Low antibiotic resistance among anaerobic Gram-negative bacteria in periodontitis 5 years following metronidazole therapy.

The objective of this study was to assess antibiotic susceptibility among predominant Gram-negative anaerobic bacteria isolated from periodontitis patients who 5 years prior had been subject to mechanical therapy with or without adjunctive metronidazole. One pooled sample was taken from the 5 deepest sites of each of 161 patients that completed the 5 year follow-up after therapy. The samples were analyzed by culture. A total number of 85 anaerobic strains were isolated from the predominant subgingival flora of 65/161 patient samples, identified, and tested for antibiotic susceptibility by MIC determination. E-tests against metronidazole, penicillin, amoxicillin, amoxicillin + clavulanic acid and clindamycin were employed. The 73/85 strains were Gram-negative rods (21 Porphyromonas spp., 22 Prevotella/Bacteroides spp., 23 Fusobacterium/Filifactor spp., 3 Campylobacter spp. and 4 Tannerella forsythia). These were all isolated from the treated patients irrespective of therapy procedures (+/-metronidazole) 5 years prior. Three strains (Bifidobacterium spp., Propionibacterium propionicum, Parvimonas micra) showed MIC values for metronidazole over the European Committee on Antimicrobial Susceptibility Testing break point of >4 µg/mL. All Porphyromonas and Tannerella strains were highly susceptible. Metronidazole resistant Gram-negative strains were not found, while a few showed resistance against beta-lactam antibiotics. In this population of 161 patients who had been subject to mechanical periodontal therapy with or without adjunct metronidazole 5 years prior, no cultivable antibiotic resistant anaerobes were found in the predominant subgingival microbiota.

Methodological issues in the quantification of subgingival microorganisms using the checkerboard technique.

The reproducibility and reliability of quantitative microbiological assessments using the DNA-DNA hybridization "checkerboard method" (CKB) were assessed. The data originated from 180 chronic periodontitis patients, who were enrolled in a clinical trial and sampled at baseline, and 3 and 12m post-therapy. The samples were divided into two portions allowing evaluation of reproducibility. In total, 531 samples were analyzed in a first run, using standard bacterial preparations of cells and 513 samples were accessible for analysis in the second, using standards based on purified DNA from the species. The microbial probe panel consisted of periodontitis marker bacteria as well as non-oral microorganisms. Three different ways of quantifying and presenting data; the visual scoring method, VSM, the standard curve method, SCM, and the percent method, PM, were compared. The second set of analyses based on the use of standard preparations of pure DNA was shown to be more consistent than the first set using standards based on cells, while the effect of storage time per se up to 2.5y seemed to be marginal. The best reproducibility was found for Tannerella forsythia, irrespective of quantification technique (Spearman's rho=0.587, Pearson's r≥0.540). The percent method (PM) based on percent of High Standard (10(6) cells) was more reliable than SCM based on a linear calibration of the High Standard and a Low Standard (10(5) cells). It was concluded that the reproducibility of the CBK method varied between different bacteria. High quality and pure specific DNA whole genomic probes and standards may have a stronger impact on the precision of the data than storage time and conditions.

Oral administration of a new soluble branched beta-1,3-D-glucan is well tolerated and can lead to increased salivary concentrations of immunoglobulin A in healthy volunteers.

The soluble branched yeast beta-1,3-D-glucan (SBG) belongs to a group of carbohydrate polymers known to exert potent immunomodulatory effects when administered to animals and humans. A new oral solution of SBG has been developed for local application to the oropharyngeal and oesophageal mucosa in order to strengthen the defence mechanisms against microbial and toxic influences. In the present study oral administration of SBG has been investigated primarily for assessment of safety and tolerability in an early phase human pharmacological study (phase I). Eighteen healthy volunteers were included among non-smoking individuals. The study was an open 1:1:1 dose-escalation safety study consisting of a screening visit, an administration period of 4 days and a follow-up period. Groups of six individuals received SBG 100 mg/day, 200 mg/day or 400 mg/day, respectively, for 4 consecutive days. The dose increase was allowed after a careful review of the safety data of the lower dose group. No drug-related adverse event, including abnormalities in vital signs, was observed. By inspection of the oral cavity only minor mucosal lesions not related to the study medication were seen in seven subjects. Repeated measurements of beta-glucan in serum revealed no systemic absorption of the agent following the oral doses of SBG. In saliva, the immunoglobulin A concentration increased significantly for the highest SBG dose employed. SBG was thus safe and well tolerated by healthy volunteers, when given orally once daily for 4 consecutive days at doses up to 400 mg.

