The use of controlled release locally delivered 10% doxycycline hyclate gel as an adjunct to scaling and root planing in the treatment of chronic periodontitis: clinical and microbiological results.

BACKGROUND: Local delivery of antimicrobial agents provides higher concentration of the drug in the periodontal site for longer periods than systemically delivered methods. In the present study an attempt is made to know the efficacy of controlled local drug delivery of doxycycline as an adjunctive treatment in the management of chronic periodontitis. MATERIALS AND METHODS: A total of 12 patients, diagnosed as Chronic Periodontitis in the age of 25 to 55 years, were selected of both the sexes in this study. They were divided into Experimental group consisted of 30 sites who received complete scaling and root planing (SRP) followed by placement of Atridox gel and control group consisted of 30 sites who received only SRP. Clinical parameters were recorded at, baseline, days 30, 90 and 180. Parameters were plaque index, gingival index, gingival bleeding index and microbial analysis, probing pocket depth and Clinical attachment level (CAL). The microbiological analysis was done at baseline visit and at 90th day. Probing pocket depth and CAL were recorded only on day 0 and 180th day. RESULTS: In 180 days study, both the groups exhibited a significant improvement in periodontal status. Significant gain in attachment level was observed in both the group. Between both the groups the clinical parameters in the experimental groups exhibited better results as compared to the control group. Both the groups exhibited significant reduction in the number of spirochetes. CONCLUSION: Combination therapy of SRP and 10% DH gel demonstrated better results at all levels suggesting that this therapy can play a significant role as an adjunct to SRP in the management of chronic periodontitis.

Impact of four local anaesthetics on growth and viability of in vitro cultured Borrelia burgdorferi sensu stricto, Borrelia bavariensis and Borrelia afzelii.

Many local anaesthetics, including lidocaine, procaine and ropivacaine inhibit bacterial growth. This study investigates potential effects of these local anaesthetics on growth of Borrelia burgdorferi sensu stricto (Bbss), Borrelia bavariensis (Bbav) and Borrelia afzelii (Ba). For this purpose, Borrelia spp. organisms were either continuously or temporarily exposed to one of four local anaesthetics preparations: 20 mg/ml procaine hydrochloride (P); 10 mg/ml ropivacaine hydrochloride (R); 20 mg/ml lidocaine hydrochloride (L1, L2). L2 also contained the preservatives methyl-benzoate and propyl-benzoate, whereas P, R and L1 did not. All four local anaesthetic preparations inhibited in vitro growth of Borrelia spp. depending on concentration and exposure time. There are differences in sensitivity among the Borrelia spp. with Bbav being more susceptible to growth inhibition than Bbss and Ba. When comparing the different local anaesthetic preparations with their regard to inhibition of growth of Borrelia spp. organisms, P showed the lowest impact. It cannot be completely excluded that preservatives present in L2, methyl-benzoate and propyl-benzoate, may be a reason for further inhibition of Borrelia spp. organisms. Concentrations of local anaesthetics used in these experiments may also be present in the skin of patients during regular medical procedures. These are preliminary findings and further experiments, preferably in vivo, are necessary. To minimize the risk to produce false negative results with cultures, we recommend using procaine in a preparation without preservatives for local anaesthesia prior to skin sampling.

Persistence of Borrelia burgdorferi following antibiotic treatment in mice.

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for the presence of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice treated with antibiotic were consistently culture negative, but tissues from some of the mice remained PCR positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, as determined based upon PCR results, and ticks from those cohorts transmitted spirochetes to naïve SCID mice, which became PCR positive but culture negative. Results indicated that following antibiotic treatment, mice remained infected with nondividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.

Decreased susceptibility to doxycycline associated with a 16S rRNA gene mutation in Brachyspira hyodysenteriae.

