Antibiotikatherapie der Parodontitis mit Metronidazol

Das Metronidazol (z.B. Clont®) gehört zu der Gruppe der Nitroimidazole, die vor allem eine sehr gute Wirksamkeit gegenüber anaerob wachsenden Bakterien, d.h. einen Stoffwechsel ohne Sauerstoffbedarf aufweisen. Metronidazol ist uns als Antibiotikum nicht geläufig, da die Behandlung von Infektionen durch solche Erreger selten ist. Sie betreffen hauptsächlich den Darm und die Parodontitis. Aber hier ist Metronidazol sehr effektiv und hat eine bakterizide, d.h. bakterienabtötende Wirkung.

Metronidazol ist gegen wichtige Markerkeime der Parodontose wirksam

Das Wirkspektrum von Metronidazol beinhaltet alle anaeroben Bakterien und Protozoen. Insbesondere ist Metronidazol ist gegen folgende Parodontitismarkerkeime wirksam:

Van-Winkelhoff-Cocktail deckt das Erregerspektrum ab

Gegen den Markerkeim Aggregatibacter actinomycetemcomitans (A.a.) ist Metronidazol nicht wirksam. Beim Nachweis dieses Erregers ist die Gabe von Amoxicillin oder Ciprofloxacin indiziert. Durch ein fast immer vorliegende Mischinfektionen verschiedener Keime ist beim Auftreten von A.a. eine Kombinationsbehandlung unumgänglich. Für die ungezielte Behandlung bei schweren Infektion wird die Kombination von Metronidazol und Amoxicillin favorisiert ("van-Winkelhoff-Cocktail"), dessen Wirksamkeit durch viele Studien belegt werden konnte. Diese beiden Antibiotika sind gegen fast alle relevanten Parodontose-Bakterien wirksam, Dosierung siehe unten.

Die Kombinaton von Metronidazol mit Ciprofloxacin ist im Falle einer Unverträglichkeit von Amoxicillin/Penicillin alternativ indiziert.

Nebenwirkungen von Metronidazol

Die Nebenwirkungen können wie bei allen Antibiotika Magen-Darm-Probleme sein. Hinzu kann es zu einem metallische Geschmack, Schwindel, Kopfschmerzen oder einer peripheren Neuritis kommen. Während der Einnahme von Metronidazol sollte man kein Alkohol trinken, weil dadurch der sogenannte "Antabus-Effekt" ausgelöst werden kann, mit heftiger Übelkeit, Kopfschmerzen, Herzrasen etc..

Empfohlene Dosierung

Metronidazol (z.B: Clont®) 3x400mg pro Tag über 7 Tage

Amoxicillin 3x500mg + Metronidazol und 3x400mg pro Tag über 7 Tage

Arie J. van Winkelhoff, Carolien J. Tijhof, and J. de Graaff, Microbiological and Clinical Results of Metronidazole Plus Amoxicillin Therapy in Actinobacillus actinomycetemcomitans-Associated Periodontitis, Journal of Periodontology, January 1992, Vol. 63, Nr. 1, 52-57

Anne D. Haffajee, Systemic Anti-Infective Periodontal Therapy. A Systematic Review, Annals of Periodontology, December 2003, Vol. 8, Nr. 1, 115-181

W. J. Loesche et al., Metronidazole in Periodontitis: I. Clinical and Bacteriological Results after 15 to 30 Weeks, Journal of Periodontology, June 1984, Vol. 55, Nr. 6, 325-335

Bildquelle: Fotolia molekuul.be

Leistungen

Qualifizierte Spezialisten in Ihrer Nähe (Deutschland).

Die Fragen in unserem Parodontose-Selbsttest sind so gewählt, dass Sie sie entweder direkt oder mit Hilfe eines Spiegels leicht beantworten können. So können Sie sich schnell über Ihr persönliches Parodontitis-Risiko informieren. Zur 1. Frage.

die Munddusche mittlerweile nicht mehr nur als Lifestyleprodukt gilt, sondern sich als gutes Hilfsmittel in der Zahnzwischenraumpflege, die bei der Parodontitisbehandlung besonders wichtig ist, etabliert hat? Mehr über die Munddusche.

