SummaryMost clinical isolates of Streptococcus pneumoniae consist of heterogeneous populations of at least two colony phenotypes, opaque and transparent, selected for in the bloodstream and nasopharynx, respectively. Microarray analysis revealed 24 orfs that demonstrated differences in expression greater than twofold between variants of independent strains. Twenty-one of these showed increased expression in the transparent variants, including 11 predicted to be involved in sugar metabolism. A single genomic region contains seven of these loci including the gene that encodes the neuraminidase, NanA. In contrast to previous studies, there was no contribution of NanA to adherence of S. pneumoniae to epithelial cells or colonization in an animal model. However, we observed NanA-dependent desialylation of human airway components that bind to the organism and may mediate bacterial clearance. Targets of desialylation included human lactoferrin, secretory component, and IgA2 that were shown to be present on the surface of the pneumococcus in vivo during pneumococcal pneumonia. The efficiency of desialylation was increased in the transparent variants and enhanced for host proteins binding to the surface of S. pneumoniae .Because deglycosylation affects the function of many host proteins, NanA may contribute to a proteaseindependent mechanism to modify bound targets and facilitate enhanced survival of the bacterium.

IgA, the major class of Ig in secretions, classically functions by interfering with microbial attachment to host tissues. Many mucosal pathogens, including Streptococcus pneumoniae, express an IgA1 protease that may circumvent the protective effects of this Ig subclass. Because these proteases are specific for human IgA1, we generated human mAbs to the major surface antigen of the pneumococcus, its capsular polysaccharide, and tested their effect in a colonization model of bacterial adherence to respiratory epithelial cells in culture. Rather than inhibiting adherence, typespecific IgA1 markedly enhanced bacterial attachment to host cells, but only when cleaved by IgA1 protease. Neither antibodies of protease-insensitive subclasses (IgA2 and IgG) nor those directed against heterologous capsules had such activity. The adherencepromoting properties of cleaved antibodies correlated with the cationic characteristics of their variable segments, suggesting that bound Fab fragments may neutralize the inhibitory effect of negatively charged capsules on adhesive interaction with host cells. Coating of pneumococci with anticapsular polysaccharide antibody unmasked the bacterial phosphorylcholine ligand, allowing for increased adherence mediated by binding to the platelet activating factor receptor on epithelial cells. In addition, our findings provide evidence for a novel function of bacterial IgA1 proteases. These enzymes may enable pathogens to subvert the antigen specificity of the humoral immune response to facilitate adhesive interactions and persistence on the mucosal surface.

Although dogs have been proposed as carriers of extraintestinal pathogenic Escherichia coli (ExPEC) with infectious potential for humans, presumed host species-specific differences between canine and human ExPEC strains have cast doubt on this hypothesis. The recent discovery that allele III of papG (the P fimbrial adhesin gene) predominates among human cystitis isolates and confers an adherence phenotype resembling that of canine ExPEC prompted the present reevaluation of the canine-human ExPEC connection. Sixteen paired pappositive urine and rectal E. coli isolates from dogs with urinary tract infection were studied. papG (adhesin) and papA (pilin) allele type, agglutination phenotypes, virulence factor genotypes, and randomly amplified polymorphic DNA and pulsed-field gel electrophoresis fingerprints were analyzed and compared with those of human ExPEC controls. The 16 canine strains contained predominantly papG allele III. Agglutination phenotypes segregated strictly according to papG allele status and were homogeneous among strains with the same papG allele profile irrespective of their human versus canine origin. Canine and human PapG variant III peptide sequences were highly homologous, without host species-specific differences. The most prevalent canine papA allele was F48, a novel variant recently identified among human urosepsis isolates. In addition to pap, human ExPEC-associated virulence genes detected among the canine strains included sfa/focDE, sfaS, fyuA, hlyA, cnf1, cdtB, kpsMT-II and -III, rfc, traT, ompT, and a marker for a pathogenicity-associated island from archetypal human ExPEC strain CFT073. Molecular fingerprinting confirmed the fecal origin of all but one canine urine isolate and showed one pair of O6 canine urine and fecal isolates to be extremely similar to an O6 human urosepsis isolate with which they shared all other genotypic and phenotypic characteristics analyzed. These data demonstrate that canine ExPEC strains are similar to, and in some instances essentially indistinguishable from, human ExPEC strains, which implicates dogs and their feces as potential reservoirs of E. coli with infectious potential for humans.

