The production of neuraminidase by food poisoning strains of Clostridium welchii (C. perfringens).

The production of neuraminidase by a classical strain of Clostridium welchii (C. perfringens) type A was studied. Good yields were produced in 5% Proteose Peptone-water medium (PPW5); the enzyme was essentially extracellular but some further neuraminidase could be released by ultrasonic disintegration of the cells. This also released N-acyl neuraminic acid-aldolase (NAN-aldolase) and the degree to which this interferes with the assay for neuraminidase was evaluated. Forty-one British reference food-poisoning strains of C. welchii type A were examined for extracellular neuraminidase production in PPW5. Twelve of 17 strains that produce so-called heat-sensitive spores were neuraminidase positive whereas 20 of 24 strains that are non-haemolytic and produce very heat-resistant sporeswere neuraminidase negative. Variation was found in the ability to produce neuraminidase among strains of a single Hobbs' serotype; four Hobbs' type-13 strains produced neuraminidase but a fifth did not. Disruption of the cells of a Hobbs' type-2 strain that did not produce any extracellular neuraminidase released NAN-aldolase but there was no evidence of cell-associated neuraminidase. British food-poisoning strains of C. welchii type A thus include some that are clearly neuraminidase positive and some that still cannot be shown to produce neuraminidase. There is no correlation between lack of neuraminidase production and the ability to cause food poisoning, although the majority of non-haemolytic heat-resistant strains do not produce neuraminidase. It remains possible that neuraminidase may play a part in C. welchii gas gangrene; it is suggested that the ability to define neuraminidase-negative strains may now be of value in investigating this possibility.     

Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice.

Seventy-five strains of Aspergillus fumigatus were screened for production of elastase in liquid and agar media containing elastin in yeast carbon base buffered with 0.05 M borate, pH 7.6. Of 71 strains which cleared elastin in agar plates, 33 produced elastase in liquid medium, as measured spectrophotometrically with elastin-Congo red. Six strains producing elastase and four nonproducers were tested for ability to cause invasive aspergillosis in immunocompromised mice (six mice per strain). All 36 mice exposed to elastase-producing strains died within 48 to 96 h. Lung tissue from dead mice showed hyphae and necrosis of the alveoli. Lungs of mice exposed to spores of strains not producing elastase showed few germinated spores and no destruction of alveoli. These results indicate that elastase may be significant in the invasion process.     

Comparative virulence of Absidia corymbifera strains in mice.

The comparative virulence of six different strains of Absidia corymbifera for cortisone-treated and untreated Swiss mice was determined. Spores of the six strains were inoculated into mice by the intravenous, intraperitoneal, and intranasal routes. All six strains were found to be virulent in cortisone-treated and untreated mice by the intravenous route. Up to a 16-fold difference in strain virulence was observed for cortisone-treated mice and up to a 10-fold difference for untreated mice. When spores were administered by the intraperitoneal route, 50% lethal dose values could be calculated only for the cortisone-treated mice, although a few deaths were seen in untreated mice challenged with 10(7) spores. Each of the six isolates of A. corymbifera, when administered in an intranasal dosage of 10(6) spores, produced death in some cortisone-treated mice. Studies made to determine the viability of spores produced by each strain revealed that germination was 90% or greater on Littman and YpSs agars at an incubation temperature of 40 degrees C in less than 12 h.     

Significance of the isolation of Clostridium welchii from routine blood cultures.

Clostridium welchii has been demonstrated in approximately 20% of contact plates taken from the antecubital fossa of 185 inpatients and outpatients and healthy staff. The highest incidence was in a group of 40 very ill patients. The isolation of the organism from blood cultures is not always of clinical significance. Skin preparation as at present practised is often inadequate to remove the spores when contamination is relatively heavy, for example, in bedridden patients.     

Incomplete anaerobiosis as a cause of metronidazole ''resistance''.

Clostridium welchii, used as a control, was found to grow well in a microaerophilic jar used for campylobacters but appeared resistant to a metronidazole disc although the campylobacter was sensitive. Minimum inhibitory concentrations for six strains of Cl. welchii were up to 64-fold higher in these conditions than when grown anaerobically. Zones of inhibition with both Cl. welchii and Bacteroides fragilis varied with the amount of air admitted to anaerobic jars.     