Intrafamilial transmission of black-pigmented, putative periodontal pathogens.

Porphyromonas gingivalis and Prevotella intermedia are black-pigmented, putative periodontopathogenic bacteria considered to cause some forms of periodontal disease. Porphyromonas gingivalis and P. intermedia can be transmitted between humans and produce periodontal disease in susceptible hosts. In this article, studies using molecular typing methods for determining the transmission of black-pigmented, putative periodontopathogens between family members are reviewed. As individuals living close to each other are more prone to transmit bacteria, the studies on transmission of periodontopathogens have been performed on family members. It has been shown that black-pigmented bacteria are not only transferred between spouses but also between parents and child. Since only a limited number of studies have been done, longitudinal and controlled studies should be carried out to elucidate further the transmittance potential of these bacteria.

The distribution and transmission of Actinobacillus actinomycetemcomitans in families with localized juvenile periodontitis.

The prevalence and distribution of A. actinomycetemcomitans in families where at least one family member (proband) suffered from localized juvenile periodontitis was investigated. 25 probands with localized juvenile periodontitis (LJP) and their 78 close family members were screened for the presence of A. actinomycetemcomitans. Among these 25 families, 10 contained at least one additional family member colonized with oral A. actinomycetemcomitans. Genomic DNA from subgingival A. actinomycetemcomitans strains from each of the probands and their family members were amplified and characterized by the polymerase chain reaction (PCR) using a single primer known to distinguish A. actinomycetemcomitans strains. The PCR products from each strain were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV light transillumination. The studies showed that 41.2% of the parents and 58% of the siblings in this LJP-based population harbored the bacterium. Comparison of the PCR generated amplitypes showed that there was a wide distribution of amplitypes among the probands and immediate relatives. No clear transmission paths were observed in this specific population.

Clinical and microbiological effects of adjunctive antibiotics in treatment of localized juvenile periodontitis. A controlled clinical trial.

The aim of this study was to assess the clinical and microbiologic effects of the combination of amoxicillin and metronidazole therapy as an adjunct to mechanical treatment in the management of localized juvenile periodontitis. Twenty-five localized juvenile periodontitis (LJP) patients from a Brazilian population were randomly allocated into an experimental group receiving mechanical treatment and antibiotics, and a control group receiving mechanical treatment and placebo. Clinical and radiographic assessments, as well as microbiologic sampling for Actinobacillus actinomycetemcomitans, were performed at baseline and one year after the end of the treatment. At the termination of the study A. actinomycetemcomitans could be isolated from the oral cavity of all patients in the control group who harbored the bacterium at baseline and in 4 out of 8 patients in the experimental group. Both treatment modalities resulted in significant benefit on an individual basis. The experimental group, however, displayed better results than did the control group regarding gingival index (GI), probing depth (PD), clinical attachment level (CAL), and radiographic analysis of crestal alveolar bone mass, but not with respect to plaque index (PI). No serious adverse effects of the antibiotic treatment were observed in the present study.

Relationship of serotype, leukotoxin gene type and lysogeny in Actinobacillus actinomycetemcomitans to periodontal disease status.

Actinobacillus actinomycetemcomitans has been associated with different forms of periodontitis, particularly with localized juvenile periodontitis (LJP). The bacterium possesses several virulence factors which have been shown to interact with the host immune system. Among these factors, leukotoxin, surface antigens (serotype) and bacteriophages have been suggested directly or indirectly to influence the course of infection. However, few studies have been able to show associations between these factors and periodontal disease, alone or in combination. Thus, the purpose of the present study was to investigate possible correlations between periodontal disease status and selected virulence factors (serotype, presence of bacteriophages, and the presence of a 530 bp deletion in the promoter region of the leukotoxin gene). 36 subjects took part in the study. Serotype c was the most frequently found serotype among periodontally affected subjects, although serotypes a and b were also present. 27 out of 36 strains harbored bacteriophages, and there was strong evidence that some of the bacteriophages were different from the previously characterized phi Aa phage. A. actinomycetemcomitans containing the F-fragment phage were more frequently associated with periodontal disease. Five strains, all serotype b, 3 from LJP patients and 2 from healthy subjects, showed a 530-bp deletion in the promoter region of the leukotoxin gene.

