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Migration of leukocytes in dental pulp in response to plaque bacteria.

Cell material from three different dental plaque bacteria (Actinomyces viscosus, Streptococcus mitis, Actinobacillus actinomycetemcomitans) were studied for their capacity to induce leukocyte migration in the dental pulp of monkeys. Altogether five animals were used to provide 123 teeth for the study. The bacterial test materials were prepared from lyophilized sonicates of pure cultures of respective bacteria mixed with crystalline bovine serum albumin (BSA) 1:1 by weight. Immediately prior to use in the experiment saline was added to render the test material into a paste-like consistency. Buccal Class V cavities were prepared in the monkey teeth leaving a thin remaining wall to the pulp. In these cavities, the test materials were enclosed for a period of 8 h. A set of control cavities received BSA alone. Placement of test and control materials were made before and after depleting the animals of serum complement by repeated injections of Cobra Venom Factor. Histologic examination of pulp tissue specimens showed that materials from two of the bacterial species (A. viscosus, Act. actinomycetemcomitans) consistently induced infiltrations of polymorphonuclear leukocytes in one to several cell layers underneath the test cavity. The material from S. mitis seemed less potent and in 11 of 22 teeth no pulpal reaction was recorded. Inactivation of complement with Cobra Venom Factor did not appear to affect the severity of the pulpal responses.

Animals↗

[A case of infective endocarditis with purpura and macroscopic hematuria as initial manifestations].

A 44-year-old female admitted to our hospital because of fever, purpura and macroscopic hematuria. She had been diagnosed as having ventricular septal defect (VSD). She noticed purpura with pain on bilateral legs and macroscopic hematuria since September 18, 1994. Three weeks later she also manifested a fever. Physical examination of admission revealed numerous purpura and leg edema. Laboratory data showed macroscopic hematuria, marked anemia (Hb 3.3 g/dl), leukocytosis, azotemia (Cr 2.7 mg/dl) and positive acute phase reactants. Increased serum immune complex level and hypocomplementemia were also found. The diagnosis of allergic purpura was made initially, but positive blood culture of Streptococcus mitis and the detection of vegetation attached to the right ventricular wall near the ostium of the VSD made the definite diagnosis of infective endocarditis (IE). Chemotherapy with PCG was started for two weeks but with no effect. The chemotherapy was altered to panipenem/betamipron with a daily dose of 3 g, Then, her fever fell and purpura, macroscopic hematuria and renal failure gradually disappeared. In this case, the cause of renal manifestations was considered to be immune complex glomerulonephritis. This is the first report of IE with macroscopic hematuria due to immune complex glomerulonephritis.

Acute Kidney Injury↗

Efficacy of trovafloxacin in treatment of experimental staphylococcal or streptococcal endocarditis.

The efficacy of trovafloxacin against Staphylococcus aureus and viridans group streptococci was investigated in vitro and in an experimental model of endocarditis. The MICs at which trovafloxacin and ciprofloxacin inhibited 90% of clinical isolates of such bacteria (MIC90s) were (i) 0.03 and 2 mg/liter, respectively, for 30 ciprofloxacin-susceptible S. aureus isolates, (ii) 32 and 128 mg/liter, respectively, for 20 ciprofloxacin-resistant S. aureus isolates, and (iii) 0.25 and 8 mg/liter, respectively, for 28 viridans group streptococci. Rats with aortic vegetations were infected with either of two ciprofloxacin-susceptible but methicillin-resistant S. aureus strains (strains COL and P8), one penicillin-susceptible Streptococcus sanguis strain, or one penicillin-resistant Streptococcus mitis strain. Rats were treated for 3 or 5 days with doses that resulted in kinetics that simulated those achieved in humans with trovafloxacin (200 mg orally once a day), ciprofloxacin (750 mg orally twice a day), vancomycin (1 g intravenously twice a day), or ceftriaxone (2 g intravenously once a day). Against the staphylococci, the activities of both trovafloxacin and ciprofloxacin were equivalent to that of vancomycin, and treatment of endocarditis with these drugs was successful (P < 0.05). However, ciprofloxacin selected for resistant derivatives in vitro and in vivo, whereas trovafloxacin was 10 to 100 times less prone than ciprofloxacin to select for resistance in vitro and did not select for resistance in vivo. Against the two streptococcal isolates, trovafloxacin significantly (P < 0.05) decreased bacterial counts in the vegetations but was less effective than the control drug, ceftriaxone. Thus, a simulated oral dose of trovafloxacin (200 mg per day) was effective against ciprofloxacin-susceptible staphylococci and was less likely than ciprofloxacin to select for resistance. The simulated oral dose of trovafloxacin also had some activity against streptococcal endocarditis, but optimal treatment of infections caused by such organisms might require higher doses of the drug.