Simplified procedure for producing Bacillus subtilis spores for the Guthrie phenylketonuria and other microbiological screening tests.

Bacillus subtilis ATCC 6051 and ATCC 6633 spores used in bacterial inhibition screening assays for genetic metabolism defects in newborn infants were produced by using liquid synthetic replacement sporulation media. These media allowed a high degree of sporulation, as judged by direct cell counts. Sporulation took place within 23 to 27 h with these media. Also, a more rational procedure for selecting the most sensitive clones of these organisms to the various inhibitors used in the microbiological screening assays is presented.     

Characterization of a catalase-deficient strain of Neisseria gonorrhoeae: evidence for the significance of catalase in the biology of N. gonorrhoeae.

We obtained a catalase-deficient (Kat-) strain of Neisseria gonorrhoeae isolated from a patient who had been unsuccessfully treated with penicillin. Quantitative enzyme assays and electrophoresis of cell extracts on native polyacrylamide gels subsequently stained for catalase and peroxidase activities failed to detect both enzymes. The strain exhibited no growth anomalies or unusual requirements when grown under ordinary laboratory conditions. However, the Kat- strain proved extremely sensitive to exogenous hydrogen peroxide, and analysis of the bacterial DNA after such exposure showed extensive single-strand breakage in both chromosomal and plasmid DNAs. Partial characterization of the gonococcal catalase from a Kat+ laboratory strain revealed that the enzyme had the physical and chemical properties of both catalase and peroxidase.     

Recovery of spores of Clostridium difficile altered by heat or alkali

The effect of heating or alkali-treatment on spore recovery in ordinary growth medium was examined for four strains of Clostridium difficile. Heating spores at 80 degrees C for 10 min produced 95.50-99.95% decreases in the recovery rates. Treatment with 0.1 N NaOH for 15 min produced 99.47 and 99.83% decreases in spore recovery rates for two of the four strains. The influence of either addition of lysozyme after treatment with sodium thioglycollate (thioglycollate-lysozyme method) or addition of sodium taurocholate (taurocholate method) on recovery of heat- or alkali-treated C. difficile spores was also examined. Viable spores of all strains altered by heating at 90 degrees C or 100 degrees C for 10 min could not be recovered at all by the taurocholate method. Nor did this method allow recovery of alkali-altered spores treated with greater than 0.2 N NaOH for 15 min. On the other hand, 10-47% of altered spores heated at 90 degrees C for 10 min were recovered by the thioglycollate-lysozyme method, and alkali-altered spores treated with 0.1-0.3 N NaOH for 15 min were as completely recovered by this method as untreated spores. These results indicate that the thioglycollate-lysozyme method is more effective than the taurocholate method for recovery of the heat- or alkali-altered C. difficile spores.     

William H. Welch, MD, and the discovery of Bacillus welchii

William H. Welch, MD, and his colleagues performed an autopsy at The Johns Hopkins Hospital in October 1891 on a 38-year-old man and discovered a new bacterium, Bacillus aerogenes capsulatus. During the postmortem examination, gas bubbles were noted within many of the patient's blood vessels. Welch's laboratory personnel determined that a previously unknown bacterium was the source of the gas. Through a series of experiments, the organism's characteristics were described and its pathophysiology was detailed, findings that proved accurate in explaining gas gangrene during World War I. Welch never followed up these initial investigations with more experimentation. His subsequent writings regarding the bacterium that came to be known, appropriately, as Bacillus welchii consisted mostly of case reports from other medical institutions and summaries of previous data.     

The isolation of an X-dependent strain of Haemophilus from otitis media identified as H. haemoglobinophilus (canis)

The growth factor requirements, together with the serological results, would seem to justify the view that strain M.G., isolated from a child, M.G., with otitis media, in pure culture has to be regarded as H. haemoglobinophilus (canis).It is interesting to note that two out of the three strains of H. haemoglobinophilus (canis) available in this laboratory agglutinate to a titre of 1:80 with the serum produced against H. aphrophilus, the only other X-dependent haemophilus we had used for the preparation of antiserum.     

