- Volume 18, Issue 3, 1984
Volume 18, Issue 3, 1984
- Articles
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Evaluation of Mycobacterial Antigens in an Enzyme-Linked Immunosorbent Assay (ELISA) for the Serodiagnosis of Tuberculosis
More LessSUMMARYFive mycobacterial antigens were compared in an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of tuberculosis. The antigens studied were an unheated sterile culture filtrate of Mycobacterium tuberculosis, tuberculin purified protein derivative (PPD) from M. tuberculosis (PPDa), purified cytoplasmic protein antigens 5 and 6 from M. tuberculosis, and a PPD prepared from M. kansasi (PPDk). Multivariate analysis of variance showed that geometric mean titres obtained with each of the antigens in ELISA were significantly different in tuberculosis patients and in control groups. The covariation of the ELISA results with the five antigens was highly interdependent. Analysis of receiver operating characteristics revealed that the most accurate test was obtained with antigen 5. M. tuberculosis PPD, M. tuberculosis antigen 6, and M. tuberculosis culture filtrate were, in descending order, less accurate.
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Sequential Study of Bacterial Clearance in Experimental Cystitis
More LessSUMMARYClearance of Escherichia coli in experimental cystitis was studied in the diuresing mouse model. Urine was collected daily; sediment was isolated by cytocentrifugation and either stained or treated with fluorescent antibodies directed against mouse immunoglobulins. During the initial phase of the infection the bacteria were either free and dispersed or adhering to epithelial cells but not generally to polymorphonuclear cells (PMNs). Subsequently, the bacteria adhered to each other, to epithelial cells and to PMNs, were phagocytosed by the latter and showed strong fluorescence. It is postulated that the appearance of opsonising and agglutinating antibodies in conjunction with activity of the PMNs is involved in bacterial clearance.
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Occurrence and Experimental Infection of Toads (Bufo Marinus and B. Granulosus) with Mycobacterium Chelonei Subsp. Abscessus
More LessSUMMARYIn a survey of 234 Amazonian toads and frogs, six strains of Mycobacterium chelonei subsp. abscessus were isolated from the liver or spleen of four of 66 Bufo marinus (61%) and from the kidney or peritoneal fluid of two of 86 B. granulosus (2.3%). There were no histopathological lesions in the viscera of the infected animals. Experimental infection of 29 captive B. marinus and B. granulosus, by the intraperitoneal route, with a pooled inoculum of M. chelonei subsp. abscessus caused five deaths near the end of a 2-month observation period. M. chelonei subsp. abscessus was isolated from the liver, spleen, kidney, gonad, heart and lung of toads killed at various intervals after inoculation, and intracellular acid-fast bacilli were seen in these organs. Histological evidence of invasion of tissues by mycobacteria became apparent from the 45th day after infection. The susceptibility to infection of B. marinus and B. granulosus suggests that these toads may serve as a fortuitous animal host for M. chelonei subsp. abscessus.
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The Microbial Flora of the Rectal Mucosa and Faeces of Patients with Crohn’s Disease Before and During Antimicrobial Chemotherapy
More LessSUMMARYThe faecal flora and mucosa-associated flora (MAF) of rectal biopsy material from 12 patients with active Crohn’s disease were studied before and during treatment with a combination of metronidazole and cotrimoxazole given orally for at least 2 weeks. The total faecal flora was greater than the MAF although the proportions of bacterial groups were similar. The changes observed during treatment were: obligate anaerobes such as Bacteroides spp. decreased in faeces (p ≪ 0.05) and in MAF (p ≪ 0.02); the total count of facultative bacteria increased in the faeces (p≪ 0.002) but not in the MAF. Steptococci, predominantly enterococci, increased significantly in faeces (p≪0.001) and in MAF (p ≪ 0.02) such that they became predominant components of these florae. Facultative gram-negative bacilli were unaltered in faeces but significantly reduced in the MAF (p≪0.05). Sporing Clostridia were infrequently isolated from the MAF but were significantly reduced in the faeces (p ≪0.01).
During the treatment period, eight of the 12 patients showed clinical improvement, but this could not be related to the site or extent of disease or to specific changes in faecal flora or MAF. This combination of antibacterial agents causes profound alterations to the bacterial flora of mucosa and faeces and these changes may help to define the role of bacteria in the pathogenesis of Crohn’s disease.
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The Systematic Serology of Neisseria Gonorrhoeae: Antigens Associated with Pathogenesis in Neisseria Spp. from Man
More LessSUMMARYSonicates of eight Neisseria species from man were analysed in a micro-Ouchterlony double-diffusion absorption assay in comparison with a gonococcal reference antiserum-antigen system. Five major gonococcal precipitin zones were identified which comprised genus-, species- and type-specific components. One antigen was found in all strains of three species with pathogenic capability—N. gonorrhoeae, N. meningitidis and N. flavescens. It was not detected in N. lactamica, N. pharyngis, N. elongata, N. cinerea or N. catarrhalis.
