Claudine E. Fasching

Antibody-enhanced pneumococcal adherence requires IgA1 protease

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, type-specific 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 adherence-promoting 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.

Killing of Streptococcus pneumoniae by capsular polysaccharide–specific polymeric IgA, complement, and phagocytes

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 Fcalpha 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-alpha. 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 pathogens entry into the bloodstream.

Therapy of lower extremity infections with ciprofloxacin in patients with diabetes mellitus, peripheral vascular disease, or both.

PURPOSE Lower extremity infections in the presence vascular insufficiency are difficult and costly to treat. Few well-controlled clinical trials evaluating the management of these infections exist. We decided to investigate the ability of a new fluoroquinolone, ciprofloxacin, to reduce the morbidity associated with these infections and the amount of in-hospital time required for the administration of antibiotic therapy. PATIENTS AND METHODS Forty-eight patients with peripheral vascular disease (46 with diabetes mellitus) who presented to the hospital for treatment of lower extremity infections were randomized in a blinded fashion to receive oral ciprofloxacin at a dosage of either 750 mg or 1,000 mg twice daily. Patients with osteomyelitis received three months of therapy and those with infections limited to soft tissues, three weeks of ciprofloxacin treatment. All subjects were followed for one year. RESULTS One patient received an amputation 24 hours after enrollment, and two patients discontinued therapy after 20 and 34 days because of adverse effects and were not evaluable. At the one-year follow-up, 27 of the 45 (60 percent) evaluable patients had a fully successful outcome defined as not requiring either repeat antimicrobial therapy for their initial infection or amputation of the involved extremity. In the group of 18 patients in whom therapy failed, a total of only nine amputations were required. In the 15 patients whose lesion closed during therapy, 93% (14 patients) experienced a long-term successful outcome. CONCLUSION Treatment with this new fluoroquinolone offers promise for the improved outcome of patients with the serious infectious complication of infected lower extremity ulcerations in peripheral vascular disease, diabetes mellitus, or both.

Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway.

Most 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 protease‐independent mechanism to modify bound targets and facilitate enhanced survival of the bacterium.

Evidence of Commonality between Canine and Human Extraintestinal Pathogenic Escherichia coli Strains That Express papG Allele III

ABSTRACT 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 pap-positive 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 topapG 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.

Analysis of the F Antigen-Specific papA Alleles of Extraintestinal Pathogenic Escherichia coli Using a Novel Multiplex PCR-Based Assay

ABSTRACT Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenicEscherichia 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 ofpapA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of thepapA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence thatpapA 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 ofpapA alleles between lineages. Sequencing ofpapA 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.

Pneumococcal vaccination in HIV-1-infected adults in Uganda: humoral response and two vaccine failures.

Objectives:To assess the feasibility of establishing a pneumococcal vaccine trial among HIV-1-infected adults in Uganda and to characterize their responses to 23-valent pneumococcal polysaccharide vaccine. Design:An open-label pilot trial to assess recruitment and compliance of HIV-1-infected adults in Uganda to vaccination and to determine the immunogenicity of the vaccine. Setting:A community clinic for HIV-1-infected adults in Entebbe, Uganda. Methods:Levels of capsule-specific IgG to four common vaccine capsular serotypes were measured before vaccination and 1 month after vaccination. Subsequent rates of disease episodes and deaths, and immunologic responses in two vaccine failures, were followed. Results:One month after-vaccination, both HIV-1-infected (n = 77) and seronegative control subjects (n = 10) demonstrated a significant rise in capsule-specific immunoglobulin G (IgG) for three of four serotypes tested, but levels were significantly lower among HIV-1-infected patients. In 149 patient-years of follow-up, two (2.6%) developed pneumococcal pneumonia, one bacteremic with serotype 1 and one non-bacteremic with serotype 13, a non-vaccine serotype; both patients showed inadequate killing of the organism in vitro. In this same follow-up period, 29 (38%) patients died. Conclusion:HIV-1-infected adults in Uganda are at high risk of pneumococcal disease and show a significant but suboptimal response to pneumococcal vaccine. Although reliable recruitment and follow-up of vaccinees is feasible, evaluation of vaccine efficacy may be compromised by limited responses to common vaccine serotypes, an unknown incidence of disease with non-vaccine serotypes, and a high rate of mortality unrelated to Streptococcus pneumoniae infection.

