Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae

In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology). Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae.

Measurement of intestinal permeability using mannitol and lactulose in children with diarrheal diseases

The excretion ratio of lactulose/mannitol in urine has been used to assess the extension of malabsorption and impairment of intestinal permeability. The recovery of lactulose and mannitol in urine was employed to evaluate intestinal permeability in children with and without diarrhea. Lactulose and mannitol probes were measured using high-performance liquid chromatography with pulsed amperometric detection (HPLC-PAD). Two groups of solutions containing 60 µM sugars were prepared. Group I consisted of glucosamine, mannitol, melibiose and lactulose, and group II of inositol, sorbitol, glucose and lactose. In the study of intra-experiment variation, a sample of 50 µl from each group was submitted to 4 successive determinations. The recovered amounts and retention times of each sugar showed a variation <2 and 1%, respectively. The estimated recovery was >97%. In the study of inter-experiment variation, we prepared 4 independent samples from groups I and II at the following concentrations: 1.0, 0.3, 0.1, 0.03 and 0.01 mM. The amounts of the sugars recovered varied by <10%, whereas the retention times showed an average variation <1%. The linear correlation coefficients were >99%. Retention (k'), selectivity (a) and efficiency (N) were used to assess the chromatographic conditions. All three parameters were in the normal range. Children with diarrhea presented a greater lactulose/mannitol ratio compared to children without diarrhea.

PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsed-field gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillin-resistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains.

Lämpötilan vaikutus oksaalihapon hapetukseen pulssitetulla koronapurkauksella

Tutkimuksen tarkoituksena oli kartoittaa lämpötilan vaikutusta veden orgaanisten haitta-aineiden hapetuksessa PCD-menetelmällä. Kokeita tehtiin näytteiden eri alkulämpötiloilla. Malliyhdisteenä kokeissa käytettiin oksaalihappoa. Teoriaosuudessa käsiteltiin pulssittaista koronapurkausta ilmiönä. Lisäksi tarkasteltiin, kuinka PCD-menetelmällä muodostuu hapettimia neste-kaasufaasissa. Syntyvistä hapettimista keskityttiin otsoniin ja hydroksyyliradikaaliin. Kokeellisessa osuudessa esiteltiin käytetty PCD-laitteisto. Esittelyn jälkeen siirryttiin hapetuskokeiden kuvaamiseen ja analyysin suorittamiseen titrauksella. Lopuksi koottiin tulokset. Tutkimuksissa prosessin hapetustehon havaittiin heikentyvän lämpötilan noustessa tutkitulla lämpötila-alueella, mikä voi selittyä kaasufaasissa muodostuvan otsonin heikentyvällä liukoisuudella. Tuloksia voidaan pitää viitteellisinä, ja selkeän mallin muodostamiseksi tarvitaan jatkotutkimuksia laajemmalla lämpötila-alueella tarkasti toistettavilla koejärjestelyillä.

Pulssimuodon vaikutus valokaaren käytettävyyteen pulssi-MIG/MAG-hitsauksessa

Käytettävyydeltään huippuluokkaa olevan pulssi-MIG/MAG-hitsausvalokaaren toteuttaminen vaatii runsaasti tietoa eri pulssiparametreista ja niiden vaikutuksista hitsaukseen. Näihin vaikutuksiin liittyvä tieteellinen tutkimus on ollut melko vähäistä. Erityisesti tieto pulssimuodon vaikutuksista hitsausääneen on perustunut lähinnä kokemuksen tuomaan tuntumaan. Tässä diplomityössä tutkittiin pulssimuodon vaikutusta valokaaren käytettävyyteen pulssi-MIG/MAG-hitsauksessa. Käytettävyys käsittää tässä tapauksessa hitsausäänen, hitsin geometrian ja hitsausominaisuudet. Tutkimuksen alussa perehdyttiin kirjallisuuteen ja tuoreimpiin tutkimuksiin, jonka jälkeen vertailtiin erilaisia pulssimuotoja keskenään hitsauskokeiden avulla. Hitsausääneen ja hitsin geometriaan liittyvät kokeet suoritettiin mekanisoidusti. Hitsausääneen liittyvät mittaukset suoritettiin luokan 1 äänitasomittarilla ja tuloksia analysoitiin tietokoneohjelmistolla. Hitsien geometrioiden vertailu suoritettiin makrohietutkimuksena. Hitsausominaisuuksia tutkittiin suurnopeuskameran ja oskilloskoopin, sekä lopulta käsinhitsauskokeiden avulla. Kaikissa koevaiheissa pulssimuodon tarkasteluun käytettiin oskilloskooppia. Lisäksi käytössä oli toinen oskilloskooppi, jolla tarkasteltiin hitsausvirran spektriä. Pulssimuodon muokkaamiseen käytettiin erillistä tietokoneohjelmaa. Työn kokeellinen osuus keskittyi pulssi-MAG-hitsaukseen. Pulssimuotoa muokkaamalla saatiin aikaan miellyttävämpi hitsausääni. Lisäksi havaittiin, että pulssimuotoa muokkaamalla hitsistä saadaan kapeampi, jolloin juuritunkeumaa saavutetaan enemmän. Käsinhitsauskokeet osoittivat muokatun pulssimuodon olevan myös hitsaajan näkökulmasta käytettävyydeltään paras pulssimuoto. Erityisesti valokaaren vakaus ja kohdistuvuus sekä suurien hitsausnopeuksien sietokyky olivat muokatun pulssimuodon etuja. Selviä haittavaikutuksia pulssimuodon muokkaamiselle ei löydetty.

