Conventional and molecular typing of Salmonella typhi strains from Brazil

Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Molecular typing methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.

Phenotypic and molecular characterization of Salmonella Enteritidis strains isolated in São Paulo, Brazil

In São Paulo State, Brazil, the epidemic increase in isolation of Salmonella Enteritidis has been observed since 1994. A total of 105 S. Enteritidis strains (72 from human and 33 from non-human sources) isolated during the period 1975-1995, previously characterized by phage typing, was analyzed by antimicrobial susceptibility, plasmid profile, and ribotyping. Over 70% of the strains were susceptible to all antimicrobial agents tested, however, multiple resistance to antimicrobials was observed among the studied strains, mainly those from hospitalized patients. Phage type 8 (PT-8) was predominant among the strains isolated during the period of 1975-1992, but in the following years, PT-4 was the most frequent phage type identified. Seven different plasmid profiles were detected and 96% of the isolates harbored a plasmid of approximately 36 MDa. Ribotyping discriminated fourteen ribotypes (R1 to R14) among the strains examined. By analysis of dendrogram the strains were included in three groups with similarity level of 60%. The obtained results indicate that, a single ribotype (R11), determined for PT-4 strains isolated from 1993, characterizes the epidemic clone of S. Enteritidis in our region.

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.

ROAZ and ROAZ II Autonomous Surface Vehicle Design and Implementation

International Lifesaving Congress 2007, La Coruna, Spain, December, 2007

Intervenção Percutânea Urgente no Enfarte Agudo do Miocárdio por Lesão do Tronco Comum

BACKGROUND: ST-elevation myocardial infarction (STEMI) with the culprit lesion in the left main artery is a rare cardiac emergency with a poor prognosis. OBJECTIVE: Review and prognosis evaluation of primary percutaneous coronary intervention (PCI) performed in the setting of STEMI with left main occlusion in a single high-volume center. METHODS: Of the 483 primary or rescue PCIs performed and followed in our hospital during a 24-month period (August 2004 to July 2006), we retrospectively evaluated those involving left main procedures and analyzed in-hospital mortality and major cardiac events (MACE) in a 12-month follow-up. We found nine patients, age 68 +/- 9 years, five male, seven with multivessel disease and two with isolated left main disease. Rescue PCI was performed in three patients and primary PCI in the others. RESULTS: Seven patients presented in cardiogenic shock and two were classified in Killip class II on admission. Inotropic drugs, intra-aortic balloon pump and abciximab were used in eight patients. Drug-eluting stents were used in six patients, bare-metal stents in two, and isolated balloon angioplasty in one. Five patients (55%) died in the hospital and the four discharged home (two of them aged 81 and 82 years) were still alive and free from MACE at 12-month follow-up. CONCLUSIONS: Clinical presentation of STEMI with the culprit lesion in the left main artery was very severe. During PCI, drug-eluting stents, intra-aortic balloon pump and abciximab were used in almost all patients. This entity had a high mortality rate even though primary PCI was performed. Those who survived had a good mid-term prognosis.

Field experiments for marine casualty detection with autonomous surface vehicles

In this paper we present a set of field tests for detection of human in the water with an unmanned surface vehicle using infrared and color cameras. These experiments aimed to contribute in the development of victim target tracking and obstacle avoidance for unmanned surface vehicles operating in marine search and rescue missions. This research is integrated in the work conducted in the European FP7 research project Icarus aiming to develop robotic tools for large scale rescue operations. The tests consisted in the use of the ROAZ unmanned surface vehicle equipped with a precision GPS system for localization and both visible spectrum and IR cameras to detect the target. In the experimental setup, the test human target was deployed in the water wearing a life vest and a diver suit (thus having lower temperature signature in the body except hands and head) and was equipped with a GPS logger. Multiple target approaches were performed in order to test the system with different sun incidence relative angles. The experimental setup, detection method and preliminary results from the field trials performed in the summer of 2013 in Sesimbra, Portugal and in La Spezia, Italy are also presented in this work.

Thermographic and visible spectrum camera calibration for marine robotic target detection

Oceans - San Diego, 2013

High-Accuracy Low-Cost RTK-GPS for an Unmanned Surface Vehicle

This work presents a low cost RTK-GPS system for localization of unmanned surface vehicles. The system is based on the use of standard low cost L1 band receivers and in the RTKlib open source software library. Mission scenarios with multiple robotic vehicles are addressed as the ones envisioned in the ICARUS search and rescue case where the possibility of having a moving RTK base on a large USV and multiple smaller vehicles acting as rovers in a local communication network allows for local relative localization with high quality. The approach is validated in operational conditions with results presented for moving base scenario. The system was implemented in the SWIFT USV with the ROAZ autonomous surface vehicle acting as a moving base. This setup allows for the performing of a missions in a wider range of environments and applications such as precise 3D environment modeling in contained areas and multiple robot operations.