The objective of this study was to assess whether nucleotide substitutions in the 16S rDNA sequence of selected Brachyspira hyodysenteriae isolates could explain differences in doxycycline minimal inhibitory concentrations (MICs). The main part of the 16S rRNA gene was sequenced and compared for 19 isolates with different doxycycline MICs. A mutation in the 16S rRNA gene at the position corresponding to 1058 in Escherichia coli has been shown to cause tetracycline resistance in other bacteria. In the B. hyodysenteriae sequences a G1058C mutation was found for all isolates with increased doxycycline MICs whereas all susceptible isolates had the wild type sequence.

The effect of fermentable carbohydrates on experimental swine dysentery and whip worm infections in pigs.

An experiment was conducted to study the effect of diets with contrasting fermentability in the large intestine on experimental infections with Brachyspira hyodysenteriae, the causative agent of swine dysentery, and the whip worm, Trichuris suis, in pigs. Two diets with organically grown ingredients were composed. Both diets were based on triticale and barley and supplemented with either rape seed cake (Diet 1) or dried chicory root and sweet lupins (Diet 2). The study had a three-factorial design, with eight groups of pigs receiving Diet 1 or Diet 2, +/-B. hyodysenteriae, and +/-T. suis. Pigs fed Diet 2 and challenged with B. hyodysenteriae did not develop swine dysentery and B. hyodysenteriae was not demonstrated in any of the pigs during the study. In contrast, 94% of the B. hyodysenteriae challenged pigs fed Diet 1 showed clinical symptoms of swine dysentery and all the pigs were shedding B. hyodysenteriae in faeces at some points in time during the experiment. The number of T. suis was lower in pigs fed Diet 2 compared to pigs fed Diet 1, but the differences were not significant. Pigs on Diet 1 and challenged with both pathogens showed clinical symptoms of SD for a longer period than pigs inoculated with B. hyodysenteriae only. The study showed that diets supplemented with highly fermentable carbohydrates from dried chicory roots and sweet lupins can protect pigs against developing swine dysentery, but do not have any significant influence on T. suis.

Variation in the Gut Microbiota of Termites (Tsaitermes ampliceps) Against Different Diets.

Termites are well recognized for their thriving on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling microbiota; however, the effects of diet changes on termite gut microbiota are poorly understood, especially for the lower termites. In this study, we employed high-throughput 454 pyrosequencing of 16S V1-V3 amplicons to compare gut microbiotas of Tsaitermes ampliceps fed with lignin-rich and lignin-poor cellulose diets after a 2-week-feeding period. As a result, the majority of bacterial taxa were shared across the treatments with different diets, but their relative abundances were modified. In particular, the relative abundance was reduced for Spirochaetes and it was increased for Proteobacteria and Bacteroides by feeding the lignin-poor diet. The evenness of gut microbiota exhibited a significant difference in response to the diet type (filter paper diets < corn stover diets < wood diets), while their richness was constant, which may be related to the lower recalcitrance of this biomass to degradation. These results have important implications for sampling and analysis strategies to probe the lignocellulose degradation features of termite gut microbiota and suggest that the dietary lignocellulose composition could cause shifting rapidly in the termite gut microbiota.

Quality-control ranges for antimicrobial susceptibility testing by broth dilution of the Brachyspira hyodysenteriae type strain (ATCC 27164T).

There are no approved standards for antimicrobial susceptibility testing of the fastidious spirochete Brachyspira hyodysenteriae. An interlaboratory study was performed to establish MIC quality control ranges for six antimicrobial agents for the type strain of B. hyodysenteriae using broth dilution. The results showed that B. hyodysenteriae B78T ATCC 27164T is a suitable quality control strain. This is a first step toward standardization of methods regarding this anaerobe.

Tiamulin resistance in porcine Brachyspira pilosicoli isolates.

There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified.

Effects of griseoviridin and viridogrisein against swine dysentery in experimental infection by using mice and pigs.

Griseoviridin, a known antibiotic produced by Streptomyces cacaoi subsp. cacaoi, was found to be active against Brachyspira hyodysenteriae--the bacterium causing swine dysentery. An in vitro synergism is observed when it is used in combination with viridogrisein--a simultaneously produced antibiotic. In mouse experiments, the effect of griseoviridin alone was less than that of lincomycin--a commercially available swine dysentery medication. However, a 1:1 mixture of griseoviridin and viridogrisein revealed a noticeable synergistic effect. In an evaluation using pigs artificially infected with B. hyodysenteriae, a large difference was not observed between the effect of griseoviridin alone and that in combination with viridogrisein. Nevertheless, griseoviridin alone exhibited a therapeutic effect superior to that of lincomycin.