Van winkelhoff cocktail

This invention was made in part with U.S. Government support under Grant Number DE04898. The United States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Periodontal diseases in man are bacterial infections in which Gram negative bacteria play an essential role. During the last decade it has become clear that the subgingival microbiota associated with this disease is not uniform, but is different in the various clinical forms. The adult form of periodontitis is often characterized by an anaerobic type of microflora in which, among others, black-pigmented Bacteroides species dominate i.e. Bacteroides gingivalis and Bacteroides intermedius. On the other hand, in the local form of juvenile periodontitis, Actinobacillus actinomycetemcomitans seems to be an etiological agent.

Scientific data have become available that show that A. actinomycetemcomitans is an important etiological factor in several forms of periodontitis. There is substantive evidence that this bacterium is not only involved in localized juvenile periodontitis, but it is also associated with severe adult periodontitis. Studies have revealed that A. actinomycetemcomitans associated periodontitis is more difficult to treat with conventional mechanical therapy than other forms of periodontitis. Conventional mechanical therapy includes supra and subgingival debridement and periodontal surgery. Furthermore, it has been shown that success of treatment in A. actinomycetemcomitans positive patients is strongly associated with the eradication of this microorganism in both juveniles and adults. Since conventional mechanical therapy is not able to arrest the disease progression in most A. actinomycetemcomitans positive patients, antibiotics have been introduced as an adjunct to mechanical and surgical treatment of A. actinomycetemcomitans associated periodontitis. The antibiotic of choice is on of the tetracyclines (tetracycline, doxycycline and minocycline). Disadvantages of the use of tetracyclines relate to the fact that their biological effects are bacteriostatic and include the relatively long period of appliance (2-7 weeks) and often the recolonization of the pockets of A. actinomycetemcomitans after some time.

Therefore, there still exists a need for an effective treatment for A. actinomycetemcomitans related periodontitis.

SUMMARY OF THE INVENTION

The invention comprises, a pharmaceutical composition for use, adjunctive with mechanical procedures, in the treatment of A. actinomycetemcomitans associated periodontal disease, which comprises an effective amount of a combination of Amoxicillin and Metronidazole. The invention also comprises a method for treating A. actinomycetemcomitans associated periodontal disease comprising treating with a pharmaceutical composition comprising the combination of Amoxicillin and Metronidazole.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A -- depicts the changes in probing attachment obtained by the previous treatment procedures.

FIG. 1B -- illustrates the results obtained three months beyond the treatment with Amoxicillin and Metronidazole.

DETAILED DESCRIPTION OF THE INVENTION

An effective amount comprises an amount of the combination of Amoxicillin and Metronidazole such that subgingival elimination of A. actinomycetemcomitans is achieved after about 7 days of administration. Preferably conventional dosages of Amoxicillin and Metronidazole are found effective. By conventional is meant about 250 mg of Metronidazole and between 250 to 375 mg of Amoxicillin three times a day.

The regime for Metronidazole is adapted according to current recommendations from the "1989 Physicians Desk Reference" (PDR) regarding infections mainly caused by anaerobic Gram-negative organisms; adult dosage is up to 30 mg/kg/24 hours (approx. 500 mg, four times a day for a 75-kg adult). Children's dose is adjusted according to weight. The same dose/kg is justified, however, children's dose is listed as up to 50 mg/kg/24 hours for Amebiasis. The treatment is extended to 7 days.

The regime for Amoxicillin is adapted according to current recommendations from PDR regarding infections of the ear, nose and throat due to streptococci, pneumococci and H. influenzae, and organisms closely related to A. actinomycetemcomitans. Adult dosage is up to 500 mg three times a day and up to 40 mg/kg/day for children. Children's dose is adjusted according to weight. The treatment is extended to 7 days.

This treatment of A. actinomycetemcomitans associated periodontitis has superior and unexpected beneficial effects. With a 7 day's therapy of this combination of antibiotics applying conventional dosages, it has been possible to eradicate A. actinomycetemcomitans from the pockets for a period of at least 1 year. Although not wishing to be bound by theory, it is believed that since the clinical improvement and the predictable eradication of A. actinomycetemcomitans was not achieved with either of the single antibiotics it seems that an in vivo synergistic effect is responsible for the effectiveness of the combination therapy.

