Piliation and colonial morphology among laboratory strains of meningococci.

Colonial morphology and piliation were studied on twelve strains from various serogroups of Neisseria meningitidis. Six different colony types (M1 to M6) were identified. Most strains elaborated only an M1 colonial type, which is similar to gonococcus T4. Several combinations of piliation and colonial morphology were observed: (i) colonial variation in which neither parent nor variant were piliated; (ii) colonial variation involving piliated and nonpiliated cells; (iii) dissociation of piliated from nonpiliated cells with no colonial change; and (iv) colonial variation in which both variants were piliated but with distinctly different pili. Results of this study demonstrate that correlations between piliation and colony morphology within N. meningitidis are exceptions rather than the rule.     

Association of type 1 pili with the ability of livers to clear Salmonella typhimurium

The role of type 1 pili in the adherence of Salmonella typhimurium strain SR-11 to hepatic sinusoidal cells was investigated. An average of 66.7% of piliated organisms was cleared by perfused livers on a single pass. Mannose and alpha-methyl-D-mannoside inhibited such trapping in a dose-dependent manner. Preincubation of the bacteria, but not the liver, with either sugar also inhibited trapping, suggesting that the sugar binds to bacterial, not hepatic, receptors. Significant numbers of previously trapped bacteria could be eluted by adding mannose to the wash medium. Bacteria with reduced piliation, obtained either by growing bacteria on agar or by using a nonpiliated variant of the parent strain, were trapped to a significantly lesser extent than the parent strain. The liver appears to selectively trap heavily piliated organisms since reperfusion of bacteria through a second liver results in significantly less trapping than occurs with the first perfusion. In vivo, the nonpiliated variant strain was cleared much more slowly than the piliated parent strain. Mannose and alpha-methyl-D-mannoside, but not glucose, decreased clearance rates of piliated organisms. Cumulatively, the data suggest that type 1 pili are a major factor in hepatic clearance of S. typhimurium.     

Colonization of porcine intestine by enterotoxigenic Escherichia coli: selection of piliated forms in vivo, adhesion of piliated forms to epithelial cells in vitro, and incidence of a pilus antigen among porcine enteropathogenic E. coli.

In contrast to K88-positive porcine enterotoxigenic Escherichia coli (ETEC), K88-negative porcine ETEC strains did not adhere to isolated intestinal epithelial cells in vitro. However, they did adhere to intestinal epithelium in vivo. Growth of one such ETEC (strain 987) in pig small intestine consistently yielded a greater percentage of piliated cells than did growth in vitro. This increase was demonstrable by electron microscopy, by change in colonial morphology, and by agglutination in specific antisera against the pili of strain 987. In contrast to the stored stock culture (which contained very few piliated cells), richly piliated forms of strain 987 did adhere to isolated intestinal epithelial cells in vitro. A series of porcine E. coli strains was tested for agglutinability in antiserum against the pili of strain 987, and several K88-negative ETEC strains were agglutinated. These data are consistent with the hypothesis that pili facilitate intestinal adhesion and colonization by K88-negative ETEC strains.     

Characteristics of Pseudomonas aeruginosa in relation to laboratory-induced resistance to gentamicin.

Pseudomonas aeruginosa strain C2 was habituated to gentamicin by serial passage in broth containing increasing concentrations of the antibiotic and up to 250 microgram/ml. The resistant progenies differed from the parent strain in antibiotic susceptibility to two other aminoglycosides, colonial morphology, lytic phage patterns, phage adsorption, and agglutination with the seven Fisher's antisera. All the progenies failed to grow at 42 degrees C and oxidised glucose in O/F tubes after incubation at 37 degrees C for three days but were catalase- and oxidase-positive. Reversion to the original properties of the parent strain was demonstrated in all cases after 10 serial subcultures in antibiotic-free broth.     

Virulence of Streptococcus mutans: revertants of mutant C4.