Animals↗

Genetic characterization of optochin-susceptible viridans group streptococci.

Two clinical isolates of viridans group streptococci (VS) with different degrees of susceptibility to optochin (OPT), i.e., fully OPT-susceptible (Opt(s)) VS strain 1162/99 (for which the MIC was equal to that for Streptococcus pneumoniae, 0.75 micro g/ml) and intermediate Opt(s) VS strain 1174/97 (MIC, 6 micro g/ml) were studied. Besides being OPT susceptible, they showed characteristics typical of VS, such as bile insolubility; lack of reaction with pneumococcal capsular antibodies; and lack of hybridization with rRNA (AccuProbe)-, lytA-, and pnl-specific pneumococcal probes. However, these VS Opt(s) strains and VS type strains hybridized with ant, a gene not present in S. pneumoniae. A detailed characterization of the genes encoding the 16S rRNA and SodA classified isolates 1162/99 and 1174/97 as Streptococcus mitis. Analysis of the atpCAB region, which encodes the c, a, and b subunits of the F(0)F(1) H(+)-ATPase, the target of optochin, revealed high degrees of similarity between S. mitis 1162/99 and S. pneumoniae in atpC, atpA, and the N terminus of atpB. Moreover, amino acid identity between S. mitis 1174/97 and S. pneumoniae was found in alpha helix 5 of the a subunit. The organization of the chromosomal region containing the atp operon of the two Opt(s) VS and VS type strains was spr1284-atpC, with spr1284 being located 296 to 556 bp from atpC, whereas in S. pneumoniae this distance was longer than 68 kb. In addition, the gene order in S. pneumoniae was IS1239-74 bp-atpC. The results suggest that the full OPT susceptibility of S. mitis 1162/99 is due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae and that the intermediate OPT susceptibility of S. mitis 1174/97 correlates with the amino acid composition of its a subunit.

Amino Acid Sequence↗

Efficacy of garenoxacin in treatment of experimental endocarditis due to Staphylococcus aureus or viridans group streptococci.

