Implications of extubation failure and prolonged mechanical ventilation in the postoperative period following elective intracranial surgery

Patients undergoing neurosurgery are predisposed to a variety of complications related to mechanical ventilation (MV). There is an increased incidence of extubation failure, pneumonia, and prolonged MV among such patients. The aim of the present study was to assess the influence of extubation failure and prolonged MV on the following variables: postoperative pulmonary complications (PPC), mortality, reoperation, tracheostomy, and duration of postoperative hospitalization following elective intra-cranial surgery. The study involved a prospective observational cohort of 317 patients submitted to elective intracranial surgery for tumors, aneurysms and arteriovenous malformation. Preoperative assessment was performed and patients were followed up for the determination of extubation failure and prolonged MV (>48 h) until discharge from the hospital or death. The occurrence of PPC, incidence of death, the need for reoperation and tracheostomy, and the length of hospitalization were assessed during the postoperative period. Twenty-six patients (8.2 percent) experienced extubation failure and 30 (9.5 percent) needed prolonged MV after surgery. Multivariate analysis showed that extubation failure was significant for the occurrence of death (OR = 8.05 [1.88; 34.36]), PPC (OR = 11.18 [2.27; 55.02]) and tracheostomy (OR = 7.8 [1.12; 55.07]). Prolonged MV was significant only for the occurrence of PPC (OR = 4.87 [1.3; 18.18]). Elective intracranial surgery patients who experienced extubation failure or required prolonged MV had a higher incidence of PPC, reoperation and tracheostomy and required a longer period of time in the ICU. Level of consciousness and extubation failure were associated with death and PPC. Patients who required prolonged MV had a higher incidence of extubation failure.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Atypical phenotypic characteristics of klebsiella pneumoniae isolates from an outbreak in a neonatal intensive care unit in Brazil

Extended-spectrum beta-Lactamase-producing (ESBL) Klebsiella sp.isolates from an outbreak in a Neonatal Intensive Care Unit (NICU) at a teaching hospital in Londrina, Paraná State, Brazil, presented atypical phenotypic characteristics that hampered their identification and the distinction between Klebsiella and Enterobacter species. Ten isolates were identified as K. pneumoniae due to negative reactions for motility and inducible beta-lactamase test (ESBL and AmpC) despite being positive for ornithyne descarboxilase. These isolates were genotyped by ribotyping and polymerase chain reaction (PCR) with repetitive extragenic palindromic sequences (REP). Ribotyping by means of an automated instrument and EcoRI and Pvu II as restriction enzymes resulted indetection of K. pneumoniae subspecie pneumoniae RIBO1 222-36-S-5 ribotype. Typing by REP-PCR showed that the 17 isolates from the outbreak were highly similar, belonging to one cluster with 100 percent of similarity, and that they presented more than 70 percent of similarity with K. pneumoniae ATCC 13883 and ATCC 10031, and 25 percent of similarity with E. aerogenes CDC 1680. In conclusion, the isolates of the outbreak were identified as Klebsiella pneumoniae, despite presenting ornithyne descarboxilase enzyme, which is an atypical characteristic of this Klebsiella species.

Isolados de Klebsiella sp. produtora de beta-lactamase de espectro estendido (ESBL), responsável por um surto na Unidade Neonatal de Terapia Intensiva (UNTI) do Hospital Universitário de Londrina, Paraná, Brasil apresentaram características fenotípicas atípicas que dificultaram sua identificação e a diferenciação entre as espécies Klebsiella pneumoniae e Enterobacter aerogenes. Dez isolados foram identificados como K. pneumoniae devido às reações negativas para motilidade e produção de enzimas beta-lactamases (ESBL e AmpC). Embora apresentassem teste positivo para ornitina descarboxilase. Estes isolados foram genotipados por ribotipagem e por reação em cadeia da polimerase (PCR) com oligonucleotídeos para "repetitive extragenic palindromic sequences" (REP). A ribotipagem com as enzimas de restrição EcoRI e Pvu II detectou o ribotipo de K. pneumoniae subespécie pneumoniae RIBO1 222-36-S-5. A técnica de REP-PCR mostrou que os isolados do surto foram similares, pertencentes a um grupo com 100 por cento de similaridade, e apresentaram mais de 70 por cento de similaridade com amostras padrão de K. pneumoniae (ATCC 13883 e 10031), e 25 por cento de similaridade com E. aerogenes CDC 1680. Concluindo, os isolados do surto da NICU mostraram se geneticamente relacionados e foram identificados como Klebsiella pneumoniae, embora apresentassem ornitina descarboxilase, característica atípica para esta espécie de Klebsiella.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinantclone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post- immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens

Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect of Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1). No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. Coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis.