Absence-like seizures in adult rats following pilocarpine-induced status epilepticus early in life

Administration of pilocarpine causes epilepsy in rats if status epilepticus (SE) is induced at an early age. To determine in detail the electrophysiological patterns of the epileptogenic activity in these animals, 46 Wistar rats, 7-17 days old, were subjected to SE induced by pilocarpine and electro-oscillograms from the cortex, hippocampus, amygdala, thalamus and hypothalamus, as well as head, rostrum and vibrissa, eye, ear and forelimb movements, were recorded 120 days later. Six control animals of the same age range did not show any signs of epilepsy. In all the rats subjected to SE, iterative spike-wave complexes (8.1 ± 0.5 Hz in frequency, 18.9 ± 9.1 s in duration) were recorded from the frontal cortex during absence fits. However, similar spike-wave discharges were always found also in the hippocampus and, less frequently, in the amygdala and in thalamic nuclei. Repetitive or single spikes were also detected in these same central structures. Clonic movements and single jerks were recorded from all the rats, either concomitantly with or independently of the spike-wave complexes and spikes. We conclude that rats made epileptic with pilocarpine develop absence seizures also occurring during paradoxical sleep, showing the characteristic spike-wave bursts in neocortical areas and also in the hippocampus. This is in contrast to the well-accepted statement that one of the main characteristics of absence-like fits in the rat is that spike-wave discharges are never recorded from the hippocampal fields.

Kaarityypin ja suojakaasun vaikutus roiskeisuuteen teräksen MAG-hitsauksessa

Tässä kandidaatintyössä tutkitaan suojakaasun ja kaarityypin vaikutuksia roiskeisuuden muodostumiselle teräksen MAG-hitsauksessa (nro. 135). Roiskeisuuden tutkimisessa tutkitaan kolmea (3) eri kaarityyppiä ja kahta (2) eri suojakaasua ja niiden keskenäisiä vaikutuksia roiskeiden syntymiselle. Tutkittavat kaarityypit ovat pulssi-, kuumakaari sekä Kemppi Oy:n WiseFusion erikoiskaarisovellus. Suojakaasuina käyetään AGAn Corgon 3 ja Mison 18 suojakaasuja. Hitsauskokeita suoritetaan kuusi (6) kappaletta alapienana jalkoasennossa (PA). Työn alussa perehdytään MAG-hitsauksen laitteistoon ja teoriaan sekä perehdytään mitkä eri asiat vaikuttavat roiskeiden muodostumiseen. Työn lopussa verrataan tieteellisistä julkaisuista löydettyjä tietoja ja tuloksia itsetehtyihin koetuloksiin.

Clonal dissemination of VanA-type glycopeptide-resistant Enterococcus faecalis between hospitals of two cities located 100 km apart

Nosocomial dissemination of glycopeptide-resistant enterococci represents a major problem in hospitals worldwide. In Brazil, the dissemination among hospitals in the city of São Paulo of polyclonal DNA profiles was previously described for vancomycin-resistant Enterococcus faecium. We describe here the dissemination of VanA phenotype E. faecalis between two hospitals located in different cities in the State of São Paulo. The index outbreak occurred in a tertiary care university hospital (HCUSP) in the city of São Paulo and three years later a cluster caused by the same strain was recognized in two patients hospitalized in a private tertiary care hospital (CMC) located 100 km away in the interior of the state. From May to July 1999, 10 strains of vancomycin-resistant E. faecalis were isolated from 10 patients hospitalized in the HCUSP. The DNA genotyping using pulsed-field gel electrophoresis (PFGE) showed that all isolates were originated from the same clone, suggesting nosocomial dissemination. From May to July 2002, three strains of vancomycin-resistant E. faecalis were isolated from two patients hospitalized in CMC and both patients were colonized by the vancomycin-resistant Enterococcus in skin lesions. All isolates from CMC and HCUSP were highly resistant to vancomycin and teicoplanin. The three strains from CMC had minimum inhibitory concentration >256 µg/ml for vancomycin, and 64 (CMC 1 and CMC 2) and 96 µg/ml (CMC 3) for teicoplanin, characterizing a profile of VanA resistance to glycopeptides. All strains had the presence of the transposon Tn1546 detected by PCR and were closely related when typed by PFGE. The dissemination of the E. faecalis VanA phenotype among hospitals located in different cities is of great concern because E. faecalis commonly colonizes the gastrointestinal tract of patients and healthy persons for periods varying from weeks to years, which, together with the persistence of vancomycin-resistant Enterococcus in hospital rooms after standard cleaning procedures, increases the risk of the dissemination and reservoir of the bacteria.

