Bacterial agents isolated from cerebrospinal fluid of patients with Acquired Immunodeficiency Syndrome (AIDS) and neurological complications

Cerebrospinal fluid (CSF) samples from 2083 patients with acquired immunodeficiency syndrome (AIDS) and neurological complications were bacteriologically examined during a period of 7 years (1984-1990). The percentage of patients who had at least one bacterial agent cultured from the CSF was 6.2%. Mycobacterium tuberculosis was the most frequently isolated agent (4.3%), followed by Mycobacterium avium complex or MAC (0.7%), Pseudomonas spp (0.5%), Enterobacter spp (0.4%), and Staphylococcus aureus (0.3%). Among 130 culture positive patients, 89 (68.5%) had M. tuberculosis and 15 (11.6%) had MAC. The frequency of bacterial isolations increased from 1988 (5.2%) to 1990 (7.2%), partly due to the increase in MAC isolations. Bacterial agents were more frequently isolated from patients in the age group 21-30 years and from women (p<0.05).

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1 M EDTA pH 7.5 at 90°C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Bacterial antigen detection in cerebrospinal fluid by the latex agglutination test

Eighty purulent cerebrospinal fluid (CSF) samples from patients with clinical evidence of meningitis were studied using the Directigen latex agglutination (LA) kit to determine the presence of bacterial antigen in CSF. The results showed a better diagnostic performance of the LA test than bacterioscopy by Gram stain, culture and counterimmunoelectrophoresis (CIE), as far as Neisseria meningitidis groups B and C, and Haemophilus influenzae type b are concerned, and a better performance than bacterioscopy and culture considering Streptococcus pneumoniae. Comparison of the results with those of culture showed that the LA test had the highest sensitivity for the Neisseria meningitidis group C. Comparing the results with those of CIE, the highest levels of sensitivity were detected for N. meningitidis groups B and C. Regarding specificity, fair values were obtained for all organisms tested. The degree of K agreement when the LA test was compared with CIE exhibited better K indices of agreement for N. meningitidis groups B and C.

Immunological status of ENL (Erythema Nodosum Leprosum) patients: its relationship to bacterial load and levels of circulanting IL-2R

Recent data suggest that the clinical course of reactional states in leprosy is closely related to the cytokine profile released locally or systemically by the patients. In the present study, patients with erythema nodosum leprosum (ENL) were grouped according to the intensity of their clinical symptoms. Clinical and immunological aspects of ENL and the impact of these parameters on bacterial load were assessed in conjunction with patients' in vitro immune response to mycobacterial antigens. In 10 out of the 17 patients tested, BI (bacterial index) was reduced by at least 1 log from leprosy diagnosis to the onset of their first reactional episode (ENL), as compared to an expected 0.3 log reduction in the unreactional group for the same MDT (multidrug therapy) period. However, no difference in the rate of BI reduction was noted at the end of MDT among ENL and unreactional lepromatous patients. Accordingly, although TNF-alpha (tumor necrosis factor) levels were enhanced in the sera of 70.6% of the ENL patients tested, no relationship was noted between circulating TNF-alpha levels and the decrease in BI detected at the onset of the reactional episode. Evaluation of bacterial viability of M. leprae isolated from the reactional lesions showed no growth in the mouse footpads. Only 20% of the patients demonstrated specific immune response to M. leprae during ENL. Moreover, high levels of soluble IL-2R (interleukin-2 receptor) were present in 78% of the patients. Circulating anti-neural (anti-ceramide and anti-galactocerebroside antibodies) and anti-mycobacterial antibodies were detected in ENL patients' sera as well, which were not related to the clinical course of disease. Our data suggest that bacterial killing is enhanced during reactions. Emergence of specific immune response to M. leprae and the effective role of TNF-alpha in mediating fragmentation of bacteria still need to be clarified.

EVALUATION OF A RAPID SCREENING ASSAY FOR BACTERIAL IDENTIFICATION (DOT-ELISA) IN FECAL SAMPLES FROM CHILDREN

With the objective of standardizing a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA) to detect antigens of fecal bacterial enteropathogens, 250 children, aged under 36 months and of both sexes, were studied; of which 162 had acute gastroenteritis. The efficacy of a rapid screening assay for bacterial enteropathogens (enteropathogenic Escherichia coli "EPEC", enteroinvasive Escherichia coli "EIEC", Salmonella spp. and Shigella spp.) was evaluated. The fecal samples were also submitted to a traditional method of stool culture for comparison. The concordance index between the two techniques, calculated using the Kappa (k) index for the above mentioned bacterial strains was 0.8859, 0.9055, 0.7932 and 0.7829 respectively. These values express an almost perfect degree of concordance for the first two and substantial concordance for the latter two, thus enabling this technique to be applied in the early diagnosis of diarrhea in infants. With a view to increasing the sensitivity and specificity of this immunological test, a study was made of the antigenic preparations obtained from two types of treatment: 1) deproteinization by heating; 2) precipitation and concentration of the lipopolysaccharide antigen (LPS) using an ethanol-acetone solution, which was then heated in the presence of sodium EDTA

Epidemiological analysis of bacterial strains involved in hospital infection in a University Hospital from Brazil

Hospital infections cause an increase in morbidity and mortality of hospitalized patients with significant rise in hospital costs. The aim of this work was an epidemiological analysis of hospital infection cases occurred in a public University Hospital in Rio de Janeiro. Hence, 238 strains were isolated from 14 different clinical materials of 166 patients hospitalized in the period between August 1995 and July 1997. The average age of the patients was 33.4 years, 72.9% used antimicrobials before having a positive culture. The most common risk conditions were surgery (19.3%), positive HIV or AIDS (18.1%) and lung disease (16.9%). 24 different bacterial species were identified, S. aureus (21%) and P. aeruginosa (18.5%) were predominant. Among 50 S. aureus isolated strains 36% were classified as MRSA (Methicillin Resistant S. aureus). The Gram negative bacteria presented high resistance to aminoglycosides and cephalosporins. A diarrhea outbreak, detected in high-risk neonatology ward, was caused by Salmonella serovar Infantis strain, with high antimicrobial resistance and a plasmid of high molecular weight (98Mda) containing virulence genes and positive for R factor.

Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis

FEBS journal, Volume 278, Issue 14, pages 2511-2524, July 2011

Bacterial inter-species communication mediated by the autoinducer-2 signal

Dissertation presented to obtain the Ph.D degree in Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica, Instituto Gulbenkian de Ciência.

Recycling old screen-printed electrodes with newly designed plastic antibodies on the wall of carbon nanotubes as sensory element for in situ detection of bacterial toxins in water

Using low cost portable devices that enable a single analytical step for screening environmental contaminants is today a demanding issue. This concept is here tried out by recycling screen-printed electrodes that were to be disposed of and by choosing as sensory element a low cost material offering specific response for an environmental contaminant. Microcystins (MCs) were used as target analyte, for being dangerous toxins produced by cyanobacteria released into water bodies. The sensory element was a plastic antibody designed by surface imprinting with carefully selected monomers to ensure a specific response. These were designed on the wall of carbon nanotubes, taking advantage of their exceptional electrical properties. The stereochemical ability of the sensory material to detect MCs was checked by preparing blank materials where the imprinting stage was made without the template molecule. The novel sensory material for MCs was introduced in a polymeric matrix and evaluated against potentiometric measurements. Nernstian response was observed from 7.24 × 10−10 to 1.28 × 10−9 M in buffer solution (10 mM HEPES, 150 mM NaCl, pH 6.6), with average slopes of −62 mVdecade−1 and detection capabilities below 1 nM. The blank materials were unable to provide a linear response against log(concentration), showing only a slight potential change towards more positive potentials with increasing concentrations (while that ofthe plastic antibodies moved to more negative values), with a maximum rate of +33 mVdecade−1. The sensors presented good selectivity towards sulphate, iron and ammonium ions, and also chloroform and tetrachloroethylene (TCE) and fast response (<20 s). This concept was successfully tested on the analysis of spiked environmental water samples. The sensors were further applied onto recycled chips, comprehending one site for the reference electrode and two sites for different selective membranes, in a biparametric approach for “in situ” analysis.

Seroprevalence of selected viral, bacterial and parasitic infections among inpatients of a public psychiatric hospital of Mexico

We sought to determine the frequency of serological markers of selected infections in a population of psychiatric patients in Durango City, Mexico, and to determine whether there are any epidemiological characteristics of the subjects associated with the infections. One hundred and five inpatients of a public psychiatric hospital of Durango were examined for HBsAg, anti-HCV antibodies, anti-HIV antibodies, anti-Brucella antibodies, rapid plasma reagin and anti-Cysticercus antibodies by commercially available assays. Anti-Cysticercus antibodies were confirmed by Western blot and HBsAg by neutralization assay. Epidemiological data from each participant were also obtained. Seroprevalences of HBsAg, anti-HCV, anti-HIV, anti-Brucella, rapid plasma reagin and anti-Cysticercus antibodies found were 0.0%, 4.8%, 0.9%, 0.0%, 1.9%, and 0.9%, respectively. Overall, 9 (8.6%) inpatients showed seropositivity to any infection marker. We concluded that our psychiatric inpatients have serological evidence of a number of infections. HCV is an important pathogen among our psychiatric inpatients. Health care strategies for prevention and control of infections in Mexican psychiatric patients should be considered.

Perinatal Bacterial Infection: Screenning of Vertical Transmitted Infections

Perinatal bacterial infection may be caused by any microorganism colonizing the vaginal tract. Neonatologists and paediatricians are especially concerned about group B Stretpococcus (GBS). However, Enterobactereacea, mainly E.coli and Proteus, are also responsible for infection. GBS screening may be accomplished in over 90% of pregnant women. In our maternity in 2007-2008, 85% of the mothers had been screened. Screening and prophylaxis were responsible for a decreasing incidence of neonatal infection - from 0.6/1000 to 0.15/1000 live births in Portugal, from 2002 to 2007. However there are some difficulties related to screening. In the second Portuguese study 16/57 NB with early-onset infection (28%) were born to “negative” mothers. Several factors illustrate how difficult is to draw national screening policies: a wide range of carrier’s state rate throughout a country - in Portugal from 12% to 30%. The success of any screening policy may also be affected by additional technical and organizational problems. In countries where home delivery is a tradition or a trend intrapartum GBS prophylaxis requires a very well organized assistance.. Moreover factors usually accepted as protective are not so effective. In the Portuguese study 24/57 infected newborns (42%) were delivery by caesarean section. Another subject deals with the workload in the postnatal ward generated by deficient compliance to the guidelines a problem not confirm by a study of our group. Decreasing the importance of GBS, highlight the importance of E. coli in perinatal infection. From the 16 340 registrations of the National Registry 1676 were newborns with mother-related infection. Applying the same reasoning to E.coli as to GBS and Listeria monocytogenes – that is considering all of them are of maternal origin - 6.7% of these infections were due to E. coli, 4.6% to SGB and 0.5% to Listeria monocytogenes. In conclusion screening and prophylaxis may be not the best way to prevent all GBS neonatal infections but by now it is the only available procedure. The other bacteria continue to demand a high suspicion level and immediate intervention.

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

Studies on BolA and ribonuclease R: two important factors in the control of bacterial gene expression

Dissertation presented to obtain the Ph.D degree in Biology