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Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and death worldwide. Immunization with conjugated pneumococcal vaccine has lowered the colonization rate and consequently invasive diseases by inducing serotype-specific antibodies. However, many of the current pneumonia cases result from infection by serotype strains not included in the vaccine. In this study, we asked if cross-protection against lung infection by heterologous strains can be induced, and investigated the underlying immune mechanism. We found that immune mice recovered from a prior infection were protected against heterologous Sp strains in the pneumonia challenge model, as evident by accelerated bacterial clearance, reduced pathology, and apoptosis of lung epithelial cells. Sp infection in the lung induced strong T-helper type 17 (Th17) responses at the lung mucosal site. Transfer of CD4+T cells from immune mice provided heterologous protection against pneumonia, and this protection was abrogated by interleukin-17A (IL-17A) blockade. Transfer of memory CD4+T cells from IL-17A-knockout mice failed to provide protection. These results indicate that memory Th17 cells had a key role in providing protection against pneumonia in a serotype-independent manner and suggest the feasibility of developing a broadly protective vaccine against bacterial pneumonia by targeting mucosal Th17 T cells.

The genus Mycobacterium contains two of the most important human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, the etiological agents of tuberculosis and leprosy, respectively. Other mycobacteria are mostly saprophytic organisms, living in soil and water, but some of them can cause opportunistic infections. The increasing incidence of tuberculosis as well as infections with non-tuberculous mycobacteria (NTM) in immuno-compromised patients has renewed interest in molecular mechanisms of drug resistance in these pathogens. Mycobacteria show a high degree of intrinsic resistance to most common antibiotics. For instance, species from the M. tuberculosis complex (MTC) are intrinsically resistant to macrolides. Nevertheless, some semi-synthetic macrolides as clarithromycin, azithromycin and most recently the ketolides, are active against NTM and widely used for infection treatment. However, shortly after the introduction of these new drugs, resistant strains appeared due to mutations affecting the macrolide target, the ribosome. The mycobacterial cell wall is considered to be a major factor in promoting the natural resistance of mycobacteria to various antibiotics. However, recent data show that specific macrolide resistance determinants (erm genes) are present in some species. This mini-review summarizes the current knowledge on the natural and acquired macrolide resistance in mycobacteria, gives an overview of potential mechanisms implicated in the intrinsic resistance and of macrolide resistance determinants in mycobacteria.

Using the crystal structure of an inhibitor complexed with the serine protease thrombin (PDB code 1UVT) and the functional group definitions contained within the Catalyst software, a representation of the enzyme's active site was produced (structure-based pharmacophore model). A training set of 16 homologous non-peptide inhibitors whose conformations had been generated in continuum solvent (MacroModel) and clustered into conformational families (XCluster) was regressed against this pharmacophore so as to obtain a 3D-QSAR model. To test the robustness of the resulting QSAR model, the synthesis of a series of non-peptide thrombin inhibitors based on arylsuphonyl derivatives of an aminophenol ring linked to a pyridyl-based S1 binding group was undertaken. These compounds served as a test set (20−24). The crystal structure for the novel symmetrical disulfonyl compound 24, in complex with thrombin, has been solved. Its calculated binding mode is in general agreement with the crystallographically observed one, and the predicted Ki value is in close accord with the experimental value.

The genus Mycobacterium contains two of the most important human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, the etiologic agents of tuberculosis and leprosy, respectively. Other mycobacteria are mostly saprophytic organisms, living in soil and water, but some of them can cause opportunistic infections. The increasing incidence of tuberculosis as well as infections with non-tuberculous mycobacteria (NTM) in AIDS patients has renewed interest in molecular mechanisms of drug resistance in these pathogens. Mycobacteria show a high degree of intrinsic resistance to most common antibiotics. For instance, species from the M. tuberculosis complex (MTC) are intrinsically resistant to macrolides. Nevertheless, some semi-synthetic macrolides as the erythromy cin derivatives clarithromycin, azithromycin and most recently the ketolides, are active against NTM, particularly Mycobacterium avium, and some of them are widely used for infection treatment. However, shortly after the introduction of these new drugs, resistan strains appeared due to mutations in the macrolide target, the ribosome. The mycobacterial cell wall with it specific composition and structure is considered to be a major factor in promoting the natural resistance of mycobacteria to various antibiotics. However, to explain the difference in macrolide sensitivity between the MTC and NTM, the synergistic contribution of a specific resistance mechanism might be required, in addition to possible differences in cell wall permeability. This mini-review summarizes the current knowledge on the natural and acquired macrolide resistance in mycobacteria, gives an overview of potential mechanisms implicated in the intrinsic resistance and brings recent data concerning a macrolide resistance determinant in the MTC.

Nasopharyngeal carriage is the reservoir from which most disease with Streptococcus pneumoniae arises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes, htrA and spoJ, that was specifically and strongly downregulated in a DeltaciaRH-null mutant. A S. pneumoniae strain lacking the htrA gene encoding a putative serine protease, but not one lacking spoJ, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the DeltaciaRH strain.