Alzheimer's Disease: Assessing the Role of Spirochetes, Biofilms, the Immune System, and Amyloid-ß with Regard to Potential Treatment and Prevention.

Alzheimer's disease (AD) is an infectious disease caused by spirochetes, and these spirochetes form biofilms, which attract the innate immune system. The innate immune system first responder, Toll-like receptor 2, generates both NF-κB and TNF-α which try to kill the spirochetes in the biofilm, but cannot penetrate the "slime". NF-κB is also responsible for the generation of amyloid-ß (Aß) which itself is anti-microbial. Aß cannot penetrate the biofilm either, and its accumulation leads to destruction of the cerebral neurocircuitry. Treatment with penicillin (as in tertiary syphilis, the comparator to AD) is outlined; a biofilm dispersing agent may need to be added to the protocol.

Cooperation of Doxycycline with Phytochemicals and Micronutrients Against Active and Persistent Forms of Borrelia sp.

Phytochemicals and micronutrients represent a growing theme in antimicrobial defense; however, little is known about their anti-borreliae effects of reciprocal cooperation with antibiotics. A better understanding of this aspect could advance our knowledge and help improve the efficacy of current approaches towards Borrelia sp. In this study, phytochemicals and micronutrients such as baicalein, luteolin, 10-HAD, iodine, rosmarinic acid, and monolaurin, as well as, vitamins D3 and C were tested in a combinations with doxycycline for their in vitro effectiveness against vegetative (spirochetes) and latent (rounded bodies, biofilm) forms of Borrelia burgdorferi and Borrelia garinii. Anti-borreliae effects were evaluated according to checkerboard assays and supported by statistical analysis. The results showed that combination of doxycycline with flavones such as baicalein and luteolin exhibited additive effects against all morphological forms of studied Borrelia sp. Doxycycline combined with iodine demonstrated additive effects against spirochetes and biofilm, whereas with fatty acids such as monolaurin and 10-HAD it produced FICIs of indifference. Additive anti-spirochetal effects were also observed when doxycycline was used with rosmarinic acid and both vitamins D3 and C. Antagonism was not observed in any of the cases. This data revealed the intrinsic anti-borreliae activity of doxycycline with tested phytochemicals and micronutrients indicating that their addition may enhance efficacy of this antibiotic in combating Borrelia sp. Especially the addition of flavones balcalein and luteolin to a doxycycline regimen could be explored further in defining more effective treatments against these bacteria.

Isolation and characterisation of intestinal spirochaetes.

Faeces or rectal swabs from 1527 subjects were examined for the presence of intestinal spirochaetes by anaerobic culture on blood agar incorporating spectinomycin (400 mg/l). Twenty three specimens (1.5%) were positive, and only one of these came from a patient with diarrhoea. All positive specimens came from either Asians or known homosexuals. Comparative tests showed a close phenotypic similarity between the human isolates and non-pathogenic porcine intestinal spirochaetes. These organisms differ from Brachyspira aalborgi, a spirochaete isolated from subjects with histologically confirmed intestinal spirochaetosis.

Antimicrobial resistance in Brachyspira pilosicoli with special reference to point mutations in the 23S rRNA gene associated with macrolide and lincosamide resistance.