Identification of a novel enterotoxigenic activity associated with Bacillus cereus.

A strain of Bacillus cereus isolated from a food poisoning outbreak characterized by vomiting has been shown to be capable of causing vomiting when cultures grown on rice, but not other media, were fed to Rhesus monkeys. In contrast, a strain isolated from a diarrhoeal outbreak produced diarrhoea, but not vomiting, when grown on various media in similar feeding trials. Furthermore, culture filtrates from the diarrhoeal strain caused fluid accumulation in ligated rabbit ileal loops whereas those from the vomiting strain did not. It is proposed that at least two enterotoxins are involved, one responsible for the vomiting and one for the diarrhoeal symptoms.     

Regulation of extracellular slime production by Actinomyces viscosus.

Extracellular slime polysaccharides produced two Actinomyces viscosus strains, T14V and T14AV, were compared. In various media containing glucose, T14Av produced abundant extracellular viscous slime polysaccharide, whereas T14V produced lower levels. Furthermore, fractionation of these polysaccharides showed that the two extracellular polysaccharides differed in molecular size and net charge. Since there was a significant difference in the relative abilities of chemically defined medium and chemically defined tissue culture medium to support slime production by T14Av, the nutritional factors influencing the production of extracellular slime were examined. Sodium bicarbonate was demonstrated to stimulate both cellular growth and the production of extracellular slime. In chemically defined medium with and without sodium bicarbonate, strain T14Av produced large quantities of viscous slime in glucose and sucrose media. In contrast, relatively low levels of slime were produced in fructose, lactose, raffinose, and inositol media, even though sodium bicarbonate stimulated the growth of T14Av in these latter media.     

一株新菌的发现及定位研究

从胆囊炎败血症患者血中分离出一株新菌,该菌为兼性厌氧G~-球菌,无鞭毛、无芽胞、无荚膜,发酵葡萄糖产气,还原硝酸盐成亚硝酸盐,氧化酶试验阴性,不具有氨基酸脱羧酶,对绝大多数糖不利用,具有β半乳糖苷酶,不产生吲哚,甲基红试验阳性,利用丙二酸盐,不利用枸橼酸盐。电镜表明该菌为芽殖细菌,G C含量为47.58mol％。16S rRNA序列测定结果经计算机检索基因库所有序列进行比较表明和目前所有菌序列都不同,根据相似性和表型特征选择最接近的15个种16S rRNA序列得出全连锁聚类分析树状谱,亲缘关系相差甚远。结果表明该菌不属于已知的科、属,根据该菌发酵代谢和特有的芽殖特点,应建立新科。拟定为发酵芽殖菌科(Fermentobadaceae fam.nov.),命名Guhaiyingella gen.nov.guhaiyingii sp.nov。 该菌已收藏在中国微生物菌种保藏中心,CGMCC No.0205~T(T=type strain)。     

Modification of methods used in bacteriophage typing of Mycobacterium tuberculosis isolates.

A procedure in which soft agar overlays were used in bacteriophage-typing Mycobacterium tuberculosis is presented. This safer method uses commercially available media, whereas media presently used must be prepared in the laboratory. Single plaque isolations of the phage BG-1 specifying phage type A and B of M. tuberculosis were readily made by using the modified procedures. This purification and the use of prototype strain Myc 1415 as the indicator host strain have significantly enhanced the ability to discriminate among strains of phage types A and B.     

Assessment of calcium hypochlorite for Bacillus anthracis spore surface's decontamination

Contamination with microorganisms occurs in laboratories but is also of high concern in the context of bioterrorism. Decontamination is a cornerstone that promotes good laboratory practices and occupational health and safety. Among the most resistant structures formed by microorganisms are spores, produced notably by Clostridium and Bacillus species. Here, we compared six products containing four different molecules (hydrogen peroxide, peracetic acid, sodium and calcium hypochlorite) on B. anthracis Sterne spores. We first selected the most efficient product based on its activity against spore suspensions using French and European standards. Four products showed sporicidal activity, of which only two did so in a time frame consistent with good laboratory practices. Then, we tested one of these two products under laboratory conditions on fully virulent B. anthracis spores, during common use and after contamination through a spill of a highly concentrated spore suspension. We, thus, robustly validated a decontaminant based on calcium hypochlorite not only on its ability to kill spores but also on its effectiveness under laboratory conditions. At the end, we were able to assure a complete disinfection in 1 min after spillover and in 2 min for common use.     