ABSTRACTNasopharyngeal carriage is the reservoir from which most disease with Streptococcus pneumoniaearises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes, htrAand spoJ, that was specifically and strongly downregulated in a ΔciaRH-null mutant. A S. pneumoniaestrain lacking the htrAgene encoding a putative serine protease, but not one lacking spoJ, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the ΔciaRHstrain.

Most isolates of Streptococcus pneumoniae are mixed populations of transparent (T) and opaque (O) colony phenotypes. Differences in the production of capsular polysaccharide (CPS) between O and T variants were accentuated by changes in the environmental concentration of oxygen. O variants demonstrated a 5.2- to 10.6-fold increase in amounts of CPS under anaerobic compared to atmospheric growth conditions, while CPS production remained low under all conditions for T variants. Increased amounts of CPS in O compared to T pneumococci were associated with increased expression of cps-encoded proteins. The inhibitory effect of oxygen on expression of CPS in O variants correlated with decreased tyrosine phosphorylation of CpsD, a tyrosine kinase and regulator of CPS synthesis. Modulation of CpsD expression and its activity by tyrosine phosphorylation may allow the pneumococcus to adapt to the requirements of both colonization, where decreased CPS allows for adherence, and bacteremia, where increased CPS may be required to escape from opsonic clearance. In patients with invasive infection, paired isolates from the same patient were shown to have predominantly a T colony phenotype without phosphotyrosine on CpsD when cultured from the nasopharynx, and an O phenotype that phosphorylates CpsD in response to oxygen when cultured from the blood. Differences in the availability of oxygen, therefore, may be a key factor in allowing for the selection of distinct phenotypes in these two host environments.

The method of structure-based pharmacophores for use in 3D-QSAR as implemented by Gillner and Greenidge (6) is further examined. Conformational models are generated using both Catalyst (3) and Macromodel (7). K i estimates obtained with the pharmacophore models are compared with observed values for a set of 4-aminopyridine thrombin inhibitors (8).

The method of structure-based pharmacophores for use in 3D-QSAR as implemented by Gillner and Greenidge (6) is further examined. Conformational models are generated using both Catalyst (3) and Macromodel (7). K i estimates obtained with the pharmacophore models are compared with observed values for a set of 4-aminopyridine thrombin inhibitors (8).

ABSTRACTPhase variation in the colonial opacity of Streptococcus pneumoniaehas been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal disease. Additionally, the synergistic effects of influenza A virus and S. pneumoniaein the development of otitis media (OM) have been reported. This study examined the ability of opaque or transparent S. pneumoniaefrom the same strain in combination with an antecedent influenza A virus infection to colonize the nasopharynx and invade the middle ear in the chinchilla model. Our data indicated that there was no significant difference in the level of nasopharyngeal colonization and induction of OM between the opaque and transparent variants unless there was a prior challenge with influenza A virus. Subsequent to influenza A virus infection, there was a significant difference between the variants in the ability to colonize and persist in the nasopharynx and middle ear. The concentrations of the opaque variant in nasopharyngeal-lavage samples and middle-ear fluid remained consistently higher than those of the transparent variant for 10 days postinoculation. Data from this study indicate that the effects of influenza A virus on the pathogenesis of experimental S. pneumoniae-induced OM differ depending on the opacity phenotype involved.

Streptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of Helicobacter pylori. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM of Lactocobacillus paracasei, a member of the family of peptidyl-prolyl cis/trans isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since a spxB-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803-813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.

An inverse correlation between colonization of the human nasopharynx by Streptococcus pneumoniae and Haemophilus influenzae, both common upper respiratory pathogens, has been reported. Studies were undertaken to determine if either of these organisms produces substances which inhibit growth of the other. Culture supernatants from S. pneumoniae inhibited growth of H. influenzae, whereas culture supernatants from H. influenzae had no effect on the growth of S. pneumoniae. Moreover, coculture of S. pneumoniae and H. influenzae led to a rapid decrease in viable counts of H. influenzae. The addition of purified catalase prevented killing of H. influenzae in coculture experiments, suggesting that hydrogen peroxide may be responsible for this bactericidal activity. H. influenzae was killed by concentrations of hydrogen peroxide similar to that produced by S. pneumoniae. Hydrogen peroxide is produced by the pneumococcus through the action of pyruvate oxidase (SpxB) under conditions of aerobic growth. Both an spxB mutant and a naturally occurring variant of S. pneumoniae, which is downregulated in SpxB expression, were unable to kill H. influenzae. A catalase-reversible inhibitory effect of S. pneumoniae on the growth of the respiratory tract pathogens Moraxella catarrhalis and Neisseria meningitidis was also observed. Elevated hydrogen peroxide production, therefore, may be a means by which S. pneumoniae is able to inhibit a variety of competing organisms in the aerobic environment of the upper respiratory tract.