The following examples and preparations describe the manner and process of using the invention and set forth the best mode contemplated by the inventors of carrying out the invention, but are not to be construed as limiting.

Patients who previously had received varying amounts of treatment (including systemic antibiotic) and tested positive for A. Actinomycetemcomitans, received 250 mg of Metronidazole plus 375 mg of Amoxicillin, three times a day for a period of 7 days.

A. actinomycetemcomitans was suppressed below detection level in all patients who had received treatment with the combination of Metronidazole plus Amoxicillin (Table 1 below) with the one exception of patient no. 11, who stopped treatment due to serious diarrhea two days after the start of the treatment.

The finding that A. actinomycetemcomitans was still undetectable 2-4 months after therapy, suggests a subgingival elimination of this micro-organism. This elimination of A. actinomycetemcomitans was not only observed in the group of LJP patients, but also in the group of RPP patients. Microbiological re-examination of 16 patients 9-11 months after therapy revealed that A. actinomycetemcomitans was still undetectable in the subgingival area. B. gingivalis could not be isolated after therapy, with the exception of patient no. 11. B. intermedium was recovered from 6 patients after therapy ranging from less than 1% up to 5% of the anaerobically cultivated microflora. The clinical condition after treatment improved markedly. In the group of patients with no history of periodontal treatment, the mean probing pocket depths of the sampled sites was reduced from 7.4 mm to 5.5 mm. In the group of patients which had received periodontal treatment in the past, the mean probing pocket depth of the sampled sites decreased from 7.2 mm to 5.3 mm. The number of pockets showing bleeding on pocket probing decreased from 98% pretreatment to 48% after treatment. Moreover, the majority of the residual bleeding pockets after therapy exhibited marked decrease in the degree of bleeding on probing.

Six patients with LJP, ages 17-22, previously treated for A. actinomycetemcomitans associated severe juvenile periodontal disease were treated with a combination of 500 mg Metronidazole four times a day and 250 mg Amoxicillin three times day. All patients were initially severely infected with subgingival A. actinomycetemcomitans in deep pockets despite previous comprehensive treatment including the administration o systemic tetracycline 250 mg four times a day for up to four weeks.

Localized Juvenile Periodontitis (LJP) is generally recognized as difficult to treat, and several clinical studies indicate an initial failure rate of up to 25%. The association between remaining Actinobacillus actinomycetemcomitans after therapy and continuing periodontal deterioration is also well established. They received additional subgingival scaling and root planing to confirm removal of all detectable subgingival deposits.

Immediately following the 7 days of administration of the treatment, all patients were negative for subgingival A. actinomycetemcomitans by microbiologic culture methods. They remained negative for A. actinomycetemcomitans for the 3 month follow up period, when the clinical conditions were critically evaluated by probing pocket depth, probing attachment level measurements and computer assisted radiographic methods. The improvements for the affected teeth can be summarized as follows: a mean pocket reduction of 2.1 mm/subject; a mean increased attachment level of 1.0 mm/subject; and an average reduction of bleeding on probing by 20%. FIG. 1A depicts the changes in probing attachment obtained by the previous treatment procedures (scaling and rootplaning + systemic tetracycline), and FIG. 1B illustrates the results obtained 3 months beyond the treatment with the combination of Amoxicillin and Metronidazole.

This study demonstrates marked reduction of subgingival A. actinomycetemcomitans and resolution of clinical signs of disease in LJP patients through the use of a combination of systemic Metronidazole and Amoxicillin.

Comparative studies were conducted in 70 A. actinomycetemcomitans positive patients, whom previously were administered a variety of treatment regimes.

Fifteen of these patients were treated previously by conventional mechanical treatment (subgingival scaling & rootplaning), including 4 patients with full mouth surgery (all refractory periodontitis patients); 12 patients were treated conventionally with an adjunct tetracycline therapy (3 weeks) without clinical success; 43 A. actinomycetemcomitans positive patients were previously untreated. All patients were treated conventionally with an adjunct Metronidazole plus Amoxicillin therapy (seven days of 250 mg Metronidazole and 375 mg Amoxicillin, three times a day for 7 days -- same regimen for all groups).

The results of the combination therapy are summarized in the attached Tables 2, 3 and 4.