A point mutation in the 23S rRNA gene causes macrolide and lincosamide resistance in Brachyspira hyodysenteriae. The possible occurrence of a similar mutation in Brachyspira pilosicoli was studied and the MICs of six antimicrobial agents for Swedish field isolates of B. pilosicoli were determined. Of 10 isolates with high MICs of macrolide and lincosamide antibiotics, six had a mutation in nucleotide position 2058 or 2059 in the 23S rRNA gene as compared to the wild type of Escherichia coli, whereas none of 10 tylosin-susceptible isolates were mutated in this region. The mutations found in position 2058 were A --> T transversions, and in position 2059 either A --> G transitions or A --> C transversions. The MICs at which 90% of the B. pilosicoli field isolates were inhibited by tylosin, erythromycin, clindamycin, virginiamycin, tiamulin, and carbadox, were >256, >256, >4, 4, 2, and 0.125 microg/ml, respectively. In conclusion, point mutations in positions 2058 and 2059 of the 23S rRNA gene can cause macrolide and lincosamide resistance in B. pilosicoli. Macrolide resistance is widespread among Swedish field isolates of B. pilosicoli. Notably also a few isolates with elevated MICs of tiamulin were found.

Comparison of methods for antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for Brachyspira hyodysenteriae isolated in Germany.

In Germany treatment of swine dysentery is hampered by Brachyspira hyodysenteriae strains showing elevated MIC values to the few antibiotics licensed. Therefore, susceptibility testing of clinical isolates is an important service to the swine practitioner. This study compares the established agar dilution procedure for antimicrobial susceptibility testing of this fastidious anaerobe to the broth microdilution test newly developed [Anim. Health Res. 2 (2001) 59; Vet. Microbiol. 84 (2002) 123; J. Clin. Microbiol. 41 (2003) 2596]. A total of 221 isolates were examined twice with either test procedure using tiamulin and valnemulin as antibiotics. Both methods gave reproducible results, and the MIC values for the reference strains B. hyodysenteriae B204 and Staphylococcus aureus ATCC 29213 corresponded to previously published data. However, the results for individual strains differed significantly for both tests (P < 0.001) with MIC values being on average one dilution step lower in the broth dilution method. The 221 strains used for comparing test procedures were isolated between 1989 and 2001. An additional 102 strains isolated in 2002 were tested only with the broth dilution procedure. A significant rise in the average MIC value for both pleuromutilins could be demonstrated when comparing earlier isolates to those from 2000 to 2001 (P < 0.05), while in 2002 the average MIC significantly decreased when compared to the value in 2000 (P < 0.05). However, strains with MIC values for tiamulin as high as 8 microg/ml (broth dilution) could still be isolated.

Dietary enzyme and zinc bacitracin reduce colonisation of layer hens by the intestinal spirochaete Brachyspira intermedia.

Brachyspira intermedia strain HB60 was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 control birds were sham-inoculated with sterile broth. All chickens received an experimental layer diet based on wheat. The infected birds were randomly divided into four groups of 10, with the diet for each group containing either 50 ppm zinc bacitracin (ZnB), 100 ppm ZnB, 256 ppm of dietary enzyme (Avizyme), 1302), or no additive. Birds were kept for 6 weeks after infection, and faecal excretion of B. intermedia, faecal water content, egg numbers, egg weights and body weights were recorded weekly. Control birds remained uninfected throughout the experiment. B. intermedia was isolated significantly less frequently from the groups of experimentally infected birds receiving ZnB at 50 ppm or enzyme than from those receiving 100 ppm ZnB or no treatment. Infected birds had a transient increase in faecal water content in the week following challenge, but no other significant production differences were detected amongst the five groups of birds in subsequent weeks. It was not established how the ZnB at 50 ppm and the dietary enzyme reduced the ability of the spirochaete to colonise, but it may have been by bringing about changes in the intestinal microflora and/or the intestinal microenvironment.

Effects of metronidazole on periodontal treatment needs.