Localized juvenile periodontitis and Actinobacillus actinomycetemcomitans in a Brazilian population.

Localized juvenile periodontitis (LJP) has been used as a model for studying periodontal disease, and its prevalence is considered to be higher in third-world countries (0.3-8%) than in industrialized countries (0.1%). Mostly, the disease has been associated with Actinobacillus actinomycetemcomitans (A.a.) but lack of association has also been reported. The aim of this study was to identify LJP patients in geographically different Brazilian populations and assess the presence of A.a. in their periodontal lesions. 7843 children, 12-19-years of age, from the cities of Rio de Janeiro, Votorantim and Belo Horizonte were screened, and LJP patients were identified by strict clinical and radiographical criteria. A final LJP prevalence of 0.3%, with a 99% confidence interval between 0.16% to 0.47%, was found. The prevalence in the subpopulations varied between 0.1-1.1% in the different areas. Subgingival bacterial samples were obtained from the oral cavity of 25 patients and their family members. 80% of these patients, 39.5% of their family members, 35.3% of their parents, and 43.9% of all siblings were culture positive for A.a. All but one of the families had at least one member in addition to the patient who was culture positive for A.a. In 3 families, > 1 member showed radiographic and clinical signs of LJP. 30% of non-LJP subjects coming from one of the areas with higher LJP prevalence harbored A.a. We conclude that LJP is highly associated with A.a. in this Brazilian population.

Attachment loss and serum antibody levels against autologous and reference strains of Actinobacillus actinomycetemcomitans in untreated localized juvenile periodontitis patients.

Immunological data have been suggested to be a potential tool in the diagnosis, classification and monitoring of periodontal diseases. However, the role of circulating antibodies in periodontal patients is poorly understood. Patients suffering from localized juvenile periodontitis (LJP) are often reported to show high titers of serum IgG antibodies against Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), but several affected patients do not. Most studies use well-known reference strains of the bacterium for testing against the patients' sera. The aim of the present investigation was to study the relationship between serum IgG antibody levels to autologous A. actinomycetemcomitans strains and clinical attachment loss (CAL). In addition, we wanted to assess the patients' serum titers against 4 well-known reference strains of the bacterium as well as their general potential immunoglobulin response. Intravenous blood samples were taken from 23 LJP patients and 10 healthy individuals, and autologous A. actinomycetemcomitans strains were cultured from 18 of the LJP patients. CAL was measured at 4 different sites around all present teeth and assessed as a % of teeth with at least 1 site moderately > or = 2 < 5 mm) or severely (> or = 5 mm) involved. An enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the serum titers of IgG antibodies to A. actinomycetemcomitans antigens. No significant correlation was found between serum IgG antibody titers to autologous strains and CAL. However, there was a trend that low responders had more moderately affected teeth than had high responders and patients with undetectable A. actinomycetemcomitans levels, which is in agreement with a hypothetically protective role of the antibodies. The total counts of immunoglobulin assessed in all participants showed that the predominant class was IgG and the reference group displayed significantly less (p < 0.05) IgG and IgG1 counts than the LJP patients. Both the reaction pattern against reference and autologous strains varied widely. We conclude that the specific antibody response against A. actinomycetemcomitans shows a weak correlation to clinical attachment levels in LJP patients.

Microbiological and immunohistological findings in a patient with Papillon-Lefèvre syndrome.