The activity of garenoxacin was investigated in rats with experimental endocarditis due to staphylococci and viridans group streptococci (VGS). The staphylococci tested comprised one ciprofloxacin-susceptible and methicillin-susceptible Staphylococcus aureus (MSSA) isolate (isolate 1112), one ciprofloxacin-susceptible but methicillin-resistant S. aureus (MRSA) isolate (isolate P8), and one ciprofloxacin-resistant mutant (grlA) of P8 (isolate P8-4). The VGS tested comprised one penicillin-susceptible isolate and one penicillin-resistant isolate (Streptococcus oralis 226 and Streptococcus mitis 531, respectively). To simulate the kinetics of drugs in humans, rats were infused intravenously with garenoxacin every 24 h (peak and trough levels in serum, 6.1 and 1.0 mg/liter, respectively; area under the concentration-time curve [AUC], 63.4 mg. h/liter) or levofloxacin every 12 h (peak and trough levels in serum, 7.3 and 1.5 mg/liter, respectively; AUC, 55.6 mg. h/liter) for 3 or 5 days. Flucloxacillin, vancomycin, and ceftriaxone were used as control drugs. Garenoxacin, levofloxacin, flucloxacillin, and vancomycin sterilized >/=70% of the vegetations infected with both ciprofloxacin-susceptible staphylococcal isolates (P < 0.05 versus the results for the controls). Garenoxacin and vancomycin also sterilized 70% of the vegetations infected with ciprofloxacin-resistant MRSA isolate P8-4, whereas treatment with levofloxacin failed against this organism (cure rate, 0%; P < 0.05 versus the results obtained with the comparator drugs). Garenoxacin did not select for resistant derivatives in vivo. In contrast, levofloxacin selected for resistant variants in four of six rats infected with MRSA isolate P8-4. Garenoxacin sterilized 90% of the vegetations infected with both penicillin-susceptible and penicillin-resistant isolates of VGS. Levofloxacin sterilized only 22 and 40% of the vegetations infected with penicillin-susceptible S. oralis 226 and penicillin-resistant S. mitis 531, respectively. Ceftriaxone sterilized only 40% of those infected with penicillin-resistant S. mitis 531 (P < 0.05 versus the results obtained with garenoxacin). No quinolone-resistant VGS were detected. In all the experiments successful quinolone treatment was predicted by specific pharmacodynamic criteria (D. R. Andes and W. A. Craig, Clin. Infect. Dis. 27:47-50, 1998). The fact that the activity of garenoxacin was equal or superior to those of the standard comparators against staphylococci and VGS indicates that it is a potential alternative for the treatment of infections caused by such bacteria.

Animals↗

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.

Ammonia↗

Bacteriocin (mutacin) production by Streptococcus mutans genome sequence reference strain UA159: elucidation of the antimicrobial repertoire by genetic dissection.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).

Bacterial Proteins↗

Phenotypic and genotypic selection of microbiota surviving under dental restorations.

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, beta-N-acetylglucosaminidase, and beta-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P = 0.002 and P = 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.

Acetylglucosaminidase↗

Bacteremia after tooth extractions studied with the aid of prereduced anaerobically sterilized culture media.

Both prereduced molten agar and broth and aerobic molten agar and broth were inoculated with blood samples collected from patients with periodontitis, but in otherwise good health, both before and after extraction of two or more teeth. Postoperative blood samples from 23 of 25 patients sampled yielded anaerobic and facultative species. Colony counts from nine samples yielded from less than 1 to over 100 colonies per ml of blood. Organisms detected were species belonging to the genera Bacteroides, Fusobacterium, Peptostreptococcus, Leptotrichia, Propionibacterium, Peptococcus, Veillonella, plus Streptococcus mitis, S. salivarius, vibrio forms, and strains resembling S. mutans. The data indicate that prereduced anaerobically sterilized culture medium with polyanethol sulfonate is effective for detecting anaerobic species in bacteremia and that anaerobic species can be prevalent in bacteremias immediately after tooth extraction in patients with periodontitis.

Aerobiosis↗

Production, purification, and properties of a bacteriocin from Staphylococcus aureus isolated from saliva.

Staphylococci from samples of human saliva were isolated on staphylococcal-selective agar plates. These strains were tested for the inhibition of the growth of Staphylococcus aureus FDA209P. The frequency of inhibitory strains among all of the staphylococcal isolates was 5.2%. Strain IYS2, which formed the biggest inhibitory zone against the growth of the indicator strain, was used as the producer of bacteriocin. IYS2 was identified to be S. aureus, based on its biological properties. The bacteriocin was purified by sequential procedures, including ammonium sulfate precipitation, fractionation with ethanol, ion-exchange chromatography, and gel filtration. Its molecular weight was determined to be 5,000. The isoelectric point was 10.0. In amino acid composition, lysine, histidine, aspartic acid, valine, and phenylalanine were predominant. The bacteriocin was heat stable but inactivated by proteases or peptidase. The bacteriocin had a bactericidal effect on susceptible cells. An analysis of the inhibitory spectrum among typical oral indigenous bacteria showed that Streptococcus salivarius, Propionibacterium acnes, Corynebacterium parvulum, and Actinomyces israelii were susceptible to the bacteriocin. Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, Actinomyces naeslundii, Fusobacterium nucleatum, Bacterionema matruchotii, and Bacteroides melaninogenicus were resistant. The majority of S. aureus tested were susceptible, and all Staphylococcus epidermidis strains tested were resistant.