The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galalpha1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of H. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galalpha1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occurring bactericidal activity for unencapsulated H. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

A number of pathogens of the upper respiratory tract express an unusual prokaryotic structure, phosphorylcholine (ChoP), on their cell surface. We tested the hypothesis that ChoP, also found on host membrane lipids in the form of phosphatidylcholine, acts so as to decrease killing by antimicrobial peptides that target differences between bacterial and host membranes. In Haemophilus influenzae, ChoP is a phase-variable structure on the oligosaccharide portion of the lipopolysaccharide (LPS). There was a bactericidal effect of the peptide LL-37/hCAP18 on a nontypeable H. influenzae strain, with an increasing selection for the ChoP(+) phase as the concentration of the peptide was raised from 0 to 10 microgram/ml. Moreover, constitutive ChoP-expressing mutants of unrelated strains showed up to 1,000-fold-greater survival compared to mutants without ChoP. The effect of ChoP on resistance to killing by LL-37/hCAP18 was dependent on the salt concentration and was observed only when bacteria were grown in the presence of environmental choline, a requirement for the expression of ChoP on the LPS. Further studies established that there is transcription of the LL-37/hCAP18 gene on the epithelial surface of the human nasopharynx in situ and inducible transcription in epithelial cells derived from the upper airway. The presence of highly variable amounts of LL-37/hCAP18 in normal nasal secretions (<1.2 to >80 microgram/ml) was demonstrated with an antibody against this peptide. It was concluded that ChoP alters the bacterial cell surface so as mimic host membrane lipids and decrease killing by LL-37/hCAP18, an antimicrobial peptide that may be expressed on the mucosal surface of the nasopharynx in bactericidal concentrations.

Background & Aims: Adenomatous polyps are by definition dysplastic and pathologically indistinguishable from the dysplasia-associated lesion or mass (DALM) described in 1981. Yet, adenomatous polyps in noncolitic colons are usually removed definitively endoscopically, whereas DALMs are regarded as harbingers of colon cancer, mandating colectomy. Methods: Since 1988, all of our patients with chronic ulcerative or Crohn's colitis and dysplastic polyps and no coexistent dysplasia in flat mucosa underwent colonoscopic polypectomy. Biopsy specimens were obtained also adjacent to polypectomy sites, from strictures, and throughout the colon at 10-cm intervals. Follow-up colonoscopies and biopsies were performed within 6 months after polypectomy and yearly thereafter. Results: Colonoscopy in 48 patients with chronic colitis (mean duration, 25.4 years) resected 70 polyps (60 in colitic and 10 in noncolitic mucosa). Polyps were detected on screening colonoscopies (29%) and on surveillance (71%). Pathology was tubular adenoma in all polyps from noncolitic mucosa and low-grade dysplasia (57), high-grade dysplasia (2), or carcinoma (1) in polyps from colitic mucosa. Subsequent colonoscopies (mean follow-up, 4.1 years) revealed additional polyps in 48% but no carcinomas. Surgical resection (6 patients) for recurrent polyps confirmed colonoscopic findings. No dysplasia or cancers in flat mucosa were found at surgery or on follow-up colonoscopies. Conclusions: In patients with chronic colitis who have no dysplasia in flat mucosa, colonoscopic resection of dysplastic polyps can be performed effectively, just as in noncolitic colons. GASTROENTEROLOGY 1999;117:1295-1300

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.

Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.

Summary Microsporidia form in the xenomas with their hosts a special type of relation. The host cell and the parasite represent together a physiological and morphological unit. In this relation the sporonts of Pleistophora debaisieuxi cause divisions of fat body cells of blackflies to an extent that each parasite sporont is finally closed in a specific host cell, the xenocyte, with a permanent functioning nucleus and a cytoplasmic coat which remains persistent during the whole sporogony of the microsporidian.

Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization.

We examined whether outer membrane protein A (OmpA) contributes to gram-negative pathogenesis by determining the effect of mutagenesis of ompA in a virulent Escherichia coli K-1 isolate. An OmpA mutant was generated by insertion of the transposon TnphoA, which was genetically modified to increase the efficiency of its delivery by conjugation. The mutant was less virulent than its parent strain in two models of E. coli K-1 infection. Equal inocula of the OmpA+ and OmpA- strains fed to neonatal rats resulted in a sevenfold-greater incidence of bacteremia at 72 h from the OmpA+ strain. The lethal effect of the OmpA- mutant was significantly less than that of the OmpA+ parent strain when inoculated onto the chorioallantoic membrane of 10-day embryonated chick eggs. There was, however, no difference between strains in growth characteristics under physiologic conditions, either in rat serum or in unembryonated chick eggs. In the presence of a 10-day chick embryo, there was a 10-fold increase in the survival and growth of the OmpA+ strain. Correction of the mutation in ompA with an E. coli K-12 ompA gene restored a level of virulence equivalent to that of the parent strain. The ompA mutant was more sensitive to the bactericidal effect of pooled human serum by the classical pathway of complement activation. These results suggest that OmpA contributes to E. coli K-1 pathogenesis by a mechanism which may involve increased serum resistance.

Abstract: Tolypocladium cylindrosporum Gams isolated fromAedes australis Erichson in New Zealand, and another strain isolated from soil in Czechoslovakia have shown pathogenicity for larvae of mosquitoes. The infection was transmited with conidia suspended in water and hyphae entered the body of larvae from the midgut. First symptoms of the infection appear 5 days after infection; in 7 days all exposed larvae were killed. Pupae and adult mosquitoes were not infected. Both strains grow and sporulate well on Sabouraud's agar. Somme morphological data show the variability of hyphae, conidiophores and conidia.

Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but is unclear what microbial factors mediate traversal of E. coli across the blood-brain barrier. Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E. coli K1 invasion of brain microvascular endothelial cells (BMEC). The invasive capability of the OmpA+ strains was 25- to 50-fold greater than that of OmpA- strains, and the invasive capability of OmpA- strains was restored to the level of the OmpA+ strain by complementation with the OmpA+ E. coli into BMEC. Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA+ E. coli invasion, suggesting that these two sequences represent the OmpA domains involved in E. coli invasion of BMEC. These findings suggest that OmpA is the first microbial structure identified to enhance E. coli invasion of BMEC, an important event in the pathogenesis of E. coli meningitis.

Zusammenfassung: Die permanenten Probleme mit dem großen achtzähnigen Fichtenborkenkäfer,Ips typographus L., in mittel- und nordeuroäischen Fichtenwäldern waren der Anlaß für qualitative und quantitative Erhebungen zum Pathogen-Komplex bei dieser Borkenkäfer-Art von einem außeralpinen Standort in SW-Deutschland. Zu diesem Zweck wurden erwachsene Käfer in Einzelsektionen im Lichtmikroskop untersucht. Zusätzlich wurde das Käfer-Geschlecht bestimmt. Bei der Auswertung wurden drei Untersuchungsgruppen unterschieden: 1. Käfer, vor dem Schlüpfen (aus den Rinden herausgelöst), 2. Käfer, die in der ersten Schlüpfwoche- und 3. Käfer, die in der zweiten Schlüpfwoche selbsttätig geschlüpft waren.Bei den Untersuchungen konnten neben den bereits bekannten Pathogen-Arten, demI. typographus-Entomopoxvirus, den MicrosporidienChytridiopsis typographis, Nosema typographi undGregarina typographi auch ovale Sporen gefunden werden, bei denen es sich vermutlich um Neogregarinen handelt. An Parasiten konnten die NematodenContortylenchus diplogaster undCryptaphelenchus macrogaster macrogaster sowie in einem Fall Hymenoptern-Larven der ArtTomicobia seitneri gefunden werden. Bei den drei Untersuchungsgrappen konnten zum Til beträchtliche Unterschiede in der Häufigkeit der einzelnen Pathogen-Arten beobachtet werden. So war die Anzahl der Pathogene, bei den selbsttätig geschlüpften Käfern erheblich höher als bei jenen, die aus den Rinden herausgelöst worden waren. Darüber hinaus war die Anzahl infizierter Käfer in der zweiten Schlüpfwoche nochmals deutlch höher als in der ersten. Ebenso war die Anzahl infizierter männlicher Käfer meist größer als die der weiblichen Käfer.Die Ergebnisse werden mit früheren Untersuchungen beiI. typographus von Standorten aus den Kern- und Randgebieten der Ostalpen in Österreich sowie solchen aus der tschechischen Republik verglichen. Die Verbreitung und Häufigkeit der Pathogene wird aus der Sicht ihrer möglichen Wirkung diskutiert.

Phosphorylcholine (ChoP) is a component of the teichoic acids of Streptococcus pneumoniae and has been recently identified on the lipopolysaccharide of Haemophilus influenzae, also a major pathogen of the human respiratory tract. Other gram-negative pathogens that frequently infect the human respiratory tract were surveyed for the presence of the ChoP epitope as indicated by binding to monoclonal antibodies (MAbs) recognizing this structure. The ChoP epitope was found on a 43-kDa protein on all clinical isolates of Pseudomonas aeruginosa examined and on several class I and II pili of Neisseria meningitidis. The specificity of the anti-ChoP MAb was demonstrated by the inhibition of binding in the presence of ChoP but not structural analogs. As in the case of H. influenzae, the expression of this epitope was phase variable on these species. In P. aeruginosa, this epitope was expressed at detectable levels only at lower growth temperatures. Expression of the ChoP epitope on piliated neisseriae displayed phase variation, both linked to pilus expression and independently of fully piliated bacteria.

Choline, although not a nutritional requirement for Haemophilus influenzae, is taken up from the growth medium and incorporated into its lipopolysaccharide (LPS). Incorporated choline is in the form of phosphorylcholine (ChoP) based on the reactivity with the monoclonal antibody with specificity for this structure, TEPC-15. Incorporation of [3H]choline from the growth medium and expression of the TEPC-15 epitope undergo high-frequency phase variation, characteristic of other LPS structures in this species. The expression and phase variation of ChoP require a previously identified locus involved in LPS biosynthesis, lic1. The first gene in lic1, licA, contains a translational switch based on variation in the number of intragenic tandem repeats of the sequence 5'-CAAT-3'. The full-length LicA polypeptide resembles choline kinases of eucaryotes, suggesting that the pathway for choline incorporation into the H. influenzae glycolipid has similarities to the pathway for choline incorporation in eucaryotic lipid synthesis. The display of ChoP, a host-like structure, renders the organism more rather than less susceptible to the bactericidal activity of human serum. The increased serum sensitivity of variants with ChoP correlates with higher serum immunoglobulin G titers to LPS containing this structure. ChoP appears to be a cell surface feature common to a number of pathogens of the human respiratory tract, including Streptococcus pneumoniae and mycoplasmas. In the case of H. influenzae, its primary contribution to pathogenesis does not appear to be antigenic variation to evade host humoral clearance mechanisms.