The results of the use of the combination therapy include eradication of A. actinomycetemcomitans in all patients except one for which no explanation is available. A possibility is a bad compliance of the medication. All other patients became free of A. actinomycetemcomitans. Moreover, recolonization of the periodontal pockets by A. actinomycetemcomitans has not been observed for up to 12 months. In addition to the elimination of A. actinomycetemcomitans, B. gingivalis was eliminated from all positive patients. Despite clinical improvement, B. intermedius was found in a substantial number of patients after therapy.

Four patients (A-D) are presented. Patients A (33 y.) and B (45 y.) had received periodontal treatment in the past, consisting of scaling and root planing in conjunction with oral hygiene instructions, however without the use of antimicrobiological agents. Despite this treatment these patients showed progressive periodontitis (refractor periodontitis). Patients C (37 y.) and D (36 y.) had no history of periodontal treatment. All four patients were referred to the Clinic for Periodontology Amsterdam for diagnosis and treatment of severe periodontitis in relation to their age. The patients were selected for microbiological investigation on the basis of the following criteria: ≧ 4 sites with probing pocket depth of ≧ 6 mm, bleeding and/or suppuration on probing of the pocket, and radiographic evidence of 50% loss of alveolar bone in several parts of the oral cavity including angular bony defects. The bleeding tendency after gentle probing was recorded as 0 (no bleeding), 1 (minor bleeding) and 2 (overt, spontaneous bleeding). None of the patients had received antibiotics 6 months prior to the initial subgingival sampling.

Subgingival plaque samples were obtained by paper points from 4 deep pockets in each patient. The samples were pooled and processed by microbiological techniqies for enumeration of A. actinomycetemcomitans and black pigmented Bacteroides species. All four patients were positive for A. actinomycetemcomitans, whereas patients C and D were also infected with B. gingivalis and B. intermedius.

Patients then received initial treatment i.e. oral hygiene instructions and scaling and root planing for 4 to 6 hours in total, with (patients A, B and C) or without (patient D) an adjunct minocycline therapy (100 mg/day for 14 days, with an initial dose of 200 mg). The clinical and microbiological results were evaluated with time intervals of approximately 3 months. The mean probing pocket depth of the four sample sites ranged from 7.5-9 mm. These sites were the same in a patient throughout the evaluation period.

Initially, all sample sites bled on probing, and 50% displayed suppuration.

Clinically none of the patients responded satisfactory to the initial treatment. No significant reduction in probing pocket depth and bleeding on probing was observed in the refractory periodontitis patients A and B. This was in line with the absence of response to initial treatment in the past. Patients C and D did respond to the initial treatment to some extent. However, the clinical situation after initial treatment was not satisfactory including the presence of deep bleeding pockets (Table 5). The initial treatment did not result in the elimination of A. actinomycetemcomitans in either of the patients, despite the use of minocycline (patients A, B and C). Moreover, three of the four patients showed an increase in the percentage and total number of A. actinomycetemcomitans cells after initial treatment (Table 5). Black pigmented Bacteroides species could no longer be isolated from patient C; whereas, patient D was still positive for these microorganisms after the initial therapy. The clinical results of the initial treatment in combination with the microbiological data were the basis for further treatment consisting of surgery (patient C) or continuous mechanical treatment in conjunction with an antibiotic therapy consisting of a combination of two antibiotics, i.e., Metronidazole plus Amoxicillin, 250 mg and 375 mg, respectively, three times a day for 7 days (patients A, B and D). In patient C, surgery resulted in shallow pockets and reduction in the number of bleeding sites. However, A. actinomycetemcomitans was not below detection level, although the number of colony forming units was reduced to only 37 (Table 5). Three months later, this patient showed again an increase in probing pockets depths, an increase in the number of bleeding sites in conjunction with a higher bleeding tendency, despite thorough maintenance care. This clinical decline was associated with a significant increase in the number of A. actinomycetemcomitans cells. At that moment, patient C also received the antibiotic therapy of Metronidazole and Amoxicillin.

In all four patients a significant clinical improvement was obtained after the antibiotic therapy with Metronidazole plus Amoxicillin. With this adjunctive therapy, further reduction in probing pocket depths and reduction in the number of bleeding sites was observed. This clinical improvement was associated with the elimination of subgingival A. actinomycetemcomitans; all four patients had no detectable levels of this microorganism after this therapy and were still negative for this microorganism on repeated sampling after several months (Table 5).

