Location of Staphylococcus aureus within the experimentally infected bovine udder and the expression of capsular polysaccharide type 5 in situ.

The objective of this study was to locate Staphylococcus aureus in the bovine udder and to investigate the expression of capsular polysaccharide type 5 (CP5) in situ in both the early and chronic stages of experimental intramammary S. aureus infections. Bovine udder tissue was obtained in early and chronic stages of intramammary infection; i.e., 24 to 96 h and 122 d after experimental intramammary infection with S. aureus Newbould 305. The presence and location of S. aureus was investigated by Gram staining of tissue sections. The expression of CP5 by S. aureus in situ was investigated by immunochemical staining of tissue sections with specific antibodies against CP5. Both in the early and chronic stages of infection, S. aureus was located within the lumen of alveoli or lactiferous ducts, in association with the epithelium, and within phagocytic cells. The staphylococci were mainly observed in clusters and often in the presence of polymorphonuclear neutrophils. Expression of CP5 by S. aureus was observed both in the early and chronic stages of infection. In general, CP5-positive S. aureus were located in alveoli and in association with the mammary epithelium. In the chronic infection, CP5-positive S. aureus were also located deep in the interstitial tissue. These results indicate that--both in early and chronic stages of experimental S. aureus mastitis--colonization of the mammary epithelia and invasion into the interstitial tissue occurs and that CP5 is expressed by S. aureus Newbould 305 in situ. The invasion of S. aureus in the interstitial tissue and the expression of CP5 probably help the bacteria to withstand the host defense mechanisms.

Use of bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains.

Adherence and invasion of epithelial cells are thought to play a role in the pathogenesis of Staphylococcus aureus mastitis. A cell culture model with primary mammary epithelial cells originating from the secretory tissue from the bovine udder was used to study adherence and invasion of S. aureus. The cells were characterized with antibodies against several cell markers that had been validated on histologic cryostat sections of bovine mammary tissue. All cells stained positively with the anticytokeratin antibodies, which are restricted to epithelial cells. The cell cultures contained a small number of alpha-smooth-muscle-actin positive cells (< 1%), probably myoepithelial cells. The use of bovine primary mammary epithelial cells and bovine S. aureus isolates, which were cultured in milk serum, results in a system similar to in vivo. Strain differences for adherence and invasion of S. aureus strains cultured in milk serum were studied. In addition, the correlation between adherence and invasion was evaluated. The number of adhered and invaded bacteria was strain dependent. The percentage of adherence after 5 min of incubation was correlated to the percentage of adherence after 3 h of incubation (r = 0.94; Pearson's correlation test). Fourteen of the 20 strains were able to invade epithelial cells. The percentage of invasion was correlated to the percentage of adherence after 5 min and to the percentage adherence after 3 h (r = 0.95 and 0.90, respectively; Pearson's correlation test). Results indicate that strain differences of adherence and invasion exist for S. aureus and that the invasion is a post adherence event.

Metronidazole susceptibility factors in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a facultative anaerobic bacterium that displays moderate susceptibility to metronidazole and this study was undertaken to identify the factors involved. A. actinomycetemcomitans appeared two to four times less susceptible to metronidazole when grown in air supplemented with 5% CO2 than under anaerobic conditions. Ferredoxin-linked pyruvate:oxidoreductase activity was absent but each strain exhibited nitroreductase activity which corresponded directly with uptake of metronidazole and susceptibility to the drug under anaerobic conditions but not in air supplemented with 5% CO2. Nitroreductase activity therefore appears responsible for the susceptibility of A. actinomycetemcomitans to metronidazole.

Amoxycillin causes an enhanced uptake of metronidazole in Actinobacillus actinomycetemcomitans: a mechanism of synergy.

We investigated the influence of sub-inhibitory concentrations amoxycillin on metronidazole uptake of the metronidazole-susceptible facultative microorganism Actinobacillus actinomycetemcomitans. The rate of metronidazole uptake by bacterial cells simultaneously incubated with amoxycillin was higher than uptake in cells incubated with metronidazole alone. This phenomenon may explain the recently reported in-vitro synergic interaction between metronidazole and amoxycillin against A. actinomycetemcomitans.

Microbiological and clinical effects of metronidazole and amoxicillin in Actinobacillus actinomycetemcomitans-associated periodontitis. A 2-year evaluation.

