Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid

A teichoic acid (TA)-like polysaccharide in Enterococcus faecalis has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from E. faecalis strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with d-Ala and kojibiose. We also detected a novel substituent at position C-2, [D-Ala-->6]-alpha-D-Glcp-(1-->2-[D-Ala-->6]-alpha-D-Glcp-1-->). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the E. faecalis strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to E. faecalis 12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.     

Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium

Enterococci are a common cause of serious infections, especially in newborns, severely immunocompromised patients, and patients requiring intensive care. To characterize enterococcal surface antigens that are targets of opsonic antibodies, rabbits were immunized with various gentamicin-killed Enterococcus faecalis strains, and immune sera were tested in an opsonophagocytic assay against a selection of clinical isolates. Serum raised against one strain killed the homologous strain (12030) at a dilution of 1:5,120 and mediated opsonic killing of 33% of all strains tested. In addition, this serum killed two (28%) of seven vancomycin-resistant Enterococcus faecium strains. Adsorption of sera with the homologous strain eliminated killing activity. The adsorbing antigens were resistant to treatment with proteinase K and to boiling for 1 h, but were susceptible to treatment with sodium periodate, indicating that the antigen inducing opsonic activity is a polysaccharide. Antibodies in immune rabbit sera reacted with a capsule-like structure visualized by electron microscopy both on the homologous E. faecalis strain and on a vancomycin-resistant E. faecium strain. The capsular polysaccharides from E. faecalis 12030 and E. faecium 838970 were purified, and chemical and structural analyses indicated they were identical glycerol teichoic acid-like molecules with a carbohydrate backbone structure of 6-alpha-D-glucose-1-2 glycerol-3-PO4 with substitution on carbon 2 of the glucose with an alpha-2-1-D-glucose residue. The purified antigen adsorbed opsonic killing activity from immune rabbit sera and elicited high titers of antibodies (when used to immunize rabbits) that both mediated opsonic killing of bacteria and bound to a capsule-like structure visualized by electron microscopy. These results indicate that approximately one-third of a sample of 15 E. faecalis strains and 7 vancomycin-resistant E. faecium strains possess shared capsular polysaccharides that are targets of opsonophagocytic antibodies and therefore are potential vaccine candidates.     

Mucosal vaccination with a multivalent, live-attenuated vaccine induces multifactorial immunity against Pseudomonas aeruginosa acute lung infection

Many animal studies investigating adaptive immune effectors important for protection against Pseudomonas aeruginosa have implicated opsonic antibody to the antigenically variable lipopolysaccharide (LPS) O antigens as a primary effector. However, active and passive vaccination of humans against these antigens has not shown clinical efficacy. We hypothesized that optimal immunity would require inducing multiple immune effectors targeting multiple bacterial antigens. Therefore, we evaluated a multivalent live-attenuated mucosal vaccination strategy in a murine model of acute P. aeruginosa pneumonia to assess the contributions to protective efficacy of various bacterial antigens and host immune effectors. Vaccines combining 3 or 4 attenuated strains having different LPS serogroups were associated with the highest protective efficacy compared to vaccines with fewer components. Levels of opsonophagocytic antibodies, which were directed not only to the LPS O antigens but also to the LPS core and surface proteins, correlated with protective immunity. The multivalent live-attenuated vaccines overcame prior problems involving immunologic interference in the development of O-antigen-specific antibody responses when closely related O antigens were combined in multivalent vaccines. Antibodies to the LPS core were associated with in vitro killing and in vivo protection against strains with O antigens not expressed by the vaccine strains, whereas antibodies to the LPS core and surface proteins augmented the contribution of O-antigen-specific antibodies elicited by vaccine strains containing a homologous O antigen. Local CD4 T cells in the lung also contributed to vaccine-based protection when opsonophagocytic antibodies to the challenge strain were absent. Thus, multivalent live-attenuated vaccines elicit multifactorial protective immunity to P. aeruginosa lung infections.     

