Influence of the aminopeptide concentration on the growth of Haemophilus influenzae, type b, and the synthesis of its capsular polysaccharide

The influence of the aminopeptide concentration on the growth of H. influenzae b culture and the synthesis of H. influenzae b capsular polysaccharide was determined. The maximum amount of capsular polysaccharide was accumulated at the concentration of aminopeptide in the culture fluid reaching 50 ml/l. An increase in the aminopeptide concentration led to a decreased amount of synthesized polysaccharide and an increased amount of biomass. The decrease of the aminopeptide concentration to 10 ml/l resulted in decreased amounts of both biomass and synthesized polysaccharide.     

Influence of nicotinamide adenine dinucleotide and hemin concentrations on the growth of Haemophilus influanzae type b and the synthesis of capsular polysaccharide

In the process the cultivation of H. influenzae, type b, in semisynthetic nutrient medium with aminopeptide base the growth of the bacteria and the synthesis of capsular polysaccharide were shown to depend on the concentrations of aminopeptide, nicotinamide adenine nucleotide (NAD) and hemin. An increase in the concentrations of NAD and hemin stimulated the growth of H. influenzae and inhibited the synthesis of capsular polysaccharide. Similar effect was observed in the simultaneous increase of NAD and hemin concentrations. At elevated concentrations of NAD and hemin and the content of aminopeptide equal to 350 mI/l the maximum weight of biomass was achieved. The increase of hemin concentration had no influence on the growth of H. influenzae, type b, and the synthesis of capsular polysaccharide.     

Isoelectric focusing of human antibody to the Haemophilus influenzae b capsular polysaccharide: restricted and identical spectrotypes in adults

Serum antibody to the capsular polysaccharide of Haemophilus influenzae b of human adults was analyzed by isoelectric focusing. Restricted antibody spectrotype patterns were commonly observed with as few as one spectrotype in some subjects after immunization with the isolated capsular polysaccharide. Some patterns were as restricted as human hybridoma antibody. There was no correlation of antibody titer and heterogeneity of patterns. The dominant spectrotype persisted unchanged for over 2 yr after immunization, and the pattern detected in preimmunization serum samples persisted unchanged after immunization. Indistinguishable patterns were commonly observed in genetically unrelated adults. Adults immunized with conjugate vaccines, which were composed of oligosaccharides prepared from the capsular polysaccharide that were covalently linked to protein carriers, also produced restricted serum antibody spectrotype patterns. Immunization with the cross-reactive polysaccharide of E. coli K100 induced a spectrotype pattern that was restricted but different from that induced by the H. influenzae b capsular polysaccharide.     

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.     

Specific characteristics of composition of synthetic nutrient media for cultivation of Hemophilus influenzae type b from the standpoint of bacterial metabolism

In many countries vaccination against Haemophilus influenzae of type b (Hib) has permitted the liquidation of severe generalized forms of infections caused by these bacteria. The vaccine is obtained on the basis of Hib capsular polysaccharide. To obtain pure capsular polysaccharide, Hib should be cultivated on synthetic nutrient media. The present review deals with the data substantiating the advantages of using synthetic nutrient media for the cultivation of these bacteria with a view to obtaining pure capsular polysaccharide.     

Common organization of chromosomal loci for production of different capsular polysaccharides in Haemophilus influenzae.

Cloned Haemophilus influenzae type b capsulation genes were used as hybridization probes to isolate DNA from the capsulation loci (cap) of other serotypes of H. influenzae. Mapping of the resulting clones and Southern hybridization analysis of chromosomal DNAs from type a, b, c, and d strains showed that in each strain cap was organized in the same way: a central DNA segment specific to each serotype flanked by DNA segments of common structure. We infer that enzymes necessary for the synthesis of specific capsular polysaccharide are encoded in the central segment of cap, while proteins involved in a more general way in the process of capsulation are encoded in the flanking segments. Studies of the function of the DNA in one of these non-serotype-specific flanking segments (J. S. Kroll, I. Hopkins, and E. R. Moxon, Cell 53:347-356, 1988) have previously identified a gene encoding a protein necessary for polysaccharide export, an event now deduced to proceed by a mechanism independent of the nature of the disaccharide subunit in the polysaccharide. The near-total duplication of cap that has been found in most type b strains was not found at the analogous locus in the other serotypes. This reinforces our previous hypothesis, based on study of type b strains alone, that while such a duplication is unnecessary for capsulation, it confers some unexplained survival advantage on the widely prevalent strains with this clinically important serotype.     