In this study, we evaluated the microbiological and clinical effects of mechanical debridement in combination with metronidazole and amoxicillin therapy in 48 patients with Actinobacillus actinomycetemcomitans-associated periodontitis, 3 months and at least 24 months after active treatment. The results of this study showed that 47 out of 48 patients were still negative for A. actinomycetemcomitans subgingivally, at the mucous membranes, the tonsillar area and in the saliva, 2 years after therapy. The clinical results showed that a reduction of probing pocket depth, probing attachment level, bleeding index and plaque index was not only seen in the time between baseline and 3 months after therapy, but further clinical improvement was observed between 3 and 24 months after active treatment. We conclude that combined mechanical debridement and metronidazole plus amoxicillin therapy is very effective in suppressing A. actinomycetemcomitans below cultivable levels over a long period of time, suggesting elimination of this organism, and that recolonization of A. actinomycetemcomitans seems to be a rare event. The elimination of A. actinomycetemcomitans is paralleled by a further improvement of the periodontal status of the patients, even up to 24 months after active treatment.

In vitro susceptibility of Helicobacter pylori to several antimicrobial combinations.

Synergy between metronidazole and its hydroxymetabolite and between each compound and amoxicillin or tetracycline-HCl was determined against Helicobacter pylori. Metronidazole plus its hydroxymetabolite and either compound combined with amoxicillin showed synergism against all 10 strains of H. pylori tested. Metronidazole plus tetracycline-HCl or the hydroxymetabolite plus tetracycline-HCl acted synergistically against seven and six strains, respectively, acted additively against three strains, and had no additional effect against one strain. These results may help to explain the in vivo efficacies of metronidazole combinations in the treatment of H. pylori-associated gastritis.

Long-standing bacteremia caused by oral Actinobacillus actinomycetemcomitans in a patient with a pacemaker.

A case of symptomatic Actinobacillus actinomycetemcomitans bacteremia in a patient with an implanted pacemaker is presented. Attacks of fever occurred for at least 1 year. Oral examination revealed a mild form of periodontitis. A. actinomycetemcomitans was isolated from several oral sites. DNA fingerprinting of strains from the blood and the oral cavity showed identical profiles. This finding strongly suggests that the oral cavity was the primary source of A. actinomycetemcomitans bacteremia in this case. The patient was treated with the combination of metronidazole plus amoxicillin for 7 days, which resulted in a rapid cure and elimination of A. actinomycetemcomitans from the blood and the oral cavity.

In vitro susceptibilities of Actinobacillus actinomycetemcomitans to a number of antimicrobial combinations.

The in vitro susceptibilities of Actinobacillus actinomycetemcomitans to 14 antimicrobial combinations were studied by using the checkerboard titration technique. The results, expressed as the range of the fractional inhibitory concentration indices, were as follows: for metronidazole or its hydroxymetabolite combined with cefixime, 0.2 to 0.6; for moxalactam, 0.2 to 0.6; for penicillin G, 0.3 to 0.6; for tobramycin, 0.8 to 2.0; for erythromycin, 0.8 to 1.7; for ciprofloxacin, 0.2 to 0.6; for tetracycline, 0.8 to 1.2. Our observations indicated that the beta-lactam antibiotics as well as ciprofloxacin act synergistically with both metronidazole and its hydroxymetabolite against A. actinomycetemcomitans. Synergistic interactions were independent of the individual MICs of the antibiotics tested. Erythromycin, tobramycin, and tetracycline combined with either metronidazole or its hydroxymetabolite showed additive to indifferent effects against the five strains of A. actinomycetemcomitans, with the fractional inhibitory concentration indices ranging from 0.8 to 2.0. A. actinomycetemcomitans was found to be highly susceptible to ciprofloxacin (MIC of ciprofloxacin for 90% of strains tested, 0.010 micrograms/ml) and cefixime (MIC of cefixime for 90% of strains tested, 0.8 micrograms/ml). The results indicate that in patients who are allergic to penicillin, cefixime and ciprofloxacin may be useful alternative antibiotics in combination with metronidazole for the treatment of A. actinomycetemcomitans-associated periodontitis.

[Chronic conjunctivitis caused by oral anaerobic germs]. / Chronische conjunctivitis veroorzaakt door orale anaëroben.