The pneumococcus expresses a protease that hydrolyzes human immunoglobulin A1 (IgA1). A gene for IgA1 protease was identified from a plasmid library of pneumococcal DNA because of the effect of its overexpression on the colony morphology of Streptococcus pneumoniae. The deduced 1,964-amino-acid sequence is highly homologous to that of the IgA1 protease from Streptococcus sanguis. The similarity to the S. sanguis enzyme and the presence of a putative zinc-binding site suggest that the pneumococcal enzyme is a metalloprotease. The two streptococcal sequences differ in a hydrophilic region with 10 tandem repeats of a 20-mer in S. sanguis, which is replaced by a similar but less repetitive sequence in S. pneumoniae. Antiserum reactive with the pneumococcal IgA1 protease was used to demonstrate that the majority of the protein is cell associated. The expression and function of this gene were confirmed by insertional mutagenesis. Interruption of the chromosomal gene resulted in loss of expression of an approximately 200-kDa protein and complete elimination of detectable IgA1 protease activity.

Phase variants in colonial opacity of pneumococci differ in the ability to colonize the nasopharynx of infant rats. To explain this observation at a cellular level, we compared the ability of opacity variants to adhere to buccal epithelial cells, type II pneumocytes, or vascular endothelial cells and to the glycoconjugates that represent the cognate receptors at each of these sites. The transparent phenotype was associated with enhanced adherence to buccal cells (approximately 100%) and their receptor relative to that of the opaque variants. Only modest differences in adherence (< 45%) were demonstrated to resting lung and vascular cells. In contrast, adherence of transparent variants increased by 90% to lung cells stimulated with interleukin-1 and by 130% to endothelial cells stimulated with tumor necrosis factor. In contrast, cytokine stimulation did not influence the adherence of opaque pneumococci. This difference correlated with the unique ability of transparent variants to adhere to immobilized GlcNAc and to cells bearing transfected platelet-activating factor receptors. These results suggest that the mechanism of enhanced colonization of the nasopharynx in vivo by transparent as compared with opaque phase variants involves a greater ability to adhere to both GlcNAc beta 1-3Gal on buccal epithelial cells and GlcNAc and PAF receptors on cytokine-activated, as opposed to resting, lung and endovascular cells.

The pneumococcus expresses a protease that hydrolyzes human immunoglobulin A1 (IgA1). A gene for IgA1 protease was identified from a plasmid library of pneumococcal DNA because of the effect of its overexpression on the colony morphology of Streptococcus pneumoniae. The deduced 1,964-amino-acid sequence is highly homologous to that of the IgA1 protease from Streptococcus sanguis. The similarity to the S. sanguis enzyme and the presence of a putative zinc-binding site suggest that the pneumococcal enzyme is a metalloprotease. The two streptococcal sequences differ in a hydrophilic region with 10 tandem repeats of a 20-mer in S. sanguis, which is replaced by a similar but less repetitive sequence in S. pneumoniae. Antiserum reactive with the pneumococcal IgA1 protease was used to demonstrate that the majority of the protein is cell associated. The expression and function of this gene were confirmed by insertional mutagenesis. Interruption of the chromosomal gene resulted in loss of expression of an approximately 200-kDa protein and complete elimination of detectable IgA1 protease activity.

Phase variants in colonial opacity of pneumococci differ in the ability to colonize the nasopharynx of infant rats. To explain this observation at a cellular level, we compared the ability of opacity variants to adhere to buccal epithelial cells, type II pneumocytes, or vascular endothelial cells and to the glycoconjugates that represent the cognate receptors at each of these sites. The transparent phenotype was associated with enhanced adherence to buccal cells (approximately 100%) and their receptor relative to that of the opaque variants. Only modest differences in adherence (< 45%) were demonstrated to resting lung and vascular cells. In contrast, adherence of transparent variants increased by 90% to lung cells stimulated with interleukin-1 and by 130% to endothelial cells stimulated with tumor necrosis factor. In contrast, cytokine stimulation did not influence the adherence of opaque pneumococci. This difference correlated with the unique ability of transparent variants to adhere to immobilized GlcNAc and to cells bearing transfected platelet-activating factor receptors. These results suggest that the mechanism of enhanced colonization of the nasopharynx in vivo by transparent as compared with opaque phase variants involves a greater ability to adhere to both GlcNAc beta 1-3Gal on buccal epithelial cells and GlcNAc and PAF receptors on cytokine-activated, as opposed to resting, lung and endovascular cells.