Evaluation of the in vitro activity of six antimicrobial agents against Neisseria gonorrhoeae

Use of antimicrobials for the treatment of gonorrhea started in 1930 with the utilization of sulfonamides. With the years other drugs were used for its treatment such as penicillin, tetracycline, spectinomycin, and others. Although highly specific in the beginning, these drugs, with time did not show anymore the expected therapeutic results because of aspects of chromosomal and plasmid-mediated resistance. The purpose of this study was to evaluate the susceptibility of Neisseria gonorrhoeae strains to six drugs used for its treatment (penicillin, tetracycline, cefoxitin, thiamphenicol, spectinomycin and ofloxacin) by the determination of minimal inhibitory concentrations of these drugs. We concluded that drugs, such as cefoxitin, thiamphenicol and spectinomycin still are excellent pharmacological agents for the treatment of gonorrhea. Penicillin, although still efficient, needs more attention regarding its use, as well as ofloxacin, because of the emergence of resistant strains. Tetracycline and its derivatives should be strongly contraindicated for the treatment of gonorrhea.

Water jet based autonomous surface vehicle for coastal waters operations

This paper presents the design of low cost, small autonomous surface vehicle for missions in the coastal waters and specifically for the challenging surf zone. The main objective of the vehicle design described in this paper is to address both the capability of operation at sea in relative challenging conditions and maintain a very low set of operational requirements (ease of deployment). This vehicle provides a first step towards being able to perform general purpose missions (such as data gathering or patrolling) and to at least in a relatively short distances to be able to be used in rescue operations (with very low handling requirements) such as carrying support to humans on the water. The USV is based on a commercially available fiber glass hull, it uses a directional waterjet powered by an electrical brushless motor for propulsion, thus without any protruding propeller reducing danger in rescue operations. Its small dimensions (1.5 m length) and weight allow versatility and ease of deployment. The vehicle design is described in this paper both from a hardware and software point of view. A characterization of the vehicle in terms of energy consumption and performance is provided both from test tank and operational scenario tests. An example application in search and rescue is also presented and discussed with the integration of this vehicle in the European ICARUS (7th framework) research project addressing the development and integration of robotic tools for large scale search and rescue operations.

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

Deciding Kleene Algebra Terms Equivalence in Coq

This paper presents a mechanically verified implementation of an algorithm for deciding the equivalence of Kleene algebra terms within the Coq proof assistant. The algorithm decides equivalence of two given regular expressions through an iterated process of testing the equivalence of their partial derivatives and does not require the construction of the corresponding automata. Recent theoretical and experimental research provides evidence that this method is, on average, more efficient than the classical methods based on automata. We present some performance tests, comparisons with similar approaches, and also introduce a generalization of the algorithm to decide the equivalence of terms of Kleene algebra with tests. The motivation for the work presented in this paper is that of using the libraries developed as trusted frameworks for carrying out certified program verification.

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.

Electrochemical characterization of Dps, a DNA-protecting protein

Dissertação para obtenção do Grau de Mestre em Biotecnologia

ABO-Incompatible Liver Transplantation in Acute Liver Failure: A Single Portuguese Center Study

INTRODUCTION: ABO-incompatible liver transplantation (ABOi LT) is considered to be a rescue option in emergency transplantation. Herein, we have reported our experience with ABOi LT including long-term survival and major complications in these situations. PATIENT AND METHODS: ABOi LT was performed in cases of severe hepatic failure with imminent death. The standard immunosuppression consisted of basiliximab, corticosteroids, tacrolimus, and mycophenolate mofetil. Pretransplantation patients with anti-ABO titers above 16 underwent plasmapheresis. If the titer was above 128, intravenous immunoglobulin (IVIG) was added at the end of plasmapheresis. The therapeutic approach was based on the clinical situation, hepatic function, and titer evolution. A rapid increase in titer required five consecutive plasmapheresis sessions followed by administration of IVIG, and at the end of the fifth session, rituximab. RESULTS: From January 2009 to July 2012, 10 patients, including 4 men and 6 women of mean age 47.8 years (range, 29 to 64 years), underwent ABOi LT. At a mean follow-up of 19.6 months (range, 2 days to 39 months), 5 patients are alive including 4 with their original grafts. One patient was retransplanted at 9 months. Major complications were infections, which were responsible for 3 deaths due to multiorgan septic failure (2 during the first month); rejection episodes (4 biopsy-proven of humoral rejections in 3 patients and 1 cellular rejection) and biliary. CONCLUSION: The use of ABOi LT as a life-saving procedure is justifiable in emergencies when no other donor is available. With careful recipient selection close monitoring of hemagglutinins and specific immunosuppression we have obtained acceptable outcomes.