The following communication is a case history of an 11 year-old female patient suffering from Papillon-Lefèvre syndrome. Since a massive occurrence of A. actinomycetemcomitans had been found in the subgingival microflora of the periodontal pockets, the patient was treated with repeated subgingival scaling, with an adjunct Amoxicillin and Metronidazol treatment. A bacteriological examination of the girl's family proved that several brothers and sisters as well as one parent also carried. A. actinomycetemcomitans, showing 3 different strains of this bacterium within the family. An immunohistological examination of the gingival tissue showed a massive inflammatory infiltrate which was dominated by plasma cells. The histological investigation of the first molars did not show morphological abnormalities of the root cementum. Posttreatment clinical and radiographical improvement of the periodontal conditions is reported despite the recurrent finding of A. actinomycetemcomitans.

The natural history of periodontal disease. The correlation of selected microbiological parameters with disease severity in Sri Lankan tea workers.

The purpose of this study was to assess the prevalence of A. actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia, and their association with periodontal disease states in a population sample from Sri Lanka. Based on clinical parameters, a total of 536 sites in 268 male Sri Lankan tea workers were categorized as healthy, or showing gingivitis only, moderate or advanced periodontitis. Bacterial samples were obtained from all sites and the three target bacteria identified by indirect immunofluorescence. P. intermedia, P. gingivalis and A. actinomycetemcomitans were found in 76%, 40% and 15% of the subjects, respectively. Of the 536 periodontal sites, 10.5% were categorized with "no disease", 14% "gingivitis only": 59% with moderate and 16% with advanced periodontitis. The prevalence of P. gingivalis and P. intermedia was significantly higher in sites with moderate and advanced periodontitis than in sites with no disease or gingivitis only. A. actinomycetemcomitans was not found in healthy sites, but occurred with equal frequency in sites with gingivitis, moderate and advanced periodontitis. The association between these three bacteria and periodontal diseases in Sri Lankan tea laborers was similar to that described for other non-industrialized and industrialized countries.

Bacterial resistance following subgingival and systemic administration of minocycline.

The aim of the present study was to compare total numbers of cultivable bacteria and prevalence of resistance to minocycline among periodontal bacteria following subgingival or systemic application of minocycline in patients suffering from periodontal disease. 10 adult patients were administered 2% minocycline ointment subgingivally into their periodontal pockets at baseline, week 2 and months 1, 3, 6 and 9. Patients had scaling/root planing at baseline and month 6. In addition, 10 patients undergoing scaling/root planing followed by a 10-day course of systemic minocycline therapy, were studied and compared with the subgingival application group. Bacterial samples were taken from the 4 deepest pockets before each subgingival application of the drug. The systemic administration group was sampled at baseline and at week 2, as well as months 1 and 3 after completing the antibiotic treatment. For each patient at each sampling, bacterial samples were pooled, diluted, seeded on parallel blood agar plates and incubated aerobically and anerobically. After incubation, 30 colonies were picked at random and transferred to blood agar plates supplemented with 10 micrograms/ml minocycline, to estimate prevalence of minocycline-resistant bacteria. The results of this study indicate that subgingival application of minocycline ointment resulted in an initial reduction in total numbers of cultivable bacteria, which then remained depressed during the full year of the study. No such observation was made in the systemic administration. Both in the subgingival and the systemic administration group, the % of cultivable aerobic and anaerobic minocycline-resistant bacterial strains increased transiently following administration of the drug, but returned to baseline levels within 3 months post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiologic tests in epidemiologic studies: are they reproducible?

Microbiologic assessments are often included in longitudinal studies to elucidate the significance of the association of certain Gram-negative bacteria and the development of periodontal diseases. In such studies, the reliability of methods is crucial. There are several methods to identify putative pathogens, and some of them are commercially available. The purpose of the present study was to compare the reproducibility of four different methods for detecting Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in order to evaluate their usefulness in epidemiologic studies. The test panel consisted of 10 young subjects and 10 adult periodontitis patients. Subgingival plaque was sampled from sites showing bone loss and "healthy" control sites. The four different methods for detecting the target bacteria were 1) cultivation, 2) Evalusite (a chair-side kit based on ELISA), 3) OmniGene, Inc, based on DNA probes, and 4) indirect immunofluorescence (IIF). The test procedure was repeated after a 1-wk interval and was performed by one examiner. Sites reported to be positive for a microorganism by any of the four methods at one or both examinations were considered to be positive for that organism and included in the analysis. The reproducibility of the four methods was low. The IIF and the cultivation methods showed somewhat higher reproducibility than did the commercial systems. A second test was done for Evalusite, three paper points for sampling being used instead of one as described in the manual. The reproducibility of the second test was improved, indicating that the detection level of the system may influence the reliability.