Multicenter evaluation of use of penicillin and ampicillin as surrogates for in vitro testing of susceptibility of enterococci to imipenem

Imipenem is approved by the U.S. Food and Drug Administration (FDA) for treatment of infections caused by Enterococcus faecalis. However, there are no NCCLS guidelines for testing susceptibility of enterococci against imipenem. To assess whether or not ampicillin or penicillin could be used as a surrogate for broth microdilution (BMD) testing of imipenem versus Enterococcus species, 633 strains of E. faecalis, E. faecium, and other enterococci isolated from blood cultures of patients at three geographically distinct university hospitals were tested by the NCCLS BMD and disk diffusion (DD) methods. Using FDA susceptibility breakpoints for imipenem and NCCLS breakpoints for penicillin and ampicillin, categorical agreement (CA) for penicillin-imipenem and ampicillin-imipenem tested with E. faecalis and E. faecium by BMD was >/=94% but was /=98% and was 92% for other enterococci; CA for penicillin-imipenem was 91% for E. faecalis, 98% for E. faecium, and 87% for other enterococci. Further analysis showed that testing E. faecalis with ampicillin resulted in no false-susceptible (FS) or false-resistant (FR) results by BMD, no FS results by DD, and a single FR result by DD (0.2%), whereas testing with penicillin resulted in no FS results by BMD or DD and two FR results by BMD (0.4%). For E. faecium and other enterococci, the combination of FS and FR results was such that surrogate testing with penicillin or ampicillin appears not to be sufficiently reliable to be used clinically. We conclude that ampicillin is an accurate predictor of the in vitro activity of imipenem against E. faecalis.     

A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge.

The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections.     

Protection against fatal Pseudomonas aeruginosa burn wound sepsis by immunization with lipopolysaccharide and high-molecular-weight polysaccharide.

A murine burn wound model was employed to evaluate the relative efficacy of purified Pseudomonas aeruginosa lipopolysaccharide (LPS) and high-molecular-weight polysaccharide as protective immunogens. LPS was found to be both highly immunogenic and protective. As little as three 0.001-microgram doses elicited good immunoglobulin M and G titers and increased the mean lethal dose more than 1,000-fold. The level of protection against a live challenge correlated with antibody titers and was found to be serotype specific. An immunizing regimen which evoked only an immunoglobulin M response was still found to offer substantial protection. Immunization with a high-molecular-weight polysaccharide was also found to be protective. However, approximately 1,000-fold more high-molecular-weight polysaccharide, as compared with LPS, was needed to protect mice to an equivalent degree. Immunization with LPS was found to promote bacterial clearance and prevent establishment of bacteremia. A multivalent LPS vaccine conferred high levels of protection (110- to 53,000-fold) against eight different challenge strains of various serotypes.     

Antibodies to capsular polysaccharide and clumping factor A prevent mastitis and the emergence of unencapsulated and small-colony variants of Staphylococcus aureus in mice

The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated “escape mutants” and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections.     

Functional activity of antibodies to the group B polysaccharide of group B streptococci elicited by a polysaccharide-protein conjugate vaccine.

Group B streptococci (GBS) are a major cause of sepsis and meningitis in infants. While antibodies directed to the type-specific GBS capsule have been shown to be protective, it is less clear whether antibodies to the group B polysaccharide, a noncapsular, cell wall-associated antigen, may play a role in immunity. To investigate the functional activity of group B polysaccharide-specific antibodies, we tested sera from rabbits vaccinated with group B polysaccharide coupled to tetanus toxoid (B-TT). Anti-B-TT was weakly opsonic in vitro for a highly encapsulated type III strain, while antiserum elicited by vaccination with type III capsular polysaccharide linked to tetanus toxoid (III-TT) was a very effective opsonin. In contrast to anti-III-TT, anti-B-TT given before or after bacterial challenge was only marginally effective in protecting newborn mice against lethal infection with type III GBS. The number of C3 molecules bound to type III GBS was augmented by anti-III-TT but not by high antibody concentrations of anti-B-TT. These results suggest that the difference in opsonic activity between anti-B-TT and anti-III-TT may be due to a difference in their ability to deposit C3. In addition, the maximum number of antibody molecules bound to the bacterial surface was greater for anti-III-TT than for anti-B-TT. That anti-B-TT binds to fewer sites than anti-III-TT may explain the differences in complement activation and in opsonic and protective efficacy of antibodies to group B polysaccharide compared with antibodies to the type-specific capsular polysaccharide.     

Isolation, structure, and immunogenicity of Pseudomonas aeruginosa immunotype 4 high-molecular-weight polysaccharide.