Use of molecular epidemiology to monitor the nosocomial dissemination of methicillin-resistant Staphylococcus aureus in a University Hospital from 1991 to 2001

Methicillin-resistant Staphylococcus aureus (MRSA) has been the cause of major outbreaks and epidemics among hospitalized patients, with high mortality and morbidity rates. We studied the genomic diversity of MRSA strains isolated from patients with nosocomial infection in a University Hospital from 1991 to 2001. The study consisted of two periods: period I, from 1991 to 1993 and period II from 1995 to 2001. DNA was typed by pulsed-field gel electrophoresis and the similarity among the MRSA strains was determined by cluster analysis. During period I, 73 strains presented five distinctive DNA profiles: A, B, C, D, and E. Profile A was the most frequent DNA pattern and was identified in 55 (75.3%) strains; three closely related and four possibly related profiles were also identified. During period II, 80 (68.8%) of 117 strains showed the same endemic profile A identified during period I, 18 (13.7%) closely related profiles and 18 (13.7%) possibly related profiles and, only one strain presented an unrelated profile. Cluster analysis showed a 96% coefficient of similarity between profile A from period I and profile A from period II, which were considered to be from the same clone. The molecular monitoring of MRSA strains permitted the determination of the clonal dissemination and the maintenance of a dominant endemic strain during a 10-year period and the presence of closely and possibly related patterns for endemic profile A. However, further studies are necessary to improve the understanding of the dissemination of the endemic profile in this hospital.

Electrical field stimulation improves bone mineral density in ovariectomized rats

Osteoporosis and its consequent fractures are a great social and medical problem mainly occurring in post-menopausal women. Effective forms of prevention and treatment of osteoporosis associated with lower costs and the least side effects are needed. Electrical fields are able to stimulate osteogenesis in fractures, but little is known about their action on osteoporotic tissue. The aim of the present study was to determine by bone densitometry the effects of electrical stimulation on ovariectomized female Wistar rats. Thirty rats (220 ± 10 g) were divided into three groups: sham surgery (SHAM), bilateral ovariectomy (OVX) and bilateral ovariectomy + electrical stimulation (OVX + ES). The OVX + ES group was submitted to a 20-min session of a low-intensity pulsed electrical field (1.5 MHz, 30 mW/cm²) starting on the 7th day after surgery, five times a week (total = 55 sessions). Global, spine and limb bone mineral density were measured by dual-energy X-ray absorptiometry (DXA Hologic 4500A) before surgery and at the end of protocol (84 days after surgery). Electrical stimulation improved (P < 0.05) global (0.1522 ± 0.002), spine (0.1502 ± 0.003), and limb (0.1294 ± 0.003 g/cm²) bone mineral density compared to OVX group (0.1447 ± 0.001, 0.1393 ± 0.002, and 0.1212 ± 0.001, respectively). The OVX + ES group also showed significantly higher global bone mineral content (9.547 ± 0.114 g) when compared to both SHAM (8.693 ± 0.165 g) and OVX (8.522 ± 0.207 g) groups (P < 0.05). We have demonstrated that electrical fields stimulate osteogenesis in ovariectomized female rats. Their efficacy in osteoporosis remains to be demonstrated.

N-terminal prohormone brain natriuretic peptide (NT-proBNP) as a noninvasive marker for restrictive syndromes