Periodontitis, a common cause of tooth loss in adult populations, is an inflammatory response to the overgrowth of anaerobic organisms such as spirochetes and bacteroides and, in some cases, micro-aerophilic organisms in the subgingival plaque. In the present investigation, using a double-blind clinical design, we sought to determine whether 1 week of metronidazole treatment plus debridement of the tooth surfaces was superior to 1 week of placebo treatment plus debridement (positive control) in reducing the subsequent amount of periodontal surgery given to the patients. Thirty-nine patients were randomly assigned to either the metronidazole or placebo (positive control) groups. All patients were given the necessary scaling and root planing and were unsupervised in their usage of the medication. After the completion of this treatment, they were reexamined and it was found that the metronidazole regimen caused a significant reduction in surgical needs of about 5 teeth per patient compared to the positive control (difference before and after treatment 8.3 +/- 6.8 teeth metronidazole versus 2.9 +/- 4.8 positive control, P = 0.007). The difference between groups was maintained during the 2 to 3 years' recall period. Metronidazole had a significant effect on the site specific reduction of spirochetes: 90% of the sites in the metronidazole group versus 64% in the positive-control group had a decrease in the percentage of spirochetes (P less than 0.05). We conclude that systemic metronidazole given 250 mg tid for 7 days in conjunction with debridement of the tooth surfaces can significantly reduce the need for periodontal surgery compared to the standard regimen which included only debridement.

Long-term treatment with subantimicrobial dose doxycycline exerts no antibacterial effect on the subgingival microflora associated with adult periodontitis.

BACKGROUND: The purpose of this study was to determine whether treatment with subantimicrobial dose doxycycline (SDD), 20 mg bid, exerted an antimicrobial effect on the microflora associated with adult periodontitis. METHODS: Following the approval of the protocol and informed consent forms by the respective IRBs at the University of Florida and West Virginia University, 76 subjects with adult periodontitis were entered and randomly assigned to receive SDD or placebo. A split-mouth design was utilized, with each subject receiving subgingival scaling and root planing (SRP) in two quadrants immediately following baseline data collection, while the remaining two quadrants were left unscaled (non-SRP). Microbial samples were collected prior to treatment, after 3, 6, and 9 months of treatment, and after 3 months of no treatment. The samples were examined by microscopy and by enumeration on selective and non-selective media. RESULTS: All treatments resulted in statistically significant decreases in the proportions of spirochetes and motile rods (P <0.05) and in an increase in the proportion of coccoid forms (P <0.0001) relative to baseline. No between-treatment differences were detected between the SDD and placebo treatments in either the SRP or non-SRP design, with the exception of the small and large spirochetal groups. The spirochetal proportions present in the SDD group were significantly lower (P<0.05) than the paired placebo group during the 9-month treatment and was preceded by a significant decrease (P<0.01) in the proportion of microbiologic sample sites that bled on probing. No between- treatment differences were detected in any of the other microbial parameters. CONCLUSION: The microbial differences observed were attributed to the anticollagenase and anti-inflammatory properties of SDD and not to an antimicrobial effect.

Metronidazole in periodontitis. I. Clinical and bacteriological results after 15 to 30 weeks.

The statistical association of certain anaerobic organisms such as black pigmented bacteroides (BPB) species and spirochetes with clinical signs of active periodontitis, i.e. bleeding upon probing and bone loss, suggests that the lesions may actually reflect a "specific infection" involving these or unidentified species. All the known oral species of BPB and spirochetes are anaerobes which suggests that antimicrobial treatment directed specifically against anaerobes might be effective in periodontal therapy. In this report, the short-term results of metronidazole treatment plus mechanical debridement in patients with extensive periodontal disease and of a double-blind clinical study in which metronidazole plus mechanical debridement is compared to placebo plus mechanical debridement are described. The findings indicate that 1 week of systemic metronidazole can optimize the clinical reduction of pockets and increase the apparent attachment in periodontitis patients who receive concurrent mechanical debridement of their root surfaces. In patients with extensive clinical involvement, metronidazole resulted in a significant reduction in the number of sites exhibiting pocket depths and attachment loss greater than or equal to 7 mm. When metronidazole plus mechanical debridement was compared with placebo plus mechanical debridement in a double-blind study, the metronidazole patients exhibited a significant improvement in those sites initially greater than or equal to 7 mm. The beneficial effect of the metronidazole was associated with a significant and sustained reduction of certain anaerobic organisms such as Bacteroides gingivalis and the large spirochetes. These data indicate that treatment aimed specifically toward the anaerobic component of the plaque flora can be associated with impressive clinical improvements 15 to 30 weeks after the initiation of treatment.