A high-molecular-weight, immunogenic form of the lipopolysaccharide O side chain of Pseudomonas aeruginosa Fisher immunotype 4 (type 4, International Antigenic Typing System 1, Lanyi O:6) was isolated and characterized. Analysis by nuclear magnetic resonance spectroscopy confirmed the structural similarity of this high-molecular-weight polysaccharide and the type 4 O side chain. The polysaccharide was immunogenic in rabbits and mice, eliciting opsonophagocytic killing antibodies. Immunization with the polysaccharide produced significant protection against homologous challenge in both burned and granulocytopenic mice. Naturally acquired opsonic killing antibodies to type 4 polysaccharide were present in sera from unimmunized normal adults at levels comparable to postimmunization levels achieved after immunization with other type-specific polysaccharides. The specificity of the naturally occurring antibodies for the O side chain was documented by immunoblot analysis and inhibition studies. Naturally occurring polysaccharide-specific antibodies were comparable in their protective activity against live challenge in neutropenic animals to immunization-induced murine antibodies with similar specificity. These data suggest that naturally occurring serum antibody to P. aeruginosa type 4 lipopolysaccharide O side chains in most adults is not distinguishable in quantity or quality from immunization-induced antibodies in mice; evaluation of type 4-specific vaccines in humans may be complicated by this finding.     

Protective immunity induced in mice by immunization with high-molecular-weight polysaccharide from Pseudomonas aeruginosa.

A high-molecular-weight alkali-labile polysaccharide (PS) isolated from the slime of immunotype 1 Pseudomonas aeruginosa was tested for its ability to protect mice from lethal challenge with the live, homologous organism. Intraperitoneal (i.p.) injection of 10 to 25 microgram of the PS protected 60 to 70% of the mice against challenge with up to 50 50% lethal dose units. Although single immunization of mice with up to 250 microgram of PS effected protective levels of only 70%, two successive immunizations provided 100% protection. Subcutaneous and intravenous immunization with PS also provided protection to i.p. challenges with immunotype 1 P. aeruginosa, but not to i.p. challenge with immunotype 4 P. aeruginosa. Although lipopolysaccharide (LPS) was found to be more immunogenic than PS in out studies, contamination of the alkali-labile PS with LPS did not account for the protection seen. Alkali treatment (0.1 N NaOH, 37 degrees C, 2 h) of the PS destroyed its protective effectiveness, while similarly treated LPS retained its capacity for inducing immunity in mice. Adsorption and passive protection studies with sera raised to either PS or a mixture of PS and LPS indicated that antibody directed to the alkali-labile PS antigen was capable of contributing to the protection of mice against challenge with P. aeruginosa.     

Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice

The efficacy and mechanisms of protection of two live vaccines and of a protective antigen (PA) vaccine against Bacillus anthracis were studied in inbred mice. Mice that differed in their natural resistance to killing by Sterne, a non-encapsulated, toxigenic vaccine strain of B. anthracis, were used. Vaccination with live Sterne spores protected Sterne-resistant mice against challenge with the virulent Vollum 1B (V1B) strain of B. anthracis, but only at doses of Sterne greater than or equal to 0.1 50% lethal dose. The live B. subtilis recombinant strain PA2, which produces the PA component of anthrax toxin, fully protected (CBA/J) or partially protected (BALB/cJ) Sterne-resistant mice against V1B. Neither immunization with the cell-free PA vaccine nor passive administration of anti-PA antiserum protected Sterne-resistant mice against V1B. Sterne-susceptible A/J mice were not protected against V1B by either live vaccine or by the PA vaccine. However, immunization with strain PA2 induced anti-PA antibody and protected A/J mice against Sterne. A/J mice passively treated with antitoxin antibodies also survived Sterne, and survivors were then partially protected against V1B. Thus, immunity to Sterne correlated with an effective anti-PA response. Immunity to fully virulent V1B also required PA but may involve mechanisms in addition to humoral immunity.     