Constrictive pericarditis (CP) and restrictive cardiomyopathy share many similarities in both their clinical and hemodynamic characteristics and N-terminal prohormone brain natriuretic peptide (NT-proBNP) is a sensitive marker of cardiac diastolic dysfunction. The objectives of the present study were to determine whether serum NT-proBNP was high in patients with endomyocardial fibrosis (EMF) and CP, and to investigate how this relates to diastolic dysfunction. Thirty-three patients were divided into two groups: CP (16 patients) and EMF (17 patients). The control group consisted of 30 healthy individuals. Patients were evaluated by bidimensional echocardiography, with restriction syndrome evaluated by pulsed Doppler of the mitral flow and serum NT-proBNP measured by immunoassay and detected by electrochemiluminescence. Spearman correlation coefficient was used to analyze the association between log NT-proBNP and echocardiographic parameters. Log NT-proBNP was significantly higher (P < 0.05) in CP patients (log mean: 2.67 pg/mL; 95%CI: 2.43-2.92 log pg/mL) and in EMF patients (log mean: 2.91 pg/mL; 95%CI: 2.70-3.12 log pg/mL) compared with the control group (log mean: 1.45; 95%CI: 1.32-1.60 log pg/mL). There were no statistical differences between EMF and CP patients (P = 0.689) in terms of NT-proBNP. The NT-proBNP log tended to correlate with peak velocity of the E wave (r = 0.439; P = 0.060, but not with A wave (r = -0.399; P = 0.112). Serum NT-proBNP concentration can be used as a marker to detect the presence of diastolic dysfunction in patients with restrictive syndrome; however, serum NT-proBNP levels cannot be used to differentiate restrictive cardiomyopathy from CP.

Clinical, epidemiological, and microbiological characteristics of bacteremia caused by high-level gentamicin-resistant Enterococcus faecalis

Enterococcus spp bacteremia is associated with high mortality and the appearance of high-level gentamicin resistance (HLGR) created additional challenges for the treatment of these infections. We evaluated the epidemiological and clinical characteristics of patients with bacteremias caused by HLGR and non_HLGR Enterococcus faecalis isolates at a teaching hospital in the State of São Paulo, Brazil. Patients with bacteremia due to E. faecalis diagnosed between January 1999 and December 2003 were included in the study. We collected clinical, epidemiological, and microbiological data from medical records. Banked isolates were typed using pulsed-field gel electrophoresis. We identified 145 cases of E. faecalis bacteremia: 66 (45.5%) were caused by HLGR isolates and 79 (54.5%) by non_HLGR. In the univariate analysis, patients with HLGR infection were older, had higher rates of bladder catheterization, and more often had treatment with cephalosporin, quinolone, and/or carbapenem compared with patients with non_HLGR infection (P < 0.05). Multivariate analysis indicated that older age, hematological malignancy, and previous use of vancomycin were independently associated with HLGR (P < 0.05). Mortality rates were not significantly different among patients with HLGR (50%) and non_HLGR (43%) infections (P = 0.40). Of the 32 genotyped isolates, 16 were distributed into 6 main electrophoresis patterns and 16 others had distinct patterns. E. faecalis bacteremia is associated with high mortality and is frequently caused by HLGR isolates at this teaching hospital. The variability among genotyped isolates suggests that endogenous infections, rather than patient-to-patient transmission of E. faecalis, are more common at this institution.

Hospital strain colonization by Staphylococcus epidermidis

The skin and mucous membranes of healthy subjects are colonized by strains of Staphylococcus epidermidis showing a high diversity of genomic DNA polymorphisms. Prolonged hospitalization and the use of invasive procedures promote changes in the microbiota with subsequent colonization by hospital strains. We report here a patient with prolonged hospitalization due to chronic pancreatitis who was treated with multiple antibiotics, invasive procedures and abdominal surgery. We studied the dynamics of skin colonization by S. epidermidis leading to the development of catheter-related infections and compared the genotypic profile of clinical and microbiota strains by pulsed field gel electrophoresis. During hospitalization, the normal S. epidermidis skin microbiota exhibiting a polymorphic genomic DNA profile was replaced with a hospital-acquired biofilm-producer S. epidermidis strain that subsequently caused repetitive catheter-related infections.

Genotyping of methicillin-resistant Staphylococcus aureus isolates obtained in the Northeast region of Brazil

Methicillin-resistant Staphylococcus aureus (MRSA) is a major agent of hospital infections worldwide. In Brazil, a multiresistant MRSA lineage (ST239-SCCmecIIIA), the so-called Brazilian epidemic clone (BEC), has predominated in all regions. However, an increase in nosocomial infections caused by non-multiresistant MRSA clones has recently been observed. In the present study, 45 clinical isolates of MRSA obtained from a university hospital located in Natal city, Brazil, were identified by standard laboratory methods and molecularly characterized using staphylococcal chromosome cassette mec (SCCmec) typing and pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was carried out using CLSI methods. The MRSA isolates studied displayed a total of 8 different pulsed-field gel electrophoresis patterns (types A to H) with predominance (73%) of pattern A (BEC-related). However, MRSA harboring SCCmec type IV were also identified, 3 (7%) of which were genetically related to the pediatric clone - USA800 (ST5-SCCmecIV). In addition, we found a considerable genetic diversity within BEC isolates. MRSA displaying SCCmecIV are frequently susceptible to the majority of non-β-lactam antibiotics. However, emergence of multiresistant variants of USA800 was detected.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.