Mutant C4, a poor plaque-forming mutant of Streptococcus mutans 6715-HSR, was employed to obtain isolates resembling the parent strain (a plaque former). Seventeen presumptive revertants, as identified by colonial morphology, were isolated from mutant C4 after enrichment cycles in a sucrose-glass beads medium. These isolates displayed properties which resembled the parent in ability to produce plaque, patterns of fermentation, and resistance to streptomycin. In a detailed study, five selected isolates were found to be similar to the parent type 6715-HSR with respect to content of the serotype antigen, sucrose- or dextran-induced cell aggregation, glucosyltransferase and adherence activities, and cariogenicity. Thus, in selection for revertants to parental colonial morphology, the pleiotropic changes in plaque formation, adherence, glycosyltransferase activity, and virulence demonstrated by C4 all concomitantly reverted to their parental phenotypes.     

Immunization of guinea pigs with epitope C-rich lipopolysaccharide from Neisseria gonorrhoea; in vivo selection of gonococcal variants

The immunogenic potential of lipopolysaccharide (LPS) of a variant of Neisseria gonorrhoeae strain Gc 40 selected by growth in vivo (vivo variant) was investigated in guinea pigs. LPS extracts obtained from the water (WLPS) and the phenol (PLPS) phases of a hot phenol-water extraction were compared for their antigenic capacity and protective effect against infection in subcutaneous chambers. Immunization with PLPS induced significant levels of anti-LPS and anti-epitope C antibodies but WLPS was not antigenic. Two days after challenge, all guinea pigs immunized with WLPS had infections similar to those seen in unimmunized control animals while most animals immunized with PLPS and challenged with either 10(3) or 10(5) gonococci per ml showed low numbers of or no viable gonococci in their chambers. Five days after challenge, however, the same animals had chamber infections with high viable counts similar to controls. Gonococci reisolated from three such animals had physically and antigenically altered lipopolysaccharide and showed patterns of serum sensitivity to pre-challenge chamber fluid from immunized animals which were different from those of the parent vivo variant used for immunization and challenge. The results demonstrate that selection of LPS variants occurs in vivo. This could constitute an immune evasion mechanism.     

Effects of freezing and thawing and storage by freezing on the biological and morphological properties of strain K-9 of Klebsiella pneumoniae and its variants

Using strain K-9 of Klebsiella pneumoniae and its variants A, B and C, which possess a large, small, or extra-small capsule or are unencapsulated, and exhibit high, middle, or low mouse virulence or avirulence in the mouse, respectively, the effect of freezing and thawing on the protection and storage by freezing in 20% skim milk solution on their biological and morphological against freezing and thawing in the order variant C, the parent strain, variants A and B. 20% skim milk solution was the most favourable for survival of the organisms compared to trypticase soy broth and 10% glycerol. Their mouse virulence was similarly sensitive to freezing and thawing experiments although no alteration of their biochemical properties was observed. When the organisms were suspended in 20% skim milk solution and stored for 2 years at –70°C, their mouse virulence was not altered. When morphological features were tested electron microscopically, cellular injuries such as vacuola or fissions of cell walls were observed in the variants but not in the parent strain. However, it was evident that conversion to unencapsulation from capsulation occurred at a high frequency in the parent strain and in variant A when determined by the colonial morphologies in soft-agar medium.     

Relationship among cell wall composition, stage of growth, and virulence of Nocardia asteroides GUH-2.