A patient with chronic, unilateral conjunctivitis is presented. The predominant bacterial flora consisted mainly of Prevotella intermedia (formerly Bacteroides intermedius) and Peptostreptococcus micros. Restriction endonuclease fingerprinting of genomic DNA demonstrated that P. intermedia most probably originated from the oral cavity. In the oral cavity this species was recovered from the dorsum of the tongue, the tonsillar area and the periodontal pockets. Topical treatment with various antibiotics had failed to cure the infection in the past. The condition was cured after systemic therapy with metronidazole and amoxicillin.

Synergistic effects between amoxicillin, metronidazole, and the hydroxymetabolite of metronidazole against Actinobacillus actinomycetemcomitans.

Interactions between metronidazole and amoxicillin, metronidazole and its hydroxymetabolite, and amoxicillin and the hydroxymetabolite of metronidazole were investigated with checkerboard titrations in combination with accurately determined MICs and MBCs. Actinobacillus actinomycetemcomitans was used as the test organism. Synergism was found for all three combinations. Fractional inhibitory concentration indices and fractional bactericidal concentration indices varied from 0.3 to 0.7. These synergistic interactions between these antibiotics may explain the efficacy of the combination of metronidazole and amoxicillin in various bacterial infections, including periodontal disease.

Chronic conjunctivitis caused by oral anaerobes and effectively treated with systemic metronidazole plus amoxicillin.

In this study, we report on a case of refractory, unilateral anaerobic conjunctivitis. The predominant anaerobic flora consisted of Prevotella intermedia (formerly Bacteroides intermedius) and Peptostreptococcus micros. By using the technique of restriction endonuclease fingerprinting of genomic DNA, it was shown that the P. intermedia likely originated from the oral cavity. Topically applied antibiotics had failed to suppress the infection in the past. Successful treatment was achieved after systemic administration of metronidazole plus amoxicillin.

Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans.

Streptococcus sobrinus is known to possess cariogenic properties in vitro. It can produce acid in large amounts and it has the capacity to adhere to enamel and other surfaces. However, most studies on cariogenicity have been performed with laboratory strains that have been subcultured over long periods of time. Therefore, the cariogenicity and acidogenicity of 9 fresh isolates of both S. sobrinus and Streptococcus mutans from human dental plaque were compared. The bacteria were inoculated into the oral cavity of rats. The rats were fed diet SSP 20/5, containing 20% sucrose and 5% glucose. After the experimental period of 42 days, the amount of caries was assessed and bacterial counts were determined using monoclonal antibodies. Four out of 9 S. sobrinus strains and 3 out of 9 S. mutans strains did not colonize the rats. Colonizing strains constituted 39-78% of the total anaerobic cultivable microflora. The numbers of advanced dentinal lesions in the fissures of the rats colonized with S. mutans were significantly lower than those colonized with S. sobrinus (p less than 0.05). S. sobrinus produced acid more rapidly than S. mutans in a pH-stat system at pH values between 6.5 and 5.0 (p less than 0.01). The results indicate that fresh isolates of S. sobrinus are more cariogenic in rats than fresh isolates of S. mutans. This is possibly due to differences in glycolytic properties of these two species.

Enumeration of mutans streptococci in clinical samples by using monoclonal antibodies.

Mutans streptococci have been strongly associated with dental caries. Two members of this group of bacteria, Streptococcus mutans and Streptococcus sobrinus, are often found in human dental plaque. Identification of mutans streptococci on the basis of sugar fermentation is troublesome and easily leads to erroneous conclusions. Furthermore, the recovery on selective media differs for different species. This causes incorrect enumeration of S. mutans and S. sobrinus in clinical samples. The aim of this study was to develop a method for simultaneous identification and enumeration of S. mutans and S. sobrinus in dental plaque and saliva samples. With this immunoblot technique (IBT), significantly more plaque samples containing S. sobrinus were detected than on the selective medium Trypticase-yeast-cysteine-sucrose-bacitracin agar (TYCSB) (P less than 0.01). The numbers of plaque samples harboring S. mutans were equal on TYCSB and by IBT. However, the numbers of CFU of S.mutans as well as of S. sobrinus detected with the IBT were significantly higher than those obtained on TYCSB (P less than 0.001). The recovery of primary isolations of S. sobrinus on TYCSB seems to have been inhibited in 26 of the 45 S. sobrinus-containing plaque samples. False-positive or false-negative reactions with the IBT were not found.