Freqüência de anticorpos contra Babesia bigemina, B. bovis e Anaplasma marginale em rebanhos leiteiros da regiäo de Londrina, Paraná / Frequency of antibodies against Babesia bigemina, B. bovis and Anaplasma marginale in dairy farms of Parana State, Brazil

Sera collected from 417 dairy cows from Londrina, PR, Brazil, were analysed by the indirect fluorescent antibody test (IFAT) for the presence of anitbody against Babesia bigemina, Babesia bovis e Anaplasma marginale. All samples were tested diluted 1:80 using rabbit anti-bovine IgG FITC conjugate diluted 1:100. Among the samples, 289 (67.30 per cent), 251 (60.19 per cent) and 281 (67.38 per cent) showed reactivity to B. bigemina, B. bovis and A. marginale, respectively. Although the number of reactive animals as relatively high, considerable percentages of negative animals (30.70 per cent, B. bigemina, 39.18 per cent, B. bovis and 32.62 per cent, A. marginale) were susceptible, characterizing the situation as enzootically instable

Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections

Strains of E. coli isolated from patients with urinary tract infection were examined for P and type 1 adhesin production by colony hybridization with pap and pil operons. The P pili probe detected 45 (46.4 per cent) of the total of 97 strains studied and the type 1 pili probe detected 83 (85.6 per cent). The pap operon was detected in 39 (53.4 per cent) of 73 strains isolated from urine of patients with urinary disease and in 6 (25.0 per cent) of 24 strains isolated from feces of healthy individuals employed as controls (P = 0.029), and the pil operon was detected in 67 (91.8 per cent) of the urinary strains and in 16 (66.6 per cent) of the fecal strains (P = 0.007). Our data did not show significant differences in frequency of P pili among isolates from pyelonephritis (78.5 per cent), cystitis (45.8 per cent) and asymptomatic bacteriuria (54.5 per cent). Type 1 pili were not associated with the different types of infection; the frequency of these pili was 100 per cent in pyelonephritis and in asymptomatic bacteriuria, and 87.5 per cent in cystitis. The incidence of pap operon in strains isolated from pyelonephritis and from asymptomatic bacteriuria was higher in 11-to 40-year old women. These data show a high frequency of pap and pil operons among uropathogenic strains of E. coli, which seems to be an important factor in the development of urinary infection.

Iron-regulated outer-membrane proteins of strains of avian septicemic Escherichia coli

1. Outer-membrane protein patterns of Escherichia coli recovered from the peritoneal cavities of infected guinea pigs and grown in medium M9 containing 2,2'-dipyridyl were studied by polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether in vivo conditions of growth affected the expression of these bacterial surface proteins. 2. Eleven strains of septicemic E. coli studied in vitro under conditions of iron restriction expressed iron-regulated outer-membrane proteins, mainly the protein of approximately 74 kDa, whereas avirulent strains grown under similar conditions did not present the 74-kDa protein. 3. These results show the distribution of iron-regulated outer-membrane proteins among avian E. coli and suggest that the protein of approximately 74 kDa may be important for the virulence of these strains.

Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli

Plasmid profile of Staphylococcus hyicus isolated from swine exudative epidermitis in Brazil

Staphylococcus hyicus isolados de leitöes com lesöes de pele foram estudados no que se refere aos seus plasmídios e resistência a agentes antimicrobianos. Plasmídios de diferentes pesos moleculares foram detectados nas seis amostras de Staphylococcus hyicus. Todas as amostras mostraram resistência à tetraciclina e um pequeno plasmídio de 4.0 Kb, com uma exceçäo. Experimentos de cura e transformaçäo demonstraram que este plasmídio abriga o determinante genético para resistência à tetraciclina nas amostras estudadas

Virulence factors in Serratia marcescens: cell-bound hemolysin and aerobactin

A total of 60 nosocomial isolates of Serratia marcescens were screened for the presence of markers related to virulence, i. e., cell-bound hemolysin and production of siderophore aerobactin. No aerobactin-producing strains were found, and the incidence of cell-bound hemolysin was 97%. Hemolysin-positive (58 strains) and hemolysin-negative (2 strains) Serratia marcescens showed the same LD50 (3 x 107) bacteria) in a test of virulence for mice. These results indicate that cell-bound hemolysin is not a main factor of virulence for mice in Serratia marcescens

Virulence factors of Eschericha coli in urinary isolates

Escherichia coli strains isolated from 100 urine samples taken from patients with urinary tract infections (UTI) and from 20 normal fecal (NF) samples were examined for serum resistance, mannose-resistant hemagglutination of human erythrocytes (MRHA) and for production of aerobactin, hemolysis and colicin. Among the UTI E. coli strains, 79% produced aerobactin, 69% showed serum resistance, 44% produced MRHA, 32% were beta-hemolytic and 22% were colicinogenic. A greater proportion of UTI E. coli strains produced aerobactin, colicin V, beta-hemolysis and MRHA when compared to NF strains. Production of MR hemagglutins was significant correlated with that of aerobactin and hemolysin. These results suggest that the presence of aerobactin may be a significant etiological factor in UTI, and that the production of MR adhesins and of hemolysin also might contribute to the virulence of these strains

Plasmid coding for aerobactin production and drug resistance is involved in virulence of Escherichia coli avian strains

Strain of avian septicemic E. coli were examined for association among the determinants of drug resistance, the genes for aerobactin production and virulence. In conjugation experiments, a single plasmid (100 Md) from a strain of septicemic E. coli (UEL 29) transferred to E. coli K12 pathogenicity for 1-day old chicks plus resistance to streptomycin and the ability to produce aerobactin and colicin. Additional evidence for the association of R-plasmid and the production of aerobactin, colicin, resistance to sulfadiazine and pathogenicity was obtained by disassociation when all traits were lost simultaneously. These data provide additional evidence for the importance of the aerobactin system for the pathogenicity of avian E. coli