Amino Acids↗

Microflora associated with experimental root surface caries in humans.

This study describes the microflora from actively progressing root surface caries lesions, in which mineral loss had been determined by quantitative microradiography. The caries lesions were produced experimentally in root surface specimens from human molars inserted in lower partial dentures carried for 3 months by six elderly individuals. A total of 780 bacterial isolates were identified from 13 plaque samples, collected with a punch technique, and six dentin samples. The composition of the microflora showed distinct individual differences. The microflora from plaque samples associated with the highest mineral loss was dominated by either Actinomyces viscosus or a combination of mutans streptococci (serotypes c, d, and f) and Lactobacillus species (L. casei and L. brevis). Plaque from root surfaces with less pronounced mineral loss harbored a more complex microflora comprising gram-positive rods, mutans streptococci, Streptococcus mitis biovar 1, Veillonella spp., gram-negative rods, and low numbers of lactobacilli. In the latter samples, individual variations in the proportions of mutans streptococci (serotypes c, d, and g), Actinomyces species (A. viscosus and A. naeslundii), and Veillonella parvula biotypes were observed. These findings suggest that certain species or combinations of species are more cariogenic than others and that dominance of single acidogenic species in particular is conducive to high caries activity.

Aged↗

Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases.

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.

Collagenases↗

Innate immune responses of human neonatal cells to bacteria from the normal gastrointestinal flora.

The hygiene hypothesis postulates that the prevalence of allergy has increased due to decreased microbial stimulation early in life, leading to delayed maturation of the immune system. The aim of this study was to examine the cytokine pattern produced from cord blood mononuclear cells relative to adult cells after stimulation with bacterial strains from the normal flora. Mononuclear cells from cord and adult blood samples were stimulated with the following bacteria: Bifidobacterium adolescentis, Enterococcus faecalis, Lactobacillus plantarum, Streptococcus mitis, Corynebacterium minutissimum, Clostridium perfringens, Bacteroides vulgatus, Escherichia coli, Pseudomonas aeruginosa, Veillonella parvula, and Neisseria sicca. The levels of interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-6 were measured by enzyme-linked immunosorbent assay. The TNF-alpha production was also analyzed after blocking CD14, Toll-like receptor 2 (TLR-2), and TLR-4 prior to stimulation with bacteria. The levels of IL-12 and TNF-alpha were similar in cord and adult cells. Gram-positive bacteria induced considerably higher levels of IL-12 and TNF-alpha than gram-negative bacteria in both cord and adult cells. The levels of IL-6 were significantly higher in newborns than in adults, whereas the levels of IL-10 were similar in newborns and adults. Gram-negative and gram-positive bacteria induced similar levels of IL-6 and IL-10 in cord cells. L. plantarum bound or signaled through CD14, TLR-2, and TLR-4, whereas E. coli acted mainly through CD14 and TLR-4. These results indicate that the innate immune response in newborns to commensal bacteria is strong and also suggest that different bacterial strains may have differential effects on the maturation of the immune system of infants.

Adult↗

One of two human lactoferrin variants exhibits increased antibacterial and transcriptional activation activities and is associated with localized juvenile periodontitis.