The role of IgA in the control of invasive mucosal pathogens such as Streptococcus pneumoniae is poorly understood. We demonstrate that human pneumococcal capsular polysaccharide-specific IgA initiated dose-dependent killing of S. pneumoniae with complement and phagocytes. The majority of specific IgA in serum was of the polymeric form (pIgA), and the efficiency of pIgA-initiated killing exceeded that of monomeric IgA-initiated killing. In the absence of complement, specific IgA induced minimal bacterial adherence, uptake, and killing. Killing of S. pneumoniae by resting phagocytes with immune IgA required complement, predominantly via the C2-independent alternative pathway, which requires factor B, but not calcium. Both S. pneumoniae-bound IgA and complement were involved, as demonstrated by a 50% decrease in killing with blocking of Fcα receptor (CD89) and CR1/CR3 (CD35/CD11b). However, IgA-mediated killing by phagocytes could be reproduced in the absence of opsonic complement by pre-activating phagocytes with the inflammatory products C5a and TNF-α. Thus, S. pneumoniae capsule-specific IgA may show distinct roles in effecting clearance of S. pneumoniae in the presence or absence of inflammation. These data suggest mechanisms whereby pIgA may serve to control pneumococcal infections locally and upon the pathogen's entry into the bloodstream.

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Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic Escherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of papA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the papA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that papA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of papA alleles between lineages. Sequencing of papA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.

A portion of the gyrA gene from amino acid codons 67 to 129 was sequenced in 34 methicillin-resistant Staphylococcus aureus strains (14 isolated in Minnesota, 10 isolated in Indiana, and 10 isolated in Tennessee). Twenty-eight of these strains were ciprofloxacin resistant. Sixteen of the strains contained a Ser----Leu mutation at codon 84; 3 contained strains a Ser----Ala mutation at codon 84; 3 strains contained two mutations, Ser----Leu at codon 84 and Ser----Pro at codon 85; and 6 strains contained a Glu----Lys mutation at codon 88. Six strains were wild type and ciprofloxacin susceptible. Several mutations from amino acid codons 84 through 88 can be associated with high-level quinolone resistance.

Pneumolysin, the major Streptococcus pneumoniae cytotoxin, contributes to the early pathogenesis of invasive pneumococcal pneumonia by facilitating intrapulmonary bacterial growth and invasion into the blood. Pneumolysin is a multifunctional toxin, with distinct cytolytic ("hemolytic") and complement-activation ("complement") activities that have been mapped to several regions of the molecule. To characterize the specific contributions of pneumolysin's hemolytic and complement properties to the pathogenesis of pneumococcal pneumonia, we compared the in vivo effects of type 2 S. pneumoniae mutant strains, which produce pneumolysins deficient in these activities. The absence of either pneumolysin's hemolytic or complement activities rendered mutant strains less virulent than the wild-type strain during pulmonary infection. Pneumolysin's hemolytic activity correlated with acute lung injury and bacterial growth at 3 and 6 h after endotracheal instillation. In contrast, pneumolysin's complement activity correlated with bacterial growth and bacteremia at 24 h after pulmonary infection. Pneumolysin's complement activity was not associated with the degree of alveolar-capillary injury or recruitment of leukocytes during initial pulmonary infection. However, pneumolysin's complement activity inhibited killing of mutant bacteria in an in vitro complement-dependent neutrophil killing assay. Thus, both pneumolysin's hemolytic and complement activities made specific contributions to the early pathogenesis of pneumococcal pneumonia at different stages of infection and by different mechanisms.