Genetic analysis of apomictic wine yeasts

The Saccharomyces cerevisiae wine yeast IFI256 was selected because of its high fermentative capacity and tolerance to ethanol. Sporulation of the IFI256 strain produced two-spore asci unable to conjugate, but able to sporulate again and the spores produced two-spore asci in all cases. That process was studied for at least five generations. The electrophoretic karyotype showed a pattern of 21 chromosomal bands, which was identical both in the parental and in all the descendants analyzed, from the first to the fifth generation. The DNA content of the parental and the descendants was of 1.7 n, which indicates that the capacity for sporulation shown by all descendants was due to apomixis rather than homothallism of the strain. Different concentrations of glucose and acetate and the addition of zinc salts to the presporulation and sporulation media increased the frequency of four-spore asci by up to 9%. However, the tetrads formed were in fact two dyads that resulted from induced endomitosis. Crosses of IFI256 with laboratory strains produced hybrids giving four-spore asci after sporulation, thus indicating the mutation to be recessive. Transformation of IFI256 with plasmids carrying either SPO12 or SPO13 functional genes and crosses with strains carrying functional or mutated SPO12 and/or SPO13 genes indicated that IFI256 carries several mutations, one of which was located to the SPO12 gene. Parasexual cycles and chromosome loss induced after crossing IFI256 with cir⁰ strains indicated that apomictic mutations were exclusively located at chromosome VIII. The high frequency of wine strains which are apomictic suggests apomixis to be an advantageous phenotype which allows the formation of stress-resistant asci but prevents the loss of favored chromosomal rearrangements.Communicated by S. Hohmann     

Production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the C-terminal region of potato virus Y coat protein

Comparing the predicted amino acid sequence between two Japanese potato virus Y (PVY) strains, necrotic strain and ordinary strain, it was found that the C-terminal regions (H₂N-HTTEDVSPSMHTLLGVKNM-COOH) of the coat proteins in the two strains were completely conserved. The conserved amino acid sequence was also found in the C-terminal region coat protein of PVY-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. Antibodies were produced against a synthetic polypeptide PVY-C19 consisting of 19 amino acids, which correspond to the C-terminal region of the coat protein, using 4 coupling combinations of polypeptide PVY-C19 to protein carriers. Carrier-free polypeptides and those coupled to ovalbumin with ECDI (ethyl-dimethylaminopropyl carbodiimide) produced high titer of antibodies and detected PVY strains from PVY-infected plants by Western blot analysis and by ELISA.     

The use of an immunoperoxidase technique to investigate by light and electron microscopy the sites of binding of Clostridium welchii type-D epsilon toxin in mice.

Mice were given an intravenous dose of formalinised C. welchii type-D epsilon prototoxin and an immunoperoxidase technique was used to demonstrate this antigen in the tissues. The antigen was found to bind to the luminal surface of the endothelial lining of certain blood vessels, to the luminal surface of the cells lining the loops of Henlé and distal convoluted tubules in the kidney, and to the hepatic sinusoids. As it has been shown previously that formalinised epsilon prototoxin and epsilon toxin can compete for the same receptor sites it is postulated that the binding sites demonstrated represent the location of the receptors for C. welchii type-D epsilon toxin.     

Lesions produced by Clostridium butyricum strain CB 1002 in ligated intestinal loops in guinea pigs

Heated spores (80 degrees C, 10 min) of Clostridium butyricum strain CB 1002 isolated from a fatal case of necrotising enterocolitis in a human neonate were inoculated into ligated intestinal loops prepared in young conventional guinea pigs. Necropsy findings 18 h later included congestion, patchy haemorrhage of the intestinal mucosa and bacteraemia. No abnormalities were observed in control loops given inocula of inactivated spores (heated at 100 degrees C for 10 min) or TYG 6 medium. The results suggest that vascular lesions are produced by C. butyricum in the intestine of young conventional guinea pigs.