Accuracy of the slide agglutination method evaluated with PCR in typing Haemophilus influenzae isolates

In 1998, the Colombian government initiated an immunization program for children under one year of age with a Haemophilus influenzae capsular type b conjugate vaccine. After two years, the surveillance program of the Colombian Instituto Nacional de Salud Microbiology Group reported a 40% decrease in meningitis cases caused by H. influenzae. This effect was attributed to the vaccination. The surveillance program uses the standardized slide agglutination technique to serotype H. influenzae. The current study validated the accuracy of the slide agglutination method by means of the PCR technique. From children under five years of age, 146 isolates were obtained. These were collected between 1999 and 2002, and were characterized by biochemical tests and serotyped by the INS as part of the surveillance program. PCR confirmed 93% of the H. influenzae serotype b and 92% of the other serotypes. When the slide agglutination technique is conducted under a strict quality control program, it remains a sensitive and specific tool for serotyping H. influenzae.     

Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.     

Development and scale-up of a fed-batch process for the production of capsular polysaccharide from Haemophilus influenzae

The production of the capsular polysaccharide, polyribosylribitolphosphate, from Haemophilus influenzae type b is important for the production of effective conjugate vaccines. Factors limiting the production of this polysaccharide from H. influenzae type b in liquid culture were investigated. A fed-batch fermentation was developed that increased cell density and PRP titer approximately four fold when compared to the batch fermentation. This fed-batch process was successfully scaled from the 1.5 l development scale to the 500 l manufacturing scale. The maximum cell density in the 500 l fermentation was 6 g dry cell weight per liter and the PRP concentration was 1.3 g l(-1).     

Haemophilus influenzae type b polysaccharide-protein conjugate vaccine

An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.     

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.     

Capsulation in distantly related strains of Haemophilus influenzae type b: genetic drift and gene transfer at the capsulation locus.

Among natural populations of capsulate Haemophilus influenzae, clones of strains with type b capsular polysaccharide are found in each of two widely separated phylogenetic divisions. The chromosomal capsulation locus found in strains from either division has a three-segment organization, with serotype-specific DNA nested between elements common to all serotypes, but pairwise comparison of the segments between the divisions suggests that they have distinct phylogenetic histories. Genes clustered in one of the non-serotype-specific segments appear to have diverged from an ancestral element, reflected in 12% nucleotide sequence divergence in one homologous pair. In contrast, genes conferring the capacity to produce type-specific polysaccharide exhibit no such divergence, and we speculate that these have been subject more recently to horizontal transfer within the bacterial population. Clinically important capsulate gram-negative bacteria share a common organization of their capsulation loci, arguing convergence on a successful arrangement of genes. In H. influenzae this appears to have allowed the occasional exchange of serotype-specific capsulation genes between strains, a event of potential clinical importance in this major bacterial pathogen.     

Cultivation of Haemophilus influenzae,serotype B,in amino peptide-based semisynthetic nutrient medium

The work shows the possibility of the cultivation of H. influenzae, serotype b, in semisynthetic nutrient medium with amino peptide as the only source of amino acids, glucose--as the main source of carbon and energy and containing, in addition, the necessary growth factors and vitamins.     

Derivation of Extracellular Polysaccharide-Deficient Variants from a Serotype A Strain of Pasteurella multocida

The production of serotype A extracellular polysaccharide is thought to be associated with expression of an approximately 40-kDa lipoprotein (Plp-40) present on the outer surface of Pasteurella multocida strains of avian origin. The tendency of certain strains to undergo colonial dissociation concomitantly with serial passaging on laboratory growth media was exploited to derive two variant strains exhibiting the capsule-deficient phenotype from a heavily capsulated parental strain. Assessments of colonial consistency, iridescence, gentian violet binding, and hyaluronidase sensitivity were consistent with cellular observations indicating little or no capsulation of derivative strains. Fluorographic analysis of electrophoretically resolved cellular lipoproteins labeled with [³H]-palmitate revealed capsular loss occurred with a concomitant diminution of Plp-40 production in the variant strains. In contrast, a phenotypically stable strain that did not undergo colonial dissociation under identical conditions exhibited no decrease in Plp-40 content. This work provides a model system for investigating the role of extracellular polysaccharide in the cell surface physiology and pathogenicity of P. multocida. The present results strongly support the notion that Plp-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis. Received: 30 October 1998 / Accepted: 4 December 1998     

Dissection of the Yersinia enterocolitica virulence plasmid pYVO8 into an operating unit and virulence gene modules

Abstract Mesophilic Aeromonas hydrophila from serotypes O:11 and O:34 grown in a glucose-rich medium produce a capsule that can be seen under light and electron microscopy. The purified capsular polysaccharide has a composition qualitatively similar for strains O:11 and O:34, but quantitatively different. The capsular polysaccharides were immunogenic in rabbits, and did not cross-react with specific antibodies against either purified lipopolysaccharide from strains O:34 or O:11 or against the S-layer characteristic of strains from serotype O:11.     

Serotypes and biotypes and antibiotic susceptibility of Haemophilus influenzae encountered in a clinical laboratory in Taiwan.

