Carbon Dioxide Requirement of Various Species of Rumen Bacteria

The carbon dioxide requirement of 32 strains of rumen bacteria, representing 11 different species, was studied in detail. Increasing concentrations of CO(2) were added as NaHCO(3) to a specially prepared CO(2)-free medium which was tubed and inoculated under nitrogen. Prior depletion of CO(2) in the inoculum was found to affect the level of requirement; however, the complexity and buffering capacity of the medium did not appear to be involved. An absolute requirement for CO(2) was observed for eight strains of Bacteroides ruminicola, three strains of Bacteroides succinogenes, four strains of Ruminococcus flavefaciens, two strains of Lachnospira multiparus, one strain of Succinimonas amylolytica, and two strains of Butyrivibrio fibrisolvens. Inconsistent growth responses were obtained in CO(2)-free media with one strain each of B. fibrisolvens, Ruminococcus albus, and Selenomonas ruminantium. Growth of six additional strains of B. fibrisolvens, and single strains of Eubacterium ruminantium and Succinivibrio dextrinosolvens was markedly increased or stimulated by increasing concentrations of CO(2). Peptostreptococcus elsdenii B159 was the only organism tested which appeared to have no requirement, either absolute or partial, for CO(2). Higher concentrations of CO(2) were required for the initiation of growth, as well as for optimal growth, by those species which produce succinic acid as one of their primary end products.     

Pneumococcal serotypes in various diseases of children

The serotyping of pneumococci isolated from different material obtained from children aged 0 to 11 years was carried out. Out of 156 patients with different diseases, hospitalized in two clinics in Moscow during February-May 1983, pneumococci were isolated from 67 patients (43%). The isolated pneumococcal strains belonged to 11 serotypes. Pneumococci of serotypes 3, 6, 9 and 19 were shown to occur most frequently in different diseases and constituted 50% of the isolated strains. The inoculation of the material by the quantitative method permitted the authors to find out the role of pneumococci as the etiological factor in the pathogenesis of some diseases. A certain dependence of diversity in the types of isolated pneumococci on the age of sick children was noted. Almost all isolated strains were found to be sensitive to penicillin, ampicillin and benzylpenicillin. But a few individual strains were sensitive only to one of these antibiotics. The data on some biological properties of pneumococci cultivated on solid culture media are presented.     

Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii

Botryococcus braunii is a green colonial fresh water microalga and it is recognized as one of the renewable resources for production of liquid hydrocarbons. CFTRI-Bb-1 and CFTRI-Bb-2 have been reported for the first time and their performance with regard to growth and biochemical profile is presented here. The present study focused on effect of carbon dioxide (CO2) on biomass, hydrocarbon, carbohydrate production, fatty acid profile, and carotenoid content in various species of B. braunii (LB-572, SAG 30.81, MCRC-Bb, N-836, CFTRI-Bb-1, and CFTRI-Bb-2) at 0.5, 1.0, and 2.0% (v/v) levels using a two-tier flask. CO2 at 2.0% (v/v) level enhanced growth of the organism, and a two-fold increase in biomass and carotenoid contents was observed in all the B. braunii strains studied compared with control culture (without CO2 supplementation). At 1% and 2% (v/v) CO2 concentrations, palmitic acid and oleic acid levels increased by 2.5 to 3 folds in one of the strains of B. braunii (LB-572). Hydrocarbon content was found to be above 20% at 2% CO2 level in the B. braunii LB-572, CFTRI-Bb-2, CFTRI-Bb-1, and N-836 strains, whereas it was less than 20% in the SAG 30.81 and MCRC-Bb strains compared with control culture. This culture methodology will provide information on CO2 requirement for growth of algae and metabolite production. B. braunii spp. can be grown at the tested levels of CO2 concentration without much influence on culture pH.     

Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae

To find reliable methods able to identify the "difficult" Streptococcus pneumoniae isolates, an in-house polymerase chain reaction (PCR) for pneumolysin gene (Ply-PCR) and a commercial RNA hybridisation test (AccuProbe) were evaluated. Selected isolates of suspected pneumococci, sent for confirmation of identification and for serotyping, were classified into four groups based on their optochin sensitivity and capsule reaction. All isolates in Group 1, which consisted of 24 typical, optochin-sensitive, encapsulated pneumococcal strains, were positive in the Ply-PCR and AccuProbe tests. In Group 2, which consisted of 25 optochin-sensitive, but unencapsulated pneumococcal strains, all the isolates were positive in the Ply-PCR test, and 23 were positive in the AccuProbe test. In Group 3, which consisted of 15 atypical, optochin-resistant but encapsulated pneumococci, 12 of the isolates were positive in the Ply-PCR and 12 in the AccuProbe test, and 11 of these 12 strains were positive in both tests. In Group 4, which consisted of 36 equivocal optochin-resistant, unencapsulated isolates, 15 strains were positive in the Ply-PCR test and 8 strains in the AccuProbe test. As a conclusion, the Ply-PCR and AccuProbe tests identified similarly typical optochin-sensitive pneumococci, but gave partly controversial results about atypical pneumococci. Thus, they did not reliably help in the identification of suspected pneumococcal isolates lacking the conventional characteristics of pneumococcus.     