The clearance and organ distribution of virulent Nocardia asteroides GUH-2P and the avirulent mutant GUH-2AI at different stages of growth was determined after intravenous inoculation into BALB/c mice. The mutant differed significantly from the parent strain in its ability to survive and grow within the murine host. Since the mutant GUH-2AI had a very different colonial morphology compared with GUH-2P, it was believed that cell surface components might be affected by the mutation that resulted in the loss of virulence. Therefore, cell walls of both GUH-2P and GUH-2AI at different stages of growth were prepared and their composition determined. There were growth-stage-dependent changes in the composition of the cell walls that appeared to correlate with concurrent alterations in virulence; however, the overall chemical composition of the cell wall of the mutant (GUH-2AI) was not significantly different from that of the parent strain (GUH-2P). Both strains demonstrated significant modifications in fatty and mycolic acid composition at different stages of growth. Furthermore, the specific composition of C54 mycolic acids was very different in virulent log-phase cells compared with less-virulent stationary-phase cells, and the avirulent mutant lacked a C54:3 mycolate that was prominent in the virulent log-phase GUH-2P. Thus, C54:3 mycolic acid represented 2.5% of the cell wall (dry weight) in log-phase GUH-2P, but it was undetectable in the walls of GUH-2AI at the stationary phase of growth. These results suggest that certain mycolic acids are associated with virulence.     

Role of Pseudomonas aeruginosa pili in acute pulmonary infection.

The role of piliation in the development and course of acute pulmonary infection was examined using infant BALB/cByJ mice inoculated by intranasal instillation of isogenic Pil+ and Pil- mutants of Pseudomonas aeruginosa PA1244, PAK, and PAO1. The piliated strains caused more cases of pneumonia, bacteremia, and mortality than the nonpiliated strains (chi-square analysis, alpha = 0.001). The piliated strains were more often associated with severe diffuse pneumonias, while the nonpiliated organisms resulted in less severe, focal pneumonias, although these differences did not achieve statistical significance. Purified pilin protein used to inoculate the mice resulted in local inflammatory changes. The nonpiliated strain PA1244-NP was as virulent as the piliated strain PAO1, suggesting that expression of other virulence factors are also important in the development of acute pneumonia. This infant mouse model of pulmonary infection appears to be a useful system for the analysis of P. aeruginosa virulence factors involved in the pathogenesis of pneumonia.     

Location of ts defects in the genome of cold-adapted recombinant influenza A virus vaccine strains

The ts phenotype and location of ts mutations were studied in the genome of parent viruses and those obtained by recombination of cold-adapted strains A/Leningrad/134/17/57 or A/Leningrad/134/47/57 with epidemic H1N1 and H3N2 influenza A virus strains. The epidemic H1N1 and H3N2 strains under study possessed a ts phenotype and contained ts mutations in one or two genes. The ts phenotype was lost following three clonings at 40 degrees C, suggesting that influenza virus strains isolated from humans may be heterogeneous and contain virions either carrying or not carrying the ts mutations in their genomes. Two cold-adapted strains possessing a distinct ts phenotype contained ts mutations in three (A/Leningrad/134/17/57 virus after 17 passages at 25 degrees C) or in five (A/Leningrad/134/47/57 variant after 30 additional passages at 25 degrees C) genes coding for non-glycosylated proteins. When compared with cold-adapted donor strains, the recombinants had either the same set or additional ts mutations. However, no ts mutation was detected in a gene which had been inherited from the donor strain. It is suggested that, in addition to the analysis of the genome composition, in cold-adapted recombinant influenza virus strains recommended as vaccine candidates it is necessary to control the number of genes containing ts mutations.     

Antibody against the capsule of Vibrio cholerae O139 protects against experimental challenge.

Antiserum to the capsular polysaccharide of an opaque variant of Vibrio cholerae O139 strain MDO-12 recognizes capsular antigen in three different colonial variants of the strain, although the amount of recognition varies with the extent of opacity. The anti-capsular-polysaccharide serum, at subagglutinating doses, protected suckling mice against challenge with both the most opaque variant and the most translucent variant. Further studies indicated that the protection was associated with inhibition of intestinal colonization by the vibrios. These results thus highlight the potential importance of the capsule in immunoprophylaxis against cholera caused by V. cholerae O139.     