The iron-binding protein lactoferrin is a ubiquitous and abundant constituent of human exocrine secretions. Lactoferrin inhibits bacterial growth by sequestering essential iron and also exhibits non-iron-dependent antibacterial, antifungal, antiviral, antitumor, anti-inflammatory, and immunoregulatory activities. All of these non-iron-dependent activities are mediated by the highly charged N terminus of lactoferrin. In this study we characterized a Lys/Arg polymorphism at position 29 in the N-terminal region of human lactoferrin that results from a single nucleotide polymorphism in exon 1 of the human lactoferrin gene. We expressed cDNAs encoding both lactoferrin variants in insect cells and purified the two proteins by ion exchange chromatography. The two lactoferrin variants exhibited nearly identical iron-binding and iron-releasing activities and equivalent bactericidal activities against a strain of the gram-negative bacterium Actinobacillus actinomycetemcomitans. When tested against the gram-positive species Streptococcus mutans and Streptococcus mitis, however, lactoferrin containing Lys at position 29 exhibited significantly greater bactericidal activity than did lactoferrin containing Arg. In addition, the Lys-containing lactoferrin stimulated bovine tracheal epithelial cells to synthesize much higher levels of tracheal antimicrobial peptide mRNA than did the Arg-containing variant. A genotyping assay that distinguished between the two alleles based on a polymorphic EarI restriction site showed that the Lys and Arg alleles had frequencies of 24% and 76%, respectively, among 17 healthy human subjects, and 72% and 28%, respectively, among nine patients with localized juvenile periodontitis. Our findings suggest that these two lactoferrin variants are functionally different and that these differences may contribute to the pathogenesis of localized juvenile periodontitis.

Aggregatibacter actinomycetemcomitans↗

Characterization of salivary immunoglobulin A responses in children heavily exposed to the oral bacterium Streptococcus mutans: influence of specific antigen recognition in infection.

The initial infection of children by Streptococcus mutans, the main pathogen of dental caries, depends on the ability of S. mutans to adhere and accumulate on tooth surfaces. These processes involve the adhesin antigen I/II (AgI/II), glucosyltransferases (GTF) and glucan-binding protein B (GbpB), each a target for anticaries vaccines. The salivary immunoglobulin A (IgA) antibody responses to S. mutans antigens (Ags) were characterized in 21 pairs of 5- to 13-month-old children. Pairs were constructed with one early S. mutans-infected and one noninfected child matched by age, racial background, number of teeth, and salivary levels of IgA. Specific salivary IgA antibody response and S. mutans infection levels were then measured during a 1-year follow-up. Robust responses to S. mutans were detected from 6 months of age. Salivary IgA antibody to AgI/II and GTF was commonly detected in salivas of all 42 children. However, GbpB-specific IgA antibody was seldom detected in the subset of infected children (38.1% at baseline). In contrast, most of the subset of noninfected children (76.2%) showed GbpB-reactive IgA antibody during the same period. Frequencies of GbpB responses increased with age, but differences in intensities of GbpB-IgA antibody reactions were sustained between the subsets. At baseline, GbpB-reactive IgA antibody accounted for at least half of the total salivary IgA S. mutans-reactive antibody in 33.3 and 9.5% of noninfected and infected children, respectively. This study provides evidence that a robust natural response to S. mutans Ags can be achieved by 1 year of age and that IgA antibody specificities may be critical in modulating initial S. mutans infection.

Antigenic Variation↗

Interleukin 1alpha increases the susceptibility of rabbits to experimental viridans streptococcal endocarditis.

Major predisposing conditions for infective endocarditis (IE) are the presence of a cardiac platelet-fibrin vegetation and of circulating bacteria with relatively low susceptibility to microbicidal activity of blood platelets. The influence of proinflammatory conditions on development of IE is unknown. We studied the effects of the presence of a catheter, inserted to induce platelet-fibrin vegetations, and of the proinflammatory cytokine interleukin-1alpha in rabbit experimental IE. Leaving the catheter in place after challenge with viridans streptococci predisposed for experimental IE. IE susceptibility rapidly decreased between 0 to 6 h after catheter removal. The catheter did not predispose for IE by providing a site for bacterial adherence, as almost all explanted catheters were culture negative. To mimic the proinflammatory influence of the catheter, rabbits were injected with interleukin-1alpha at 24 h after catheter removal and at 0, 1, and 3 h before bacterial challenge. Interleukin-1alpha injected 3 h prior to challenge significantly increased IE incidence due to a platelet releasate-susceptible Streptococcus oralis strain, with rapidly increasing numbers of bacteria within the vegetations. IE due to the Streptococcus sanguis strain less susceptible to platelet releasate was not enhanced. We conclude that proinflammatory stimuli, either a catheter or interleukin-1alpha, enhanced susceptibility to IE due to the platelet releasate-susceptible S. oralis. As with rabbits, temporary intravascular proinflammatory conditions may predispose for IE in humans at risk for this serious infection.