Forty-four serologically and biochemically typable Haemophilus influenzae isolates from clinical specimens in Taiwan were subjected to analysis in their relationship with source of isolation and age distribution. It was found that all isolates from blood and cerebrospinal fluid were serotype b, biotype I, and all were in children less than 4 years of age. Serotypes b and e, biotypes I and III were encountered to have the highest incidence of infection caused by H. influenzae in this area. All H. influenzae isolates were further tested for susceptibility to several selected antibiotics. All strains of this organism were susceptible to erythromycin and chloramphenicol. All but two strains were susceptible to tetracycline, whereas more strains were resistant to carbenicillin, gentamycin, keflin, and penicillin. Thirty-four percent strains were found to be resistant to ampicillin and all were beta-lactamase producer. No direct correlation between ampicillin resistance and serotypes or biotypes was recognized.     

肺炎链球菌5型发酵工艺的优化

目的:采用正交试验设计方法进行肺炎链球菌5型发酵工艺的研究。方法:根据正交试验设计表L9(34)设计的试验条件组合进行了9次肺炎链球菌5型的发酵,采用70升发酵罐进行发酵工艺的摸索,提取了肺炎链球菌5型荚膜多糖粗糖。结果:最佳的发酵培养条件组合为温度37℃、葡萄糖20克/升、大豆胨15克/升、pH值7.3,最佳的纯化条件组合为冷酚抽提三次、沉核酸乙醇浓度23%、超滤膜孔径50kD、最终沉糖乙醇浓度60%,在此筛选得到的最佳条件下,连续进行了5个批次肺炎链球菌5型的发酵与荚膜多糖提取,荚膜多糖粗糖的平均收率为808.6mg/L,相对标准偏差为3.84%。结论:上述发酵培养条件组合适合用于肺炎多糖疫苗的研究和生产。     

Electron microscopy of Haemophilus influenzae

The electron microscopic study of H. influenzae standard strains, serovar b, in the capsular and noncapsular forms has revealed the pronounced pleomorphism of these organisms in successive generations.     

流感嗜血杆菌的分型研究

目的分析同济医院分离的流感嗜血杆菌的生物学分型及荚膜基因分型,了解本地区分离的流感嗜血杆菌的主要流行株。方法2012年1月1日至2012年12月31日从华中科技大学同济医学院附属同济医院分离流感嗜血杆菌100株。根据脲酶、吲哚和鸟氨酸脱羧酶试验对流感嗜血杆菌进行传统的生物学分型,分为Ⅰ～Ⅷ八个生物型。回顾患者病史资料,分析生物学分型和流感嗜血杆菌所引起的疾病之间的关系。用流感嗜血杆菌荚膜编码基因（bexA）和a—f型特异性荚膜基因设计引物,采用PCR技术对流感嗜血杆菌进行荚膜基因检测。通过生物学分型和荚膜基因分型结果的比对,探讨两者之间的关联。结果分离的100株流感嗜血杆菌生物学分型结果如下：Ⅲ型6株,Ⅳ型28株,Ⅴ型1株,Ⅵ型54株,Ⅶ型11株。未分离到Ⅰ型、Ⅱ型和Ⅷ型。分析患者的临床诊断,发现主要流行株Ⅵ型流感嗜血杆菌主要引起患者肺炎（包括支气管肺炎和新生儿肺炎）和支气管炎（包括毛细支气管炎和喘息性支气管炎）。荚膜基因分型结果显示,未分离到b型和b-型流感嗜血杆菌。共分离到1株f型,其余99株均为无荚膜抗原的不可分型流感嗜血杆菌。生物学分型和荚膜分型之间无明显的相关性。结论该院分离的流感嗜血杆菌主要为生物型Ⅵ型。回顾患者病史,发现Ⅵ型主要引起肺炎和支气管炎。荚膜基因分型显示,本地区分离的流感嗜血杆菌主要为不可分型流感嗜血杆菌。生物学分型和荚膜基因分型之间无明显相关性。     

The capsular polysaccharide of Haemophilus influenzae type B prevents killing by complement and antibodies against outer membrane protein a

Abstract The ability of antibodies, raised in rabbits against purified outer membrane protein a ( M _r 47 000) of Haemophilus influenzae type b, to promote complement-dependent killing of these encapsulated organisms was investigated. Killing of encapsulated strains was not induced by these antibodies in conjunction with either human, mouse, rabbit or guinea-pig complement. Acapsular mutants were effectively killed by complement in the presence of antibodies against protein a . Killing was dependent on the presence of the 47-kDa protein a and was not influenced by the outer membrane protein subtype or lipopolysaccharide serotype of the strain. The killing-promoting activity could be absorbed from the sera with cells of strains with the same protein a , purified protein a , but not by purified lipopolysaccharide and capsular polysaccharide. Binding experiments showed that the encapsulated strain and its acapsular mutant bound antibodies against protein a with the same rate and to the same extent, indicating that the capsule probably interferes with complement activation or insertion of the membrane attack complex into the bacterial cell.