Michael F. Cole

Identification of pioneer viridans streptococci in the oral cavity of human neonates.

Three hundred and sixty-seven strains of pioneer streptococci isolated from the mouths of 40 healthy, full-term infants during the first month of life were examined by two taxonomic schemes that incorporated biochemical and physiological characteristics, IgA1 protease production and glycosidase activities. Streptococcus mitis biovar 1 and S. oralis comprised 55.0% of the pioneer streptococci isolated from neonates. S. salivarius constituted 25.3% of the isolates, while S. anginosus, S. mitis biovar 2, S. sanguis and S. gordonii accounted collectively for 11.4%. Difficulties in identifying streptococci were encountered and 8.4% of the 367 isolates could not be assigned to a recognised species.

Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis

The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (delta ura3::imm434/delta ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.

Accelerated hematopoietic toxicity by high energy (56)Fe radiation.

Purpose: There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or X-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. Methods: C57BL/6J mice were irradiated with 56Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of 56Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. Results: Although onset was more rapid, 56Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)50/30 (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy, respectively, with relative biologic effectiveness for 56Fe ions of 1.25 and 1.06 for protons. Conclusions: 56Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity.

Comparing micellar, hemolytic, and antibacterial properties of di- and tricarboxyl dendritic amphiphiles

Homologous dicarboxyl dendritic amphiphiles-RCONHC(CH(3))(CH(2)CH(2)COOH)(2), 4(n); and ROCONHC(CH(3))(CH(2)CH(2)COOH)(2), 5(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC(10)) in phosphate-buffered saline (PBS). LogCMC showed a linear dependence on chain length (n); the longest chain in each series had the lowest CMC-in triethanolamine: 4(21), 180μM and 5(22), 74μM and at pH 7.4: 4(21), 78μM and 5(22), 33μM. These two series, 4(n) and 5(n), and three series of homologous tricarboxyl dendritic amphiphiles-RCONHC(CH(2)CH(2)COOH)(3), 1(n); ROCONHC(CH(2)CH(2)COOH)(3), 2(n); RNHCONHC(CH(2)CH(2)COOH)(3), 3(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2-3.4μg/mL) against S. aureus (vancomycin, MIC=0.25μg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7-6.8μg/mL) against MRSA (vancomycin, MIC=0.25μg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC(10)s of the most active homologues ranged from 7 to 18μg/mL and 18 to 220μg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9-36μg/mL, ∼18-72μM) of ten amphiphiles were measured by microdilution (log2 progression) with dye-covered beads. The EC(10)s were similar to those in PBS. The MICs of most amphiphiles (14-72μg/mL) and vancomycin (1.1-2.2μg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1μg/mL compared to vancomycin (2.2μg/mL). With CMC (9-18μg/mL) and EC(10) (16μg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.

Clonal diversity of vancomycin-resistant enterococci from an outbreak in a tertiary care university hospital

BACKGROUND Enterococci have become important nosocomial pathogens and now account for approximately 12% of nosocomial infections. Enterococci can be transferred from patient to patient and from health care personnel to patient. We investigated the clonal diversity of vancomycinresistant enterococci (VRE) causing an outbreak of infections and attempted to determine the patterns of spread of these bacteria in a university hospital. METHODS Ribotyping was used to examine the clonal diversity of 50 VRE isolates, including 23 from wounds, 14 from urine, 8 from blood, 3 from the rectum, 1 from drainage, and 1 from the cornea. RESULTS Nine patients were infected with Enterococcus faecalis, 10 with Enterococcus faecium, 3 with both E faecalis and E faecium, and 1 with Enterococcus avium. The results suggest that the sources of the VRE infections included endogenous strains and strains acquired by transmission from attending staff or from the environment. Three patients were infected by both nosocomial and endogenous strains. CONCLUSIONS These data suggest that the collection and analysis of several isolates from repeated specimens is necessary to obtain a fuller understanding of the epidemiology and population structure of antibiotic-resistant enterococci.

Effect of IgA1 Protease on the Ability of Secretory IgA1 Antibodies to Inhibit the Adherence of Streptococcus mutans

Secretory IgA (SIgA) is the principal immunoglobulin isotype present in the mucosal secretions of humans. SIgA is thought to play a major role in host defense at these surfaces by inhibiting the colonization of potentially pathogenic microorganisms. A number of bacteria that are mucosal pathogens of humans produce a protease that specifically cleaves the IgA1 subclass of humans and great apes at the hinge region to produce Fab and Fc fragments. In order to study the effect of IgA1 protease on the ability of SIgA1 antibodies to inhibit bacterial adherence, an in vitro assay that quantifies the adsorption of radiolabeled Streptococcus mutans to hydroxyapatite (HA) beads was employed. High titer S. mutans‐specific SIgA1 and SIgA2 antibodies were induced in chimpanzee milk for use in the assay. Fabα1 fragments had significantly reduced ability to inhibit adherence of S. mutans to saliva‐coated HA compared to intact SIgA1 or SIgA2 anti‐S. mutans antibodies. These data support the potential importance of IgA1 proteases as an ecological determinant in the oral cavity and their role as a determinant of pathogenesis of pathogenic bacteria whose portal of entry is the mucosal surface.