Animals↗

ESTABLISHMENT OF HUMAN INDIGENOUS BACTERIA IN GERM-FREE MICE.

Gibbons, R. J. (Forsyth Dental Center and Harvard School of Dental Medicine, Boston, Mass.), S. S. Socransky, and B. Kapsimalis. Establishment of human indigenous bacteria in germ-free mice. J. Bacteriol. 88:1316-1323. 1964.-Thirteen strains of bacteria indigenous to the gingival crevice area of man were tested for their ability to establish as monocontaminants in germ-free mice. Three facultative organisms, Streptococcus mitis, Staphylococcus albus, and a "diphtheroid," established, as well as three anaerobes. Fusobacterium fusiforme, an anaerobic diphtheroid, and a Bacteroides strain. Seven other anaerobes (two strains of B. melaninogenicus, and one strain each of Treponema microdentium, Veillonella alcalescens, a Peptostreptococcus strain, Vibrio sputorum, and B. oralis) failed to establish. A mixture consisting of ten organisms representative of the predominant groups of cultivable bacteria present in the gingival crevice area of man was inoculated intraorally into germ-free mice. All organisms with the exception of B. melaninogenicus and T. microdentium became established as polycontaminants. Escherichia coli could be established in the above polycontaminated mice, as well as in those contaminated directly with human gingival debris. E. coli comprised approximately 50 and 6% of the fecal flora in the two groups, respectively. Diet, cecal contents, and feces of germ-free and polycontaminated mice were tested for inhibitory action against T. microdentium and B. melaninogenicus. None inhibited T. microdentium, whereas all three inhibited B. melaninogenicus. The inhibitory effect appeared to be due to dietary alfalfa. B. melaninogenicus could become established in mice monocontaminated with a facultative diptheroid and maintained on an alfalfa-free diet. These experiments indicate that human indigenous bacteria can become established in germ-free mice, and that microbial interactions and diet composition are important determinants.

Animals↗

Rapid detection of Bordetella pertussis by a monoclonal antibody-based colony blot assay.

Monoclonal antibodies to Bordetella pertussis filamentous hemagglutinin (FHA) and lipopolysaccharide (LPS) were used in a colony blot enzyme-linked immunosorbent assay designed for rapid detection of B. pertussis. Bacterial colonies from Bordet-Gengou agar plates were blotted onto nitrocellulose filter disks, lysed by immersion in chloroform, and reacted with monoclonal antibodies. Following reaction with peroxidase-conjugated rabbit anti-mouse immunoglobulin antisera and 4-chloro-1-naphthol, blue dots representing single colonies appeared on the filters. Blotting of single B. pertussis colonies could be performed after incubation for 40 h, i.e., before the colonies were visible by eye on the agar surface. Ten of ten B. pertussis strains showed positive blotting reactions with antibodies specific for B. pertussis FHA and LPS. Fourteen of fourteen B. parapertussis strains reacted with two of the FHA-specific antibodies but not with two of the LPS-specific antibodies. Strains of B. bronchiseptica showed a variable reaction pattern. No cross-reactions were observed with strains of Streptococcus mitis, S. pyogenes, S. pneumoniae, Staphylococcus aureus, Branhamella catarrhalis, or Klebsiella pneumoniae. This assay may be useful for identification of B. pertussis and B. parapertussis in suspected cases of whooping cough.

Antibodies, Bacterial↗