Isolation of colonial variants of Bacteroides gingivalis W50 with a reduced virulence

The spontaneous appearance of unusual colony forms was observed during prolonged growth of Bacteroides gingivalis W50 in a chemostat. Two variants were selected for further study which could be distinguished from the parent strain by the rate and intensity of pigmentation of their colonies. For example, after anaerobic incubation for 14 days, variant W50/BR1 produced brown colonies whereas those of the parent strain were black; in contrast, variant W50/BE1 did not show signs of pigmentation until incubation had continued for 21 days. In subsequent studies in the chemostat, variant W50/BE1 bred true even after prolonged growth whereas other colony forms appeared after incubation of variant W50/BR1 for 14 days. The relatedness of W50/BR1 and W50/BE1 to the parent strain was confirmed by comparisons of the whole-cell fatty-acid profiles, the patterns of pre-formed enzymes and by the metabolic end products after growth. However, the variants did differ from the parent strain in their virulence in a mouse pathogenicity model. The parent strain killed all mice given infective doses greater than 5 x 10(8) cfu whereas W50/BR1 was much less virulent (2 out of 10 mice killed and higher infective doses needed for higher mortality rates) and W50/BE1 was avirulent at all infective doses tested.     

Phenotypic variants of meningococci and their potential in phagocytic interactions: the influence of opacity proteins,pili, PilC and surface sialic acids

In previous studies we have examined the roles of meningococcal surface structures (capsule, lipopolysaccharides, pili and opacity proteins: Opa and Opc) in bacterial interactions with human epithelial, endothelial and mononuclear phagocytic cells In the current investigations, using defined derivatives of a serogroup A strain C751 and a serogroup B strain MC58, we studied the roles of these structures with human polymorphonuclear phagocytes (PMN) In addition, we examined the potential influence of the pilus-associated protein, PilC, previously known to affect epithelial cell interactions The data show that, as with monocytes, opacity proteins affect bacterial interactions with PMN and require surface sialic acids (on capsule and LPS) to be down-modulated in order to function Also, in contrast to their role in human epithelial and endothelial adherence, neither pili nor PilC expression had any effect on phagocytic cell interactions with respect to induction of chemiluminescence as well as phagocytic killing Examination of the relative influence of Opa and Opc indicated that Opa proteins are more effective than Opc in PMN interactions whereas the reverse was the case with monocytes These results suggest that Opa and Opc mediate interactions with phagocytic cells via distinct mechanisms Observations presented here and reported previously collectively show that the structural requirements of meningococci for interacting with phagocytes, in the absence of opsonins, are present in the phenotype which is often isolated from the nasopharynx (asialylated, piliated, Opa/Opc⁺) whereas the phenotype prevalent in the blood (sialyted, piliated, Opa/Opc⁺) retains the ability to adhere to endothelial cells (via pili) but appears to be refractory to interactions with phagocytic cells.     

Role of Pili in the Virulence of Neisseria gonorrhoeae

Gonococci of the colonial types that are associated with virulence, types 1 and 2, have pili that enable the bacteria both to attach in vitro to human epithelial cells and to resist phagocytosis by polymorphonuclear leukocytes. These piliated gonococci also agglutinate various mammalian and chicken erythrocytes. Gonococci of an avirulent colonial type, i.e., type 4, have no pili and neither attach to epithelial cells or erythrocytes nor resist phagocytosis. Like the type 4 bacteria, mechanically or enzymatically (trypsin) depiliated type 1 gonococci failed to attach to epithelial cells and erythrocytes and were susceptible to phagocytosis. Pili of types 1 and 2 gonococci were antigenically similar. Both type 1 gonococci and pili isolated from them induced in rabbits antibody that (i) precipitated gonococcal pili in immunodiffusion, (ii) reacted with piliated gonococci as tested by indirect immunofluorescent analysis, (iii) inhibited attachment of piliated gonococci to both human epithelial cells and erythrocytes, and (iv) opsonized piliated gonococci.     