Incidence of early periodontitis in a group of young individuals during 8 years: associations with selected potential predictors.

The aim of the present study was to assess the incidence of early radiographic bone loss in a birth cohort over 8 years and to assess possible associations between incidence of bone loss and reported dental behavior, ethnic background, and previous orthodontic treatment. In a case control study comprising a proportion of the study population, the detection of black pigmented Bacteroides and Actinobacillus actinomycetemcomitans and their association with early radiographic bone loss was assessed. At the beginning of the study in 1984, there were 2,767 subjects. In 1992 sets of bite-wing radiographs were obtained from 215 subjects, who also filled out a questionnaire concerning their present and past dental behavior, ethnic background, and orthodontic treatment. Radiographic alveolar bone loss was recorded if the distance from the cemento-enamel junction to the alveolar crest exceeded 2 mm. Thirteen subjects (6%) showed new sites with bone loss over the 8-year period. Subgingival plaque was sampled from these 13 subjects and from 13 control subjects. None of the independent variables could be associated with the observed incidence of radiographic bone loss in this cohort, with the possible exception of the presence of A. actinomycetemcomitans which was detected in about 50% of the new sites with bone loss.

Prevalence and distribution of bacteriophage phi Aa DNA in strains of Actinobacillus actinomycetemcomitans.

phi Aa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans. Since the discovery of phage phi Aa, additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of phi Aa or phi Aa-related temperate phages in this species, a phi Aa-specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans. Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage phi Aa probe. A bacteriophage designated phi Aa33384 was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the phi Aa probe. The phi Aa probe hybridized with the DNA extracted from bacteriophage phi Aa33384. The distribution of the phage phi Aa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage phi Aa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.

Use of a nonradioactive genetic probe identified, synthesized, and labeled in the polymerase chain reaction.

This study introduces a strategy to identify and produce sequences useful as genetic markers, or native genetic probes for DNA-DNA hybridization in bacterial strains where the genetics is not well described. Actinobacillus actinomy-cetemcomitans (A.a.) was used as an example. Fifty ng genomic DNA from A.a. ATCC 33384 and Haemophilus aphrophilus ATCC 33389 was amplified in a thermocycler using a single 10-mer primer. The PCR products were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV illumination, and the strain-specific amplitypes were compared. DNA from two bands, 0.9 and 4 kb, unique for the A.a. strain, was cut out, amplified under high stringency with the same primer and labeled by replacing 33.3 microM dTTP with digoxigenin-labeled dUTP in the reaction mixture. The labeled probe was then repeatedly used for hybridization to DNA from various A.a., H. aphrophilus, and other bacterial strains of the Pasteurellaceae family. The results showed that the 0.9-kb probe detected all A.a. tested, and distinguished it from other closely related bacterial species. We conclude that the described strategy is useful for identifying and selecting genetic sequences useful as genetic markers in A.a.

Chlorhexidine use after two decades of over-the-counter availability.

Chlorhexidine (CHX) is a compound with plaque-inhibiting effects and available only by prescription in the United States. In Norway, however, CHX has been dispensed over-the-counter for over 20 years, and this study was undertaken to evaluate dentists' perceptions regarding its indications, usefulness, and side effects. A written questionnaire was sent to a sample of 10% of dentists registered in Norway, 78% of whom (354) responded. Additionally, representatives from 2% of all dental practices in Norway were contacted by telephone. Fourteen percent (14%) of the respondents reported that they never recommended CHX to their patients. Among those recommending the compound, 85% used it frequently after surgical periodontal procedures; 74% when treating acute gingivitis; 57% following oral surgery in general; and 35% during non-surgical periodontal therapy. It was used also as an adjunct to other treatment routines. Seventy-three percent (73%) reported frequent use of CHX when treating stomatitis and 54% in herpes simplex infections. As to side effects, 77% of the dentists indicated that staining of teeth, restorations, and the tongue was a major concern to patients; 12% reported inconveniences due to the bitter taste; and 6% reported other disturbances such as dryness of the mouth and development of oral ulcerations. The majority (94%) of the dentists recommended mouth rinsing, whereas 6% recommended a gel form. Only 4% of the dentists recommended rinsing with a patient-diluted 0.1% concentration, whereas 96% recommended the standard 0.2% formulation; 88% recommended using CHX mouthwash twice a day or more often.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution and transmission of Actinobacillus actinomycetemcomitans in families with established adult periodontitis.