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The Bactericidal Action of Human Serum on Bacteroides Species
More LessSUMMARYSera obtained from 60 healthy normal subjects were tested for their inhibitory action against four clinical isolates of the Bacteroides fragilis group. All the sera inhibited clinical strains of B. vulgatus and B. ovatus, 92% of 60 sera inhibited B. thetaiotaomicron and 62% inhibited B. fragilis. Bacterial inhibition required components of uninactivated serum and was related to the serum concentrations and species of the bacteria. The viable counts of all the strains, except B. fragilis, were significantly reduced in pooled sera from patients with anaerobic systemic infections and from non-infected patients. The bactericidal activity of the sera from infected patients was more pronounced than that of the ‘normal’ sera, on all strains except B. fragilis. These observations may explain in part the predominance of B. fragilis in serious clinical infections involving anaerobes.
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Isolation of Obligate Anaerobic Bacteria from Bovine Abscesses in Sites Other Than the Liver
More LessSUMMARYA survey in Japan showed that of 2036 slaughtered cattle 58(3%) had abscesses in sites other than the liver. In 21 of the affected animals the lesions were pulmonary and in 32 abdominal (excluding hepatic); in five animals the lesions were found elsewhere (muscle 2, skin 2, bone 1). Nineteen (33%) of the 58 cattle also had abscesses in the liver.
Obligate anaerobes alone were isolated from 17(29%) of the affected animals (Fusobacterium necrophorum 14, Bacteroides spp. 2, Peptostreptococcus anaerobius 1). A mixture of obligate anaerobes and aerobes or facultative anaerobes was isolated from 31 affected cattle: of these animals 21 yielded large and five small numbers of F. necrophorum; three yielded fusobacteria other than F. necrophorum; and two yielded Propionibacterium acnes. The remaining 10 affected animals yielded only aerobes or facultative anaerobes. The numbers of viable obligate anaerobes in pus specimens were in the range 103–109/ml.
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Growth of Yersinia Pseudotuberculosis in Mouse Spleen Despite Loss of a Virulence Plasmid of Mol. WT 47 × 106
M. Simonet, D. Mazigh and P. BercheSUMMARYA highly virulent strain of Yersinia pseudotuberculosis (LD50 c. 102 bacteria/mouse) harboured two plasmids with mol. wt of 47 × 106 and 61 × 106. The role of these plasmids in virulence was studied in mice with derived strains cured of plasmids. It was confirmed that the plasmid of mol. wt 47 × 106 played a major function in virulence. This was shown both by the increase of the LD50 and the lower rate of multiplication in the spleen obtained with strains cured of the plasmid of mol. wt 47 × 106. The plasmid of mol. wt 61 × 106 did not play any role in virulence. This work also demonstrates that the strain cured of the plasmid of mol. wt 47 × 106 and the plasmid-free strain were able to multiply in the spleens of infected mice during a 7-day period. This suggests that virulence factors not associated with plasmids are also responsible for the bacterial growth in tissues in vivo.
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The Variable Response of Bacteria to Free Haemoglobin in the Tissues
More LessSUMMARYThe local enhancement of infection by exogenous ferric iron, as ferric ammonium citrate, and by ferrous iron as guinea-pig haemoglobin, was assessed in studies with 55 strains of bacteria injected into the skin of guinea-pigs. The test organisms included Staphylococcus aureus, Streptococcus spp., Klebsiella spp., Escherichia coli and Pseudomonas aeruginosa. Four strains of Bacteroides spp. were tested with haemoglobin only. As previously reported with other strains, enhancement of infection by members of a given species by ferric iron was variable; in this study infection with only 11 of 59 strains was enhanced. Haemoglobin either of equal or lesser iron content was a more potent enhancer, affecting 27 of the 59 strains. The enhancement ranged from two-fold to 80-fold, the higher figures on the whole being characteristic of haemoglobin enhancement. Some few instances of depression by both haemoglobin and ferric ammonium citrate were noted. A few tests were made with systemic haemoglobin but the concentrations attainable were largely ineffective. Enhancement of infection did not appear to be related to the capacity of a strain to lyse or digest host red blood cells.
In so far as guinea-pigs, whose antibacterial defences are lowered by ferric or ferrous iron, represent human subjects at risk of infection because of clinical circumstances characterised by excess of available iron—either exogenous or as a result of haemolysis—our results with organisms of a kind commonly associated with infection in hospitals suggest that only a small proportion of environmental bacteria can take advantage of any decreased resistance associated with iron excess.
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Production and Release of Toxins A and B by Clostridium Difficile
More LessSUMMARYThe production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated. Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells. Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay. Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle. The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed.
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Zinc and the Heat-Labile Enterotoxin of Escherichia Coli
More LessSUMMARYEnterotoxigenic Escherichia coli is a major cause of diarrhoea in man. When zinc in concentrations of 10−6 M or 10−5 M was added to the growth medium, there was a significant increase in heat-labile enterotoxin production by each of six toxigenic strains. Zinc in these concentrations did not alter bacterial growth or the activity of preformed toxin. Other heavy metals did not enhance toxin production and o-phenanthroline, a relatively specific zinc-chelating compound, blocked the enhancing effect. The significance of these findings is discussed in relation to the use of supplemental dietary zinc.