Critical role of germ tube formation in the pathogenesis of candidal vaginitis.

A variant strain of Candida albicans incapable of hyphal production at 37 degrees C was used to study the role of germ tube formation in the pathogenesis of experimental vaginal candidiasis in rats. No difference was observed in the in vitro adherence at 25 degrees C of blastoconidia of the variant strain to vaginal epithelial cells when compared with the parent wild-type, germ tube-producing strain and multiple clinical isolates of C. albicans. However, after exposure to conditions favoring germ tube production, the adherence of the variant strain to epithelial cells was significantly less than that of germinated strains (P less than 0.01). In vivo animal studies revealed that the variant strain was less likely to result in vaginal colonization and infection than the wild-type strain and the other clinical isolates. Furthermore, infection, when established, was milder, often transient, and with significantly lower titers of cultured vaginal microorganisms obtained by lavage. Electron microscopic studies confirmed the failure of the variant strain to produce hyphae in vivo. The capacity of C. albicans to produce hyphae appears to be an important but nonessential virulence factor in the pathogenesis of candidal vaginitis.     

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

Immunity conferred by Aro- Salmonella live vaccines

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).     

RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.     

In vitro adhesion of piliated Escherichia coli to small intestinal villous epithelial cells from rabbits and the identification of a soluble 987P pilus receptor-containing fraction.

Type 987P-piliated Escherichia coli adhered in vitro to small intestinal villous epithelial cells and brush borders isolated from adult female rabbits, but not from infant rabbits. K99-piliated E. coli adhered to epithelial cells and brush borders from both adult and infant rabbits. 987P-negative and K99-negative E. coli as well as CFA/I-positive and CFA/I-negative E. coli failed to adhere to epithelial cells or brush borders from either adult or infant rabbits. CFA/II-positive and CFA/II-negative E. coli did not adhere to epithelial cells from infant rabbits. Pretreatment of adult rabbit brush borders with purified 987P pili inhibited adherence of piliated strain 987. Optimal adherence occurred after 15 min when brush borders and piliated strain 987 were incubated together at 25 degrees C or 37 degrees C in buffers at pH 7 to 8 containing 0.11 to 0.21 M total salts. A receptor-containing fraction that caused aggregation of piliated strain 987 was released into solution when brush borders were stored at 4 degrees C. Receptor activity (aggregation) remained in solution when centrifuged at 10,000 X g for 15 min, but was sedimented at 226,000 X g for 1 h. Receptor activity was abolished by periodate oxidation and pronase digestion.     

Evaluation of the Immune Response Directed Against the Salmonella Antigenic Factors O4,5 and O9

A pair of O4,5,12 and O9,12 his(+) sister transductants derived from a virulent Salmonella typhimurium parent were used as intraperitoneal and oral challenge strains to determine whether immunity directed against the O9 and O4,5 antigenic components could be detected after immunization with heat-killed vaccines containing one or the other of these antigenic components. Challenge with a mixture (ca. 1:1) of the two strains and culturing of livers and spleens at intervals indicated that the O4,5,12 strain multiplied to a greater extent than the O9,12 strain after both oral and intraperitoneal challenge of CF1 and C57BL/6J control mice. Immunization with O4,5,12 or O9,12 vaccine resulted in diminished bacterial counts from the livers and spleens for the homologous strain but had little effect on the heterologous strain. The diminution of the homologous strain was more evident after intraperitoneal challenge but was clearly demonstrable after oral challenge, particularly in the C57BL/6J mice. Of several live vaccines tested, an FOR S. typhimurium (O4,5,12) strain phenotypically smooth but avirulent seemed the most promising, suppressing the multiplication of both members of the challenge pair. After oral challenge suppression of the O4,5,12 strain was greater. These results indicate that the specific immune response directed against either the O4,5 (O-acetyl abequose) or the O9 (tyvelose) antigen is a measurable component of the overall response after both intraperitoneal and oral challenge.