivência destas estirpes em ambiente hospitalar e na comunidade, para além de destacar a questão da potencial co-resistência entre biocidas e antibióticos.--------------- ABSTRACT: Drug efflux has become an important cause of multidrug resistance (MDR) in bacteria. Plasmid-encoded MDR efflux pumps, such as QacA and Smr, are implicated in reduced susceptibility to biocides, generally used in the prevention and control of nosocomial infections, including the ones caused by methicillin-resistant Staphylococcus aureus (MRSA). In this work, we aimed to evaluate the relevance of QacA and Smr to the susceptibility profile of the clinical MRSA isolates SM39 and SM52, which harbor the plasmids pSM39 and pSM52 that carry the determinants qacA and smr, respectively. Efflux activity of strain SM39 and its plasmid-free counterpart, SM39 cured, SM52 and RN4220:pSM52 (susceptible strain RN4220 transformed with pSM52) was characterized by: (1) determination of minimum inhibitory concentration (MIC) of biocides, dyes and antibiotics, in the absence and presence of the efflux inhibitors thioridazine, chlorpromazine, verapamil and reserpine; and (2) real-time fluorometry. MIC determination showed that QacA and Smr mediated efflux was involved in the reduced susceptibility profile to the biocides and dyes tested, which included hexadecyltrymethylammonium bromide, cetrimide, benzalkonium chloride, berberine, dequalinium chloride, pentamidine and ethidium bromide. Fluorometric assays confirmed the higher efflux activity present in strains harboring qacA or smr genes. Moreover, MIC determination for β-lactam antibiotics together with the nitrocefin test confirmed the presence of a β-lactamase in the plasmid carried by SM39 strain, pSM39. This work highlights the relevance of QacA and Smr to the biocide resistance in MRSA strains, and consequently to their survival and maintenance in the hospital environment and in the community. Furthermore, the presence of a β-lactamase and qacA determinants in the the same plasmid reinforces the question of the potencial biocide/antibiotic co-resistance in MRSA strains.

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.

FREQUENCY, URINALYSIS AND SUSCEPTIBILITY PROFILE OF PATHOGENS CAUSING URINARY TRACT INFECTIONS IN ENUGU STATE, SOUTHEAST NIGERIA

Objective: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture.Methods: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream ‘clean catch’ method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.Results: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin.Conclusion: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.

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.