Transport of Neisseria meningitidis cultures: growth-supporting media and freezing as an alternative.

The abilities of Transgrow (TG), Thayer-Martin (TM), and New York City (NYC) solid media to maintain the viability of 12 strains of Neisseria meningitidis under various controlled conditions were assessed. The effects of charcoal impregnation of swabs, temperature, and an enriched CO2 atmosphere were examined with holding for up to 21 days. Recovery from samples held at 35 degrees C was, in almost all instances, greater than at 22 or 4 degrees C. A strong requirement for added CO2 was demonstrated, especially at lower temperatures. No positive effect could be attributed to the use of charcoal-impregnated swabs. NYC and TM media were the best overall, with the former permitting recovery from more than 75% of all samples held on slants for 20 days at 4 degrees C in an atmosphere of 5% CO2 in air. Freezing, with holding on dry ice, was a useful alternative to the use of growth-supporting media. This latter method eliminated the requirement for an enriched CO2 atmosphere.     

Interfering Effect of Incubation in Carbon Dioxide on the Identification of Pneumococci by Optochin Discs

Incubation of pneumococci in a CO(2) environment decreases the zone sizes produced by optochin discs, with the result that pneumococcal-like organisms require further study.     

Streptococcus pneumoniae serotypes in children with chronic inflammatory diseases of the respiratory organs

During the 7-year period of observation (1982-1988) the serotypes of 276 pneumococcal strains isolated from children with chronic bronchopulmonary diseases were studied. Among the serotypes of pneumococci under study, serotypes 6, 19 and 15 held the leading place and included a half of all typed pneumococci. Dynamic observation on the serotype composition of pneumococci revealed periodic fluctuations in the occurrence of some types/groups. The regional analysis of different serotypes of pneumococci showed the common occurrence of serogroups 6 and 19, as well as some regional features in the circulation of serotypes 6, 10, 3 and rarely occurring serotypes. The study revealed that any new exacerbation of the chronic bronchopulmonary process is caused by pneumococci of some other serotype. Pneumococcal strains, resistant (3.4%) and sensitive (1.8%) to penicillin, were detected; most of them belonged to serogroup 19.     

Disk Test for the Differentiation of Pneumococci from Other Alpha-Hemolytic Streptococci

One hundred twenty-five strains of Diplococcus pneumoniae and one hundred twenty-five strains of other alpha-hemolytic streptococci were planted on horse red cell-Brucella agar, and a disk of methicillin was placed on each inoculated plate. All strains were incubated anaerobically at 37 C for 24 h and then exposed to air at 6 C for 48 h. All strains of pneumococci produced a ring of beta-hemolysis surrounding the zone of inhibition produced with the methicillin disk, whereas no strain of alpha-hemolytic streptococci produced beta-hemolysis. It is suggested that this new criterion is useful for the differentiation of pneumococci from other alpha-hemolytic streptococci in those cases where other characteristics were insufficient for an unequivocal identification.     

Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum,L. paraplantarum,L. pentosus,and L. casei: prevalence of CO(2)-dependent auxotrophs and characterization of deficient arg genes in L. plantarum

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO(2). We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO(2) requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.     

Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in "atypical" pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcal alleles. Conversely, the piaA gene was found to be specific to typical pneumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.     

Pneumococci resistant to erythromycin.

Susceptibility to erythromycin was determined for all pneumococci isolated in one laboratory from clinical specimens between 1969 and 1977. All 4724 isolates examined prior to October 1973 were susceptible to erythromycin. From October 1973 to December 1977, 64 (0.71%) of 8995 pneumococcus isolates were resistant to erythromycin. The resistant strains were isolated from 38 patients living in six widely separated communities in Alberta. The erythromycin-resistant strains were of nine capsular types, including six that often cause bacteremic disease and five for which resistance to erythromycin has not been reported hitherto. Certain strains of type 33 and of type 15 were highly resistant, the minimum inhibitory concentration (MIC) of erythromycin being 2000 microgram/mL; these strains were also highly resistant to lincomycin and clindamycin. Resistance in strains of other types was much lower, the MIC of erythromycin being 0.6 to 20 microgram/mL, and all but one of these strains were susceptible to lincomycin and clindamycin. All the erythromycin-resistant pneumococci were suspectible to penicillin.     