These data show that microbiological monitoring in severe periodontal disease can be a meaningful and practical supplement in the diagnosis, the treatment planning and treatment evaluation of these forms of periodontitis. This study also shows that subgingival elimination of A. actinomycetemcomitans may serve as a microbiological endpoint of periodontal treatment, since improvement of the periodontal condition is clearly associated with the subgingival elimination of A. actinomycetemcomitans. Minocycline was found to be inactive in eliminating A. actinomycetemcomitans from the subgingival area. The above results are also in agreement with previous studies in which it was shown that tetracyclines were not successful in suppressing A. actinomycetemcomitans in LJP patients and that further periodontal destruction is associated with continued presence of A. actinomycetemcomitans.

In patient C a significant reduction in the number of A. actinomycetemcomitans was obtained with the surgical treatment. However, the improved clinical situation of patient C appeared to be not stable after the surgery. This was strongly associated with the outgrowth of A. actinomycetemcomitans. From these data one may conclude that even 37 CFU (Colony Forming Units) recovered of this microorganism in the subgingival area is too many and may lead to further periodontal inflammation and periodontal breakdown. The combination of Metronidazole plus Amoxicillin appeared to be very active against subgingivally occurring A. actinomycetemcomitans, since eradication below detection level of this bacterium was achieved in all four patients. Repeated microbiological examinations in the patients of the present study has revealed no reinfection of the subgingival area.

As control to the claims of synergistic action for Amoxicillin and Metronidazole data were obtained for 10 A. actinomycetemcomitans positive adult periodontitis patients. They were treated by conventional techniques with adjunctive systemic antibiotics (4 with Amoxicillin 375 mg three times per day and 6 with Metronidazole 250 mg three times per day).

The results (Tables 6 and 7) clearly indicate that neither of the two antibiotics can predictably eradicate subgingival A. actinomycetemcomitans and further strengthen the claim of synergistic effect.

Other embodiments of the invention will be apparent to the skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Antibiotic treatment/therapy

Occasionally in exceptional cases it is necessary to give antibiotics in addition to the other therapies. Most of the disease can be treated with conservative treatment methods. Specific germs or bacterial range, however, should be treated with medication in addition to those. This is differentiated into local or systemic application. The local application of antibiotics is predominantly useful in localized or chronically defects, while the systemic treatment is considered, if the presence of certain bacteria could be determined.

Antibiotic treatment – course of antibiotics to treat gum diseases

The standard therapy for acute, aggressive periodontits is the so called “Winkelhoff Cocktail” that is named after van Winkelhoff.

A combination of Amoxycillin and Metronidazol: 3 x 375 – 500 mg/day Amoxycillin + 3 x 250 – 500 mg Metronidazol for 8 days.

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Release of metronidazole from electrospun poly( l -lactide-co- d / l -lactide) fibers for local periodontitis treatment

Objectives

We aimed to achieve detailed biomaterials characterization of a drug delivery system for local periodontitis treatment based on electrospun metronidazole-loaded resorbable polylactide (PLA) fibers.

PLA fibers loaded with 0.1–40% (w/w) MNA were electrospun and were characterized by SEM and DSC. HPLC techniques were used to analyze the release profiles of metronidazole (MNA) from these fibers. The antibacterial efficacy was determined by measuring inhibition zones of drug-containing aliquots from the same electrospun fiber mats in an agar diffusion test. Three pathogenic periodontal bacterial strains: Fusobacterium nucleatum , Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were studied. Cytotoxicity testing was performed with human gingival fibroblasts by: (i) counting viable cells via live/dead staining methods and (ii) by exposing cells directly onto the surface of MNA-loaded fibers.

MNA concentration influenced fiber diameters and thus w/w surface areas: diameter being minimal and area maximal at 20% MNA. HPLC showed that these 20% MNA fibers had the fastest initial MNA release. From the third day, MNA release was slower and nearly linear with time. All fiber mats released 32–48% of their total drug content within the first 7 days. Aliquots of media taken from the fiber mats inhibited the growth of all three bacterial strains. MNA released up to the 28th day from fiber mats containing 40% MNA significantly decreased the viability of F. nucleatum and P. gingivalis and up to the 2nd day also for the resistant A. actinomycetemcomitans. All of the investigated fibers and aliquots showed excellent cytocompatibility.