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is able to display an extensive repertoire of different LPS structures through the loss and acquisition of multiple oligosaccharide epitopes in various combinations. This marked heterogeneity of LPS molecules has complicated the analysis of the structure of LPS and its role in pathogenesis. A genomic library was screened for the ability to transform H. influenzae to express novel LPS epitopes defined by reactivity with oligosaccharide specific monoclonal antibodies. A chromosomal locus, lic-1, involved in expression of at least three different epitopes (recognized by monoclonal antibodies 4C4, 12D9, and 6A2), was identified on a 5.6-kilobase restriction endonuclease fragment. Transformation of H. influenzae with subclones from within lic-1 was used to generate a series of isogenic and phenotypic variants. All transformants displayed phase variation for their newly acquired epitopes. Altered binding specificities of LPS with monoclonal antibodies correlated with changes in sugar compositional analysis. The expression of two epitopes was eliminated by introduction of site-specific mutations in lic-1, confirming the role of lic-1 in oligosaccharide biosynthesis.

Phase variation of lipopolysaccharide epitopes of an Haemophilus influenzae serotype b strain (strain RM.7004) occurs through a mechanism which depends on multiple tandem repeats of the DNA sequence 5'-CAAT-3' situated within the chromosomal locus lic1. We report here that the same tetranucleotide repeats are also found in two other genomic loci (lic2 and lic3) of RM.7004. Similar to lic1, there are multiple tandem repeats of 5'-CAAT-3' present at the 5' ends of long open reading frames in lic2 and lic3. Variation in the number of repeats of CAAT, by shifting the upstream initiation codons in or out of phase with the remainder of the open reading frame, could switch on or off the translation of downstream genes. Similar to previously reported findings for lic1, site-directed mutations in the open reading frame downstream (3') from the repeats of CAAT in lic2 abolished phase variation and identified DNA sequences required for the expression of additional oligosaccharide epitopes. When we used an oligonucleotide comprising five repeats of CAAT or DNA sequences specific for lic1, lic2, and lic3 as probes, a survey of other encapsulated H. influenzae strains (serotypes a through f) and nontypable H. influenzae strains (including biotype aegyptius) showed that the chromosome of H. influenzae can have from two to five regions which contain multiple tandem repeats of CAAT in addition to other sequences which hybridize to lic1 and lic2.

Choline, although not a nutritional requirement for Haemophilus influenzae, is taken up from the growth medium and incorporated into its lipopolysaccharide (LPS). Incorporated choline is in the form of phosphorylcholine (ChoP) based on the reactivity with the monoclonal antibody with specificity for this structure, TEPC-15. Incorporation of [3H]choline from the growth medium and expression of the TEPC-15 epitope undergo high-frequency phase variation, characteristic of other LPS structures in this species. The expression and phase variation of ChoP require a previously identified locus involved in LPS biosynthesis, lic1. The first gene in lic1, licA, contains a translational switch based on variation in the number of intragenic tandem repeats of the sequence 5'-CAAT-3'. The full-length LicA polypeptide resembles choline kinases of eucaryotes, suggesting that the pathway for choline incorporation into the H. influenzae glycolipid has similarities to the pathway for choline incorporation in eucaryotic lipid synthesis. The display of ChoP, a host-like structure, renders the organism more rather than less susceptible to the bactericidal activity of human serum. The increased serum sensitivity of variants with ChoP correlates with higher serum immunoglobulin G titers to LPS containing this structure. ChoP appears to be a cell surface feature common to a number of pathogens of the human respiratory tract, including Streptococcus pneumoniae and mycoplasmas. In the case of H. influenzae, its primary contribution to pathogenesis does not appear to be antigenic variation to evade host humoral clearance mechanisms.

Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but is unclear what microbial factors mediate traversal of E. coli across the blood-brain barrier. Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E. coli K1 invasion of brain microvascular endothelial cells (BMEC). The invasive capability of the OmpA+ strains was 25- to 50-fold greater than that of OmpA- strains, and the invasive capability of OmpA- strains was restored to the level of the OmpA+ strain by complementation with the OmpA+ E. coli into BMEC. Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA+ E. coli invasion, suggesting that these two sequences represent the OmpA domains involved in E. coli invasion of BMEC. These findings suggest that OmpA is the first microbial structure identified to enhance E. coli invasion of BMEC, an important event in the pathogenesis of E. coli meningitis.

When colonies of encapsulated isolates of Streptococcus pneumoniae are viewed with oblique, transmitted light on a transparent surface, they are heterogeneous in appearance because of variation in opacity. There is spontaneous phase variation among at least three discernible phenotypes at frequencies from 10(-3) to 10(-6). The ability to detect differences in opacity varies according to serotype, but variation is independent of capsule expression. Electron microscopy shows no difference in chain length but suggests that autolysis occurs earlier in the growth of the transparent variant. There was no identifiable difference in membrane protein profiles of opaque and transparent variants of the same strain. In an infant rat model of nasopharyngeal carriage, there was no significant colonization by opaque variants. Efficient and stable colonization by the transparent variants was observed, suggesting a selective advantage for this phenotype in the nasopharynx. In contrast, there was no difference in the incidence of bacteremia or in the 50% lethal dose among the variants following their intraperitoneal inoculation. These results suggest that phase variation which is marked by differences in colonial morphology may provide insight into the interaction of the pneumococcus with its host.

Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization.

Zusammenfassung 1.Die Lungen einseitig vagotomierter Ratten bieten vollkommen normalen Sektionsbefund, die Lungen doppelseitig vagotomierter zeigen Ödem und fleckförmige Hyperämie nebst Blutungen in Alveolen und Septen.2.Hyperämie der Lungen kann unter Bedingungen, die zur Lungen-schädigung führen, Ödem zur Folge haben.3.Morphologisch läßt sich eine Alteration ber Lungen bei den Versuchstieren nicht nachweisen, funktionell auch nicht, was die Capillaren anbelangt, wohl aber die Alveolarepithelien, deren Durchlässigkeit für verschiedene Stoffe auf der Seite der Vagotomie zunimmt. Normalerweise überwiegt etwas die linke Seite, trotz kleinerer Oberfläche; bei linksseitiger Vagotomie wird dieses Verhältnis wesentlich sinnfälliger, bei rechtsseitiger umgekehrt.4.Vagotomie bewirkt also eine funktionelle Gewebsalteration, durch welche die Lunge ödembereit wird; das Ödem tritt ein, sobald die Hyperämie nach beiderseitiger Vagotomie dazu kommt.5.Unsere Ergebnisse sprechen zugunsten der Tschermakschen Hypothese einer tonischen Impermeabilisierungsinnervation der Lunge durch den Vagus und gegen die ältere Theorie einer „Vaguspneumonie“.

Zusammenfassung Die Schädigung durch Blei lokalisiert sich im Beginn an den Fasern langer Chronaxie unter Bevorzugung der am distalen Ende des Oberarms entspringenden Unterarmstreckmuskulatur.

Zusammenfassung: Der Tetaniekrampf ist durch die Calciumtheorie nicht ausreichend erkl�rt. Das regelm��ige Nebeneinander von trophischen und vegetativen St�rungen und die Abh�ngigkeit des Tonus, der Zuckung und Chronaxie von zentralen Einfl�ssen l��t an eine gemeinsame Beziehung der vegetativen St�rungen und des Krampfes zu den Regulationsstellen im Hirnstamm denken. Durch den Nachweis einer �bererregbarkeit ist der Erregungszustand des Muskels bei Tetanie noch nicht definiert. Besonderes Interesse beansprucht die Chronaxie, die schon vor dem Krampf ansteigt und diagnostische Bedeutung hat. Eine Zunahme der Chronaxie findet man bei der Magensondierung; au�er der �berventilation bei Beginn der Untersuchung und der m�glichen Alkalose durch S�ureabgabe im weiteren Verlauf spielt die blo�e Ber�hrung der Magenwand eine wesentliche Rolle f�r die �nderung der Erregungsverh�ltnisse im Muskel, die schlie�lich im manifesten Krampf sich �u�ern kann. Die Umstimmung mu� reflektorisch erkl�rt werden. Reizung des zentralen Vagusstumpfes nach beidseitiger Vagotomie f�hrt im Tierversuch zu einer Verl�ngerung der Muskelchronaxie. Die Zunahme der Muskelchronaxie kann durch vorherige Atropingaben ged�mpft werden. Bei Kranken mit Z�gen von vegetativer Neurose und bei Leberkranken sind die Chronaxien unbest�ndig, zur Erh�hung neigend oder konstant erh�ht. Nach dem klinischen Symptomenkreis und experimentellen Beobachtungen wird die verl�ngerte Chronaxie als Symptom der Vagotonie bewertet. Bei Basedow sind die Chronaxiewerte niedrig.

Zusammenfassung: In abgeklemmten, mit Paprikaaufschwemmung oder Crotonl gefllten Dnndarmabschnitten des Hundes wurden die morphologischen Vernderungen der Darmzotten untersucht. In Zeitabstnden von 1 Minute, spter in 2-Minuten-Intervallen wurden Darmstcke zur Untersuchung entnommen. Es konnte hierdurch der Ablauf der ersten Entzndungs-erscheinungen ziemlich gut erfat werden. Konzentrierte Paprikaauf-schwemmung erzeugt eine mchtige Capillarhypermie in der oberen Zottenhlfte. Mit der Hypermie geht in den betreffenden Abschnitten eine starke Vermehrung der Gewebsflssigkeit einher, wodurch die Zotten am oberen Ende anschwellen und eine keulenartige Gestalt annehmen. Auffallend ist die starke Erweiterung der zentralen Chylusgefe in den hypermischen Teilen der Zotten, whrend sie an blutleeren Stellen in der Regel zusammengeklappt sind und daher nicht hervortreten. Die Ausdehnung des Chylusgefes drfte passiv durch Aufspannung der Zotte infolge der hypermischen Erweiterung des Capillargerstes zustande kommen. Nach 7 Minuten langer Einwirkung der Paprika hat die Blutfllung in den Zottenkuppen den hchsten Grad erreicht. Zu dieser Zeit findet man auch eine vense Stauung im oberflchlichen Venennetz. An Stellen intensivster Reizeinwirkung entstehen oberflchliche Flssig-keitsblschen, die das Epithel vom Zottenstroma abheben. Sie treten schon nach 5 Minuten auf, wachsen in beilufig weiteren 6 Minuten zu ballon-artigen Blasen herab, die schlielich gegen das Darmlumen hin platzen. ber den Blasen wird das hohe Epithel zu einem ganz niedrigen Plattenepithel abgeflacht. In der Blschenflssigkeit sind oft zellige Elemente enthalten. Das Crotonl ruft hnliche Erscheinungen hervor, die aber noch frhzeitiger auftreten und rascher ihren Hhepunkt erreichen.