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A Simplified Method for Detecting the Heat-Labile Enterotoxin of Escherichia Coli
More LessSUMMARYA tissue-culture method is described that is suitable for screening large numbers of Escherichia coli isolates for heat-labile enterotoxin production. The method uses readily available laboratory media for culture of the E. coli and does not require cell-free extracts. Antibiotics are used to increase toxin production to levels that allow cultures from several different colonies to be pooled for testing, thus minimising the number of tissue-culture monolayers required. Y1 mouse adrenal cells are used in microplate cultures. The method is simple, sensitive and economical and is suitable for use in a routine diagnostic microbiology laboratory.
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Media for Isolation of Aeromonas Spp. from Faeces
More LessSUMMARYFive solid media were evaluated for isolation of Aeromonas spp. from faeces: desoxycholate citrate agar (DCA), MacConkey’s agar (MAC), xylose-desoxycholate-citrate agar (XDCA), Rogol’s medium (ROG), which contained ampicillin 20 mg/L and p-nitrophenyl-glycer-ine 25 mg/L as inhibitors, and blood agar (BA) with ampicillin 10 mg/L. False negative oxidase tests limited the usefulness of DCA and MAC and, although the use of XDCA avoided the problem of fermentation of lactose, some Aeromonas spp. failed to grow on XDCA or produced minute colonies unsuitable for oxidase tests. BA yielded the highest rate of isolation for Aeromonas spp. from 323 faecal samples—15.2% for all Aeromonas spp. and 9.3% for enterotoxigenic (ENT+) strains. This compares with 10.8% for all strains and 6.5% for ENT+ strains isolated on DCA, 7.1% for all strains and 4% for ENT+ strains on MAC and 4% for all strains and 1.5% for ENT+ strains on ROG. Blood agar with ampicillin is recommended for isolation of Aeromonas spp. from faeces.
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Vaccination Against 77 Capsular Types of Klebsiella Aerogenes with Polyvalent Klebsiella Vaccines
R. J. Jones and E. A. RoeSUMMARYA method is described for producing monovalent and polyvalent vaccines from culture filtrates of Klebsiella aerogenes. With a single injection, each monovalent vaccine protected mice against lethal intraperitoneal challenge by more than 30 capsular types; and polyvalent vaccines containing 2–12 monovalent components protected against 46–61 of the 77 capsular types of K. aerogenes. One vaccine with 12 components, administered in two doses, induced full protection against 71 types and protected half of the mice challenged with the other six types.
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Toxic Effect of Products of Oxidative Metabolism on the Yeast Form of Paracoccidioides Brasiliensis
More LessSUMMARYThe effectiveness of toxic oxygen metabolites in killing the yeast form of Paracoccidioides brasiliensis (the form that occurs in host tissues) was studied with a fluorescence method in vitro. The two isolates studied were similar in susceptibility and H2O2 alone was lethal with an LD50 of 15–25 mm. The addition of halide (5 × 10−4 M) augmented the lethality of H2O2 and in that setting H2O2 was c. 90% lethal at 5 × 10−5 M. Killing was most effective in the presence of peroxidase, when only 5 × 10−6 M H2O2 (a concentration attainable in vivo by phagocytes) was required for a 95% kill. Kinetic studies revealed that toxic concentrations of H2O2 alone or of the H2O2−halide-peroxidase (PPH) system produced significant killing in 1 min; killing was maximal in 15 min. The PPH system was the more rapid in action. The dependence of the PPH killing system on H2O2 was demonstrated by an absence of killing in the presence of catalase. The susceptibility of P. brasiliensis to H2O2 and the PPH system appeared different in some respects from that noted for other dimorphic fungal pathogens. These studies suggest that toxic oxygen metabolites are important in host defence against P. brasiliensis.
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Liposomal Encapsulation Augments Delayed Hypersensitivity Reactions to Tuberculin Proteins
More LessSUMMARYEncapsulation of purified tuberculoproteins in liposomes augmented their ability to elicit delayed hypersensitivity reactions in BCG-immune rats. The effect was most marked with a low-molecular-weight tuberculopeptide that was relatively poor at eliciting reactions when in free form. These findings indicate that, in addition to the antigenic nature of the material, the physical form of presentation of mycobacterial test antigens can influence their ability to elicit hypersensitivity reactions.
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Acute Synovitis Caused by an Organism of the Rhodochrous TAXON
More LessSUMMARYA 36-year-old man with systemic lupus erythematosus developed synovitis of the right wrist caused by an organism of the Rhodochrous taxon. The capacity of this pathogen to produce a granulomatous inflammatory reaction in human tissues is discussed. The isolate was sensitive to chloramphenicol, gentamicin, carbenicillin and colistin in in-vitro tests and the infection responded promptly to treatment with chloramphenicol.
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