Responsiveness of Human Immunodeficiency Virus Type 1-Infected Kenyan Women with or without Prior Pneumococcal Disease to Pneumococcal Vaccine

In East Africa, Streptococcus pneumoniae is a common and serious, but potentially preventable, human immunodeficiency virus type 1 (HIV-1)-associated pathogen. For 54 HIV-1-infected women, baseline levels of capsule-specific antibody to 2 of 4 pneumococcal serotypes were lower than levels in 15 seronegative women (P < .05). After immunization, specific antibody to all 4 serotypes increased in HIV-1-infected and -uninfected women (P < .05). Convalescent levels for 2 of 4 serotypes were greater in seronegative women, but the levels were not different between HIV-1-infected women with (n = 21) or without (n = 33) prior invasive pneumococcal disease. The baseline functional activity to kill S. pneumoniae type 14 was lower in HIV-1-infected than -uninfected women but also rose significantly in all groups after immunization. It is concluded that HIV-1 infection in Kenyan women is associated with decreased levels of natural antibody to selected pneumococcal capsular serotypes, but the vaccine is immunogenic in these patients who are at high risk of invasive pneumococcal disease.

Induction of Functional Secretory IgA Responses in Breast Milk, by Pneumococcal Capsular Polysaccharides

Capsule-specific secretory IgA (s-IgA) in breast milk may enhance protection against pneumococcal disease in infants. After immunization of 3 lactating mothers with 23-valent polysaccharide vaccine, specific s-IgA, but not IgG, increased by >2-fold in milk of at least 1 subject for 6 of 7 serotypes. The s-IgA was predominantly IgA1, in secretory form, and highly specific with avidity distinct from serum IgA and IgG. Milk whey from 2 immunized women supported dose- and complement-dependent killing of Streptococcus pneumoniae serotypes 19F and 14 by human neutrophils, as did purified s-IgA to serotype 19F. These data reveal that capsule-specific human s-IgA in breast milk can initiate killing of S. pneumoniae, providing proof of concept that vaccine-induced human mucosal s-IgA can support functional bactericidal activity. Determining the biologic role for s-IgA in killing and inhibiting adherence of S. pneumoniae in vivo will contribute to the development of mucosal vaccines against S. pneumoniae.

Comparison of azlocillin, ceftizoxime, cefoxitin, and amikacin alone and in combination against Pseudomonas aeruginosa in a neutropenic-site rabbit model.

The efficacy of beta-lactam antibiotics and amikacin alone and in various combinations against Pseudomonas aeruginosa was studied in a rabbit model simulating a closed-space infection in a locally neutropenic site. Six strains of P. aeruginosa were studied in semipermeable chambers placed subcutaneously in rabbits. Therapy was begun 4 h after inoculation of 5 X 10(4) CFU of bacteria per ml of pooled rabbit serum into the chambers. Antibiotics were administered intramuscularly every 6 h for 16 doses. Quantitative bacteriology was measured at the start of therapy and at 20, 44, and 92 h thereafter. Antibiotic concentrations were measured in blood and chamber fluid. Results were compared with in vitro tests of susceptibility and synergy. No single-agent therapy eradicated any of the six test organisms. Azlocillin (100 mg/kg per dose) plus amikacin (20 mg/kg per dose) eliminated five of six organisms by 92 h, and ceftizoxime (100 mg/kg per dose) plus amikacin (20 mg/kg per dose) eliminated three of six test strains. Azlocillin plus ceftizoxime (each 100 mg/kg per dose) failed to eliminate any of the six strains. To eliminate P. aeruginosa in this model, two drugs were required, with one being an aminoglycoside. In vitro susceptibility tests of synergy were predictive of successful therapy whenever the antibiotic concentrations (free and total) at the infection site exceeded the MBC for both the aminoglycoside alone and the beta-lactam when tested in combination with amikacin.