Zusammenfassung 1.Auch für die Lunge des Frosches besteht die HypotheseTschermaks vom dichtenden Einfluß des Vagus auf die Lunge zu Recht (verstärkte Durchlässigkeit für Trypanblau nach Vagusdurchschneidung).2.Wenn überhaupt, so schwächt die Ausschaltung von sympathischen Fasern, die etwa im Vagusstamm zur Lunge verlaufen, den Ausfall der reinen Vagotomie ab, was auf einen Antagonismus gegenüber der dichtenden Vaguswirkung schließen ließe. Doch kann das letzte Wort hierüber noch nicht gesprochen werden.3.Der Verlauf der die Lungenpermeabilität mindernden, dichtenden Eigenfasern durch den Nervus vagus darf als gesichert, der Verlauf von permeabilitätssteigernden, entdichtenden Fasern durch Grenzstrang, G. jugulare und Vagusstamm als möglich bezeichnet werden.4.Es wird auf erne vorwiegende Permeabilitätsänderung der Lungenepithelien geschlossen, ohne daß dabei eine Alteration auch des übrigen Lungengewebes geleugnet werden kann.5.Auch das Verhalten anderer Organe gegenüber Trypanblau wird durch Vagotomie beeinflußt.

Zusammenfassung: Bei der Injektion von hypertonischen Lsungen spielt nicht nur die Art der gelsten Substanz, sondern auch der physikalische Charakter der Lsung eine Rolle. Manche Wirkungen sind von der Art der injizierten Substanz unabhngig und treten bei anderer Medikationsform als in hypertonischer Lsung nicht auf. Im Blut sinkt auf die Injektion von kleinen Mengen hypertonischer Lsungen (Kochsalz, Glucose, Calciumsalze usw.) die Alkalireserve ab. Der Serumcalciumgehalt zeigt die Tendenz zu sinken. Am Skeletmuskel ndert sich der Erregungszustand: In normalen Fllen und in pathologischen Fllen mit verlngerten Chronaxien bei anscheinend normalem Blutchemismus (vegetative Neurose) steigen die Werte fr die Chronaxien auf die Injektion hin an. Im hypoglykmischen Zustand und bei Hypochlormie durch Chlorverlust nach auen sind die Werte erhht und sinken bei i.v. Injektion von Glucose bzw. Kochsalz zur Norm ab, nicht dagegen bei Verwendung anderer Lsungen. Bei Hypocalcmie scheint ein hnlicher Effekt durch die Injektion von Calciumsalz in hypertonischer Lsung nicht zu bestehen. Durch berventilation steigen die Chronaxien; in diesem Falle werden die abnorm verlngerten Werte auch durch Lsungen von Substanzen herabgesetzt, die im Blute nicht vermindert sind. Nach Tierversuchen, in denen der motorische Nerv durchschnitten wurde, und anderen nach Durchschneidung des Rckenmarks scheint der Angriffspunkt dieser die Erregung umstimmenden Wirkung supramedullr gelegen zu sein.

Zusammenfassung: 1. Zahlreiche Vorgnge an der Darmschleimhaut sind nur in vivo im Mikroskop zu verfolgen, dagegen am fixierten Objekt wegen der Art und der Intensitt der Vernderungen schwer oder gar nicht zu fassen. 2. Man kann mittels Capillarmikroskop nicht nur die Ttigkeit der Zotten, sondern auch das Verhalten der Schleimhaut und der Gefe bei Einwirkung verschiedenster Reize schr wohl verfolgen, und bei dauernder Beobachtung ein fortlaufendes Bild ber die Einwirkung des zu prfenden Reizes auf die Schleimhaut und die Gefe gewinnen. 3. Calcium verhindert bei lokaler Applikation das Auftreten der Hypermie auf entsprechende Reize.4. Calcium verzgert bei lokaler Applikation das Eintreten der Surewirkung und setzt gleichzeitig die Reizschwelle hinauf.5. Calcium intravens appliziert hat wohl noch die Gefwirkung, wie bei lokaler Applikation, aber keine Schutzwirkung gegenber Sure.

The degrees of competence of opaque and transparent colony variants of Streptococcus pneumoniae were compared. The transparent variants were transformed at 9- to 670-fold-higher rates than the opaque variants, independent of the DNA incorporated, due to decreased expression of capsular polysaccharide. Genetic transformation, therefore, tends to select for a less-encapsulated subpopulation.