Streptococcus pneumoniae serotypes in children with acute pneumonia and pleurisy

The study of pneumococci of different serotypes, isolated from patients with acute pneumonia and pleuritis and from healthy children was carried out. Among the pneumococcal serotypes causing pneumonia and pleuritis in children serotypes 1, 6, 19, 14 and 3 were most widely spread and constituted 62.3% of all isolated pneumococci. In young children cases of acute pneumonia and pleuritis were more often induced by serotypes 6 and 14 and in older children, by serotypes 1 and 3. In patients with uncomplicated pneumonia and pleuritis differences in the detected serotypes of pneumococci were observed, and the disease course differed in severity. Serotypes 14, 3 and 3 induced destructive processes in the lungs more often than other serotypes. Monitoring of the sensitivity of pneumococci to antibiotics showed that most of the strains retained high sensitivity to penicillin and ampicillin. In most cases the detected resistant pneumococcal strains belonged to serogroup 19.     

中国肺炎链球菌标准菌株的筛选和建立

对我国自行分离的737株肺炎链球菌,依据生物学特性、地区分布及血清学分型进行筛选,选出297株具有典型特征的肺炎链球菌,建立了我国肺炎链球菌标准菌株。共分成42个群(型),其中有我国自行分离并保藏的二株国际上首次发现的新型10C和16A致病性肺炎链球菌,以及国际上多年来未见到的19C型、22A型和仅在亚洲分离到的33C型菌株。研究肺炎链球菌的血清学分型(群),建立标准分型菌株不仅有分类学的重要价值,而且对研究肺炎链球菌疾病的防冶具有重要意义。     

Formate auxotroph of Methanobacterium thermoautotrophicum Marburg.

A formate-requiring auxotroph of Methanobacterium thermoautotrophicum Marburg was isolated after hydroxylamine mutagenesis and bacitracin selection. The requirement for formate is unique and specific; combined pools of other volatile fatty acids, amino acids, vitamins, and nitrogen bases did not substitute for formate. Compared with those of the wild type, cell extracts of the formate auxotroph were deficient in formate dehydrogenase activity, but cells of all of the strains examined catalyzed a formate-carbon dioxide exchange activity. All of the strains examined took up a small amount (200 to 260 mumol/liter) of formate (3 mM) added to medium. The results of the study of this novel auxotroph indicate a role for formate in biosynthetic reactions in this methanogen. Moreover, because methanogenesis from H2-CO2 is not impaired in the mutant, free formate is not an intermediate in the reduction of CO2 to CH4.     

Altered Diaphorase Activity in Optochin-resistant Pneumococci

Methylene blue (MB) reductase activities of washed cell suspensions and of cell-free extracts prepared from optochin-resistant mutant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of similar preparations from the optochin-sensitive parent strains. With whole cells, optochin hydrochloride was approximately four times more potent than quinine hydrochloride in inhibiting MB reductase activity. However, with cell-free extracts, both drugs had similar inhibitory activities. Glucose uptake was not affected by optochin hydrochloride, and both optochin-sensitive and optochin-resistant pneumococci had similar glucose uptake patterns. Diaphorase activities of cell-free extracts prepared from optochin-resistant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of cell-free extracts prepared from the optochin-sensitive parent strains. Flavin concentrations of cell-free extracts prepared from optochin-sensitive and optochin-resistant strains were similar.     

The abortion issue

The capsular type of 160 strains of pneumococci isolated from blood or cerebrospinal fluid of patients in Alberta and Ontario between June 1978 and August 1980 was determined. Of the 83 known serotypes 36 were represented, and the type distribution was similar to that reported from the United Kingdom and the United States. Although only 111 (69.3%) of the strains belonged to the serotypes represented in the licensed pneumococcal vaccine, if related types within the same serogroup are also included 132 (82.5%) of the strains belonged to the types or groups represented in the vaccine, However, because the vaccine is not recommended for persons aged less than 2 years, from whom 30 strains were isolated, and because 28 strains from those 2 years of age and older were of nonvaccine types or groups, one can presume that 58 (36.3%) of the 160 bacteremic and meningitic infections would not have been prevented by prior vaccination, even if the vaccine were completely effective.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.