Role of monoclonal O-antigen antibody epitope specificity and isotype in protection against experimental mouse typhoid

A panel of 14 monoclonal antibodies with specificity for the O-antigenic polysaccharide chain of the lipopolysaccharide of the cell envelope of Salmonella typhimurium was established. The specificity of each antibody clone was determined against a set of Salmonella saccharide antigens, natural and synthetic, in passive hemagglutination and enzyme immunoassays. The monoclonal antibodies could be classified into at least five different groups: (i) O4 epitope specific, (ii) O4,12 specific, (iii) O4,12(2) specific, (iv) O5 specific, and (v) O12 specific. These specificities correspond to different structural and conformational domains of the polysaccharide chain, and often extend over more than one repeating unit (tetrasaccharide) of the polymer. The passive protection afforded by these antibodies was estimated in an experimental mouse typhoid model using S. typhimurium SH2201 for intraperitoneal challenge. Monoclonal antibodies of the IgG3 isotype were available for four of the epitope groups and were protective in the following order of activity O4 greater than O4,12 greater than O4,12(2) greater than or equal to O12. The difference between O4 and 012 antibodies was greater than 2500 fold in protective activity. Antibodies of the IgM class were highly protective irrespective of being of the O4,12 or O12 epitope specificity. Two IgA antibodies with O5 epitope specificity were not protective. The results show that both isotype and epitope specificity can be of importance for the protective ability of antibodies generated by the host.     

Comparative Evaluation of Penicillin, Ampicillin, and Imipenem MICs and Susceptibility Breakpoints for Vancomycin-Susceptible and Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium

Although imipenem has in vitro activity against Enterococcus faecalis and Food and Drug Administration-approved indications for treatment of infections caused by this microorganism, there are no NCCLS guidelines for susceptibility testing of imipenem versus enterococci. Therefore, the in vitro activities of penicillin, ampicillin, imipenem, and vancomycin against 201 blood isolates of E. faecalis and 24 blood isolates of Enterococcus faecium were compared. The susceptibility of isolates to penicillin or ampicillin accurately predicted the in vitro activity of imipenem. Since the susceptibility of enterococci to imipenem can be predicted by the results obtained by testing of penicillin or ampicillin, testing of imipenem by clinical laboratories probably is not necessary.     

Evaluation of MicroScan for identification of Enterococcus species.

Emerging drug resistance of the enterococci necessitates differentiation from group D streptococci and accurate species identification. MicroScan (Baxter Healthcare Corp., West Sacramento, Calif.) has recently developed a microdilution system for identification and antibiotic susceptibility testing of gram-positive cocci. To evaluate the ability of this system to identify Enterococcus species, 100 isolate identified as enterococci by MicroScan were tested by conventional media and 60 isolates of streptococci were tested by MicroScan. Incubation times for conventional and MicroScan methods were 96 and 18 to 24 h, respectively. For 94 strains of enterococci (77 Enterococcus faecalis, 14 Enterococcus faecium, and 3 Enterococcus durans), identification by conventional media and MicroScan agreed. Of the remaining six isolates, four were identified as E. faecalis and two were identified as E. durans by MicroScan, whereas by conventional media the four E. faecalis isolates were identified as Enterococcus solitarius and the two E. durans isolates were identified as Enterococcus hirae. None of the 60 streptococci were identified as enterococci. MicroScan is a reliable method for identification of the commonly encountered enterococcal species E. faecalis and E. faecium; however, modifications of the system are necessary for identification of other Enterococcus species.     

Saliva-mediated aggregation of Enterococcus faecalis transformed with a Streptococcus sanguis gene encoding the SSP-5 surface antigen.

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.     

Sex pheromone response, clumping, and slime production in enterococcal strains isolated from occluded biliary stents

Bile-resistant bacteria, particularly gram-positive Enterococcus faecalis and Enterococcus faecium, play an important role in biliary stent occlusion, because their sessile mode of growth protects them against host defenses and antimicrobial agents. Twelve E. faecalis and seven E. faecium strains isolated from occluded biliary stents have been investigated for slime production, presence of aggregation substance genes, and ability to adhere to Caco-2 cells. Ten isolates were strong producers of slime, and seven isolates produced clumps when exposed to pheromones of E. faecalis JH2-2 and/or OG1RF. The small E. faecium clumps differed from the large clumps of E. faecalis and were similar to those of E. faecium LS10(pBRG1) carrying a pheromone response plasmid. After induction with pheromones, the adhesion to Caco-2 cells of clumping-positive strains was found to increase from two- to fourfold. Amplicons of the expected size were detected in three clumping-positive and three clumping-negative E. faecalis isolates by using primers (agg) internal to a highly conserved region of the E. faecalis pheromone response plasmids pAD1, pPD1, and pCF10 and primers internal to prgB of the E. faecalis plasmid pCF10. The agg/prgB-positive E. faecalis strains were also positive in Southern hybridization experiments with a prgB-specific probe. No PCR products were obtained with the same primers from four clumping-positive isolates (one E. faecalis and three E. faecium strains), which were also Southern hybridization negative. Our results demonstrate that slime production and pheromone response are both present in isolated enterococci, suggesting that clinical strains with these features might have a selective advantage in colonizing biliary stents.     