The prevalence and genotype distribution of Actinobacillus actinomycetemcomitans strains in families where at least one adult family member (proband) suffered from periodontal disease was investigated to better understand how this periodontal organism is acquired or transmitted. Fifteen probands with severe (established) periodontal disease (EPD) and their 46 immediate family members were sampled for A. actinomycetemcomitans. Among the 15 families, 10 contained at least one additional family member colonized with oral A. actinomycetemcomitans. Genomic DNA from 3 subgingival A. actinomycetemcomitans strains from each of the 10 probands and their 17 family members were amplified and characterized by the polymerase chain reaction (PCR) using a single arbitrary primer known to distinguish A. actinomycetemcomitans strains. The PCR products from each strain were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV light transillumination. The amplification products migrated to form readily distinguishable bands and, since the banding patterns were characteristic of strains of A. actinomycetemcomitans, these patterns were called "amplitypes." The culture studies showed that 51% of all patients suffering from EPD carried oral A. actinomycetemcomitans. Moreover, 50% of their spouses and 30% of their children harbored the bacterium. Comparison of the PCR-generated amplitypes showed that 26 out of 27 individuals had strains exhibiting a single amplitype of A. actinomycetemcomitans, the 27th being colonized by 2 different amplitypes. They also showed that in 6 out of 7 families, the husband and wife did not harbor the same A. actinomycetemcomitans amplitype. Furthermore, most often children carried an an amplitype identical to one of the parents.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of strains of Actinobacillus actinomycetemcomitans by arbitrarily primed polymerase chain reaction.

The present study describes a method for amplifying DNA in Actinobacillus actinomycetemcomitans by using short, synthetic oligonucleotides of random sequence as primers in the polymerase chain reaction. Genomic DNA from each of 20 human isolates of A. actinomycetemcomitans was successfully amplified in a thermal cycler with a single synthetic primer (GGGTAACGCC) and reproducibly produced 14 different DNA amplification profiles (amplitypes). A. actinomycetemcomitans isolates from the same subject revealed the same amplitype. The arbitrarily primed polymerase chain reaction appears to be useful in characterizing human isolates of A. actinomycetemcomitans for studies of epidemiology and bacterial transmission.

Prevention of transmission of resistant bacteria between periodontal sites during subgingival application of antibiotics.

This study was designed to investigate whether antibiotic resistant micro-organisms are able to contaminate and survive on syringe tips used for subgingival deposition of antibiotics, and to test simple and effective means of disinfecting the syringe tip between applications. In the first part of the study, syringe tips used for application of Minocycline subgingival formula in 20 adult periodontitis patients were cultured for bacteria resistant to this drug before and after disinfecting them with ethanol. The results showed that 80% of the unwashed syringes were culture positive for minocycline resistant bacteria, whereas only 1 ethanol washed syringe tip was contaminated. In part II of the study, after dispensing minocycline periodontal formula in 20 patients, 10 of the syringe tips were washed with ethanol while 10 were left untreated. All syringes were stored in a refrigerator for 8 days, whereafter the tips were sampled for resistant bacteria. 20% of the unwashed tips were contaminated after 8 days incubation at 4 degrees C. None of the ethanol washed syringe tips were culture positive. We conclude that syringe tips may be contaminated with antibiotic resistant bacteria after dispensing the antibiotic in periodontal pockets. The transmission of these bacteria to other periodontal sites may be avoided by disinfecting the syringe tip with ethanol between applications. We have also shown that antibiotic resistant bacteria may survive on the syringe tip following 8 days storage in a refrigerator, suggesting that syringes used for subgingival deposition of an antibiotic should not be stored for reuse.