Humoral Immunity to Commensal Oral Bacteria: Quantitation, Specificity and Avidity of Serum IgG and IgM Antibodies Reactive with Actinobacillus actinomycetemcomitans in Children

The levels, specificity and avidities of serum IgM and IgG antibodies reactive with Actinobacillus actinomycetemcomitans (Aa) serotypes a, b and c were determined in periodontally healthy (PH) children and compared with subjects with localized juvenile periodontitis (LJP). All PH children exhibited IgM and IgG Aa‐reactive antibodies whether or not Aa was detected subgingivally but the antibodies were not specific for Aa. In contrast, LJP sera contained high concentrations of IgM and IgG antibodies reactive with Aa that were largely specific for this bacterium. IgM and IgG antibodies in both PH and LJP subjects were of low avidity. With one exception, the avidities of IgG anti‐Aa antibodies were significantly greater than those of IgM antibodies in both PH and LJP subjects. However, although the LJP subjects had as much as 115‐fold more Aa‐reactive IgG antibody than did the PH subjects the avidities of their IgG antibodies were no greater than those of the PH group. The induction by the host of low‐avidity antibodies, that are ineffective in immune elimination, may be a reason why commensal bacteria persist at mucosal surfaces and why persons with LJP fail to eliminate Aa from their periodontal pockets.

Humoral immunity to commensal oral bacteria in human infants: evidence that Streptococcus mitis biovar 1 colonization induces strain-specific salivary immunoglobulin A antibodies

To define the relationship between salivary SIgA antibodies and commensal oral bacteria, we examined the reactivity of SIgA antibodies from the saliva of four infants with their own colonizing Streptococcus mitis biovar 1 (S. mitis bv 1) clones (ribotypes). Immunoblot analysis was used to examine reactivity of these antibodies with persistent ribotypes isolated from the mouths of the infants over the first year postpartum. Results showed that the pattern of SIgA antibody reactivity with the majority of clones increased in complexity after colonization but that most additional bands were common to other clones, indicating that they represented shared antigens. However, unique bands were identified in 75% of the selected persistent clones. We hypothesized that if strain-specific SIgA antibody was induced in response to colonization of a particular clone and contributed to its elimination from the mouth, then the appearance of unique bands would immediately precede the disappearance of the strain. Seventy-three percent of all unique bands identified in the study fulfilled this criterion. Because the mouth is an open, dynamic environment and multiple factors are believed to play a role in the immune response at mucosal surfaces, it may not be possible to conclusively define the relationship between SIgA antibody and commensal bacteria. However, our data provide evidence that SIgA antibody, reactive with unique antigens of their own colonizing strains, is produced in infants and may point to a role of this antibody in regulating colonization by S. mitis bv 1.

Study of humoral immunity to commensal oral bacteria in human infants demonstrates the presence of secretory immunoglobulin A antibodies reactive with Actinomyces naeslundii genospecies 1 and 2 ribotypes.

ABSTRACT The mouths of three human infants were examined from birth to age 2 years to detect colonization of Actinomyces naeslundii genospecies 1 and 2. These bacteria did not colonize until after tooth eruption. The diversity of posteruption isolates was determined by ribotyping. Using immunoblotting and enzyme-linked immunosorbent assay, we determined the reactivity of secretory immunoglobulin A (SIgA) antibodies in saliva samples collected from each infant before and after colonization against cell wall proteins from their own A. naeslundii strains and carbohydrates from standard A. naeslundii genospecies 1 and 2 strains. A. naeslundii genospecies 1 and 2 carbohydrate-reactive SIgA antibodies were not detected in any saliva sample. However, SIgA antibodies reactive with cell wall proteins were present in saliva before these bacteria colonized the mouth. These antibodies could be almost completely removed by absorption with A. odontolyticus, a species known to colonize the human mouth shortly after birth. However, after colonization by A. naeslundii genospecies 1 and 2, specific antibodies were induced that could not be removed by absorption with A. odontolyticus. Cluster analysis of the patterns of reactivity of postcolonization salivary antibodies from each infant with antigens from their own strains showed that not only could these antibodies discriminate among strains but antibodies in saliva samples collected at different times showed different reactivity patterns. Overall, these data suggest that, although much of the salivary SIgA antibodies reactive with A. naeslundii genospecies 1 and 2 are directed against genus-specific or more broadly cross-reactive antigens, species, genospecies, and possibly strain-specific antibodies are induced in response to colonization.

Characterization of the mucosal immune response in breast milk after peroral immunization of chimpanzees (Pan troglodytes) with Streptococcus mutans.

The characteristics of the mucosal immune response to Streptococcus mutans cells, antigen A, antigen B, glucosyltransferases and glucan-binding proteins were examined in four pregnant chimpanzees that had been immunized perorally with Strep. mutans. Six pregnant chimpanzees served as non-immunized controls. None of the chimpanzees harbored S. mutans. Samples of milk were collected from all animals throughout the experiment. Peroral immunization resulted in an overall 17-fold median increase in SIgA in milk. Although SIgA1 comprised almost two-thirds of milk SIgA, Strep. mutans whole-cell antibody activity was contained predominantly in the SIgA2 subclass. The difference between the specific activities of anti-Strep. mutans SIgA1 and SIgA2 antibodies compared over time reached the borderline of statistical significance (p = 0.08). The avidity of anti-Strep. mutans antibodies was low in three of four chimpanzees and there was no evidence of affinity maturation. SIgA antibodies from the milk of all four immunized chimpanzees recognized antigen A. In three animals these antibodies were restricted to the SIgA1 subclass and, in one animal, anti-A antibodies were confined to SIgA2. Antibodies from all of the immunized chimpanzees recognized degradation products of antigen B in both the SIgA1 and the SIgA2 subclasses. Only two of four immunized chimpanzees responded to glucosyltransferases and these antibodies were restricted to the SIgA1 subclass. None of the chimpanzees responded to the 74-kDa glucan-binding protein. However, three animals produced SIgA1 antibodies against the 59-kDa glucan-binding protein and two of these also produced SIgA2 antibodies against this protein.