Characteristics of the protective response of BALB/c mice immunized with a purified Plasmodium yoelii schizont antigen

The response of BALB/c mice to immunization with a 230,000 mol. wt protective antigen purified from the blood stages of Plasmodium yoelii was analysed. The protective response to immunization was adjuvant-dependent, and saponin was found to be the most effective adjuvant tested. The intraperitoneal route was found to be superior to the subcutaneous route for protective immunization. Maximal protection was achieved using two immunizations with greater than 12.5 micrograms of antigen plus saponin, but even under these conditions challenge infection developed normally for 5 days, followed by the appearance of crisis forms and rapid clearance of parasites. Effective immunization induced high antibody titres, but serum from immunized mice was not protective on passive transfer. On the other hand, hyperimmune serum from mice recovered and rechallenged with live P. yoelii was protective on passive transfer, even though the titre of antibody specific for the 230,000 mol. wt antigen in this serum was the same as that in the serum from mice immunized with the purified antigen. It was concluded that the 230,000 mol. wt antigen may provide protection against P. yoelii via the cell-mediated immune effector pathway described by Playfair et al. (1979), and that hyperimmune serum contains protective antibodies specific for P. yoelii antigens other than the 230,000 mol. wt antigen.     

Fatal meningitis caused by vancomycin-resistant enterococci: report of two cases from south India

Vancomycin-resistant enterococci rarely cause meningitis and present a therapeutic challenge. Antimicrobial susceptibility testing was done for strains of Enterococcus species isolated from CSF samples of patients with meningitis by phenotypic methods. Multiplex polymerase chain reaction was performed to determine the genetic basis of vancomycin resistance of such isolates. We report here two cases of enterococcal meningitis caused by vancomycin-resistant Enterococcus species. One of the isolates was identified as Enterococcus faecalis and the other as Enterococcus gallinarum. We also report the simultaneous presence of vanC1 and vanA resistance genes in the strain of E. gallinarum. To the best of our knowledge, this is the first report of vanA resistance gene in an isolate of E. gallinarum from the Indian subcontinent. This is also the first Indian report of vancomycin-resistant Enterococcus causing meningitis.     

Immunology of the infant rat experimental model of Haemophilus influenzae type b meningitis.

The age-related acquisition of serum anticapsular and bactericidal antibodies to Haemophilus influenzae type b observed in rats was similar to that of humans. The antigenic source for this "natural" immunity was not identified since neither pharyngeal infection with H. influenzae b nor enteric colonization by cross-reacting bacteria was detected. Infant rats surviving H. influenzae b bacteremia failed to respond immunologically to the capsular polysaccharide. However, surviving rats demonstrated no impairment of immune responsiveness to this antigen after subsequent immunization with live bacteria in adulthood. In passive protection experiments, antibodies directed against the type b capsular polysaccharide represented the major protective specificity. However, a small protective effect of antibodies to noncapsular antigens also appeared to have been demonstrated.     

In vitro resistance to human platelet microbicidal protein among urethral staphylococcal and enterococcal isolates with its correlation with prostatitis

The study was carried out to test the in vitro activity of human platelet microbicidal protein (hPMP) on most commonly isolated urethral pathogens and compare the same with clinical isolates from cases of chronic prostatitis (CP). Urethral isolates of Staphylococcus aureus (n=19), coagulase negative staphylococci (n=40) and Enterococcus faecalis (n=16) from patients with or without CP were tested. The hPMP susceptibility of bacterial strains was determined by exposing bacterial cells to serial dilutions of hPMP. A significantly higher proportion of CP-strains of coagulase negative staphylococci (91.3% vs 5.88%) was resistant to hPMP than was that of non-CP strains (P S.aureus studied, 77.8% were considered resistant to the bactericidal action of hPMP. All nine CP-strains of E.faecalis were highly resistant to hPMP. Most non-CP urethral isolates of S.aureus, coagulase negative staphylococci and E.faecalis were susceptible to the bactericidal action of hPMP, while CP isolates of all species were significantly more resistant to hPMP. Data from the present study may have significant implications in understanding the pathogenesis of CP.