Significance

This study shows that MNA-loaded electrospun fiber mats represent an interesting class of resorbable drug delivery systems. Sustained drug release properties and cytocompatibility suggest their potential clinical applicability for the treatment of periodontal diseases.

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Topic 3 Antibiotics in Periodontology

ИГРАТЬ

2 - alter cell membrane integrity (NOT in perio)

3 - inhibit ribosomal protein synthesis

4 - suppress DNA synthesis

5 - inhibit folic acid synthesis (NOT in perio)

2 - slow growth and stress response - slow cellular turnover, less susceptible to antibiotics

3 - heterogeneity - same bacteria are not equal in a biofilm, different phenotype/genotype/response to ab's

2 - choice of ab

3 - admin routes (systemic, local)

4 - clinical evidence of effectiveness

5 - should NOT be used as monotherapy for tx

Inhibits cell wall synthesis

Adverse effects - allergy, diarrhoea, vomiting, nausea, thrush, skin rash

Long half life (3.2 days)

Inhibits protein synthesis

Adverse effects - GI complications, prolonged QT interval (beware heart conditions)

Amoxicillin (500mg) + Metronidazole (400mg)

Interferes with DNA synthesis

Adverse effects - GI complications, hypersensitive reaction, ALCOHOL interaction, metallic taste

2 - adverse effects with systemic ab and local delivery

3 - patient compliance

4 - long term effectiveness?

5 - microbial resistance

Localised refractory perio pockets

Localised recurrent perio pockets

Patients with good compliance

if all else fails)

Pt with good compliance

Size of perio pocket (small, not enough can get in there without being diluted)

Studies show "marginal improvement"

Periodontology

According to Dr. Bjarne Klausen from Esbjerg, Danish National Clinical Guidelines for the use of antibiotics in dental practice had recently been finalized. See a quick guide here [pdf]. As regards periodontal infection, he writes,

We formulated a focused question that was in line with the Scandinavian consensus: Should prescription of antibiotics be considered in patients with sufficient oral hygiene, if their periodontal condition does not respond to conventional treatment?

Well, they did not find a single relevant study. How’s that? Isn’t it so that dozens if not hundreds of studies had been performed since the early discovery by Jørgen Slots in Copenhagen identifying an unidentified gram-negative rod in abundance in what was then localized juvenile periodontitis? Soon later it was clear that this bug was Actinobacillus (now Aggregatibacter) actinomycetemcomitans, and that it could best be targeted by systemic antibiotics in conjunction with traditional mechanical/surgical periodontal treatment. Soon after a couple of case series by van Winkelhoff et al. in 1989 and 1992, Amoxicillin plus Metronidazole (the infamous “van Winkelhoff cocktail”) became extremely popular and has since then been mentioned in national guidelines for the treatment of severe periodontitis where A. actinomycetemcomitans could be found in abundance.

Despite the fact that any, in general reasonable, additional treatment is likely to have some additional, albeit transient, beneficial effects on surrogate clinical outcomes such as clinical attachment level, I have criticized some arguments by proponents of adjunct antibiotic use, in particular as regards minimizing the need for periodontal surgery, on several occasions here on this blog. The current global threat by increasing numbers of resistant, to antibiotics, pathogens may soon prevent dentists from light-heartedly prescribing antibiotics for their patients who might indeed suffer from severe chronic or aggressive periodontitis (a vastly exaggerating term for a subpopulation of patients with the same disease but more rapid progression).

But anyway, how is it possible that the consultant to the Danish Health Authority on formulating national guidelines on the use of antibiotics in connection with dental treatment, Dr. Bjarne Klausen, now denies any evidence for benefits of systemic antibiotics in periodontitis?

at least among Scandinavian periodontologists, a consensus has evolved that antibiotics should only be used as a last resort in patients who do not benefit from ordinary treatment including oral hygiene instruction, scaling, root planing and possibly periodontal surgery. And we certainly do not prescribe antibiotics to patients who cannot brush their teeth properly. That is what the students are taught in the dental schools, and that is what most of us do in our clinics.

When the Danish committee applied the above focused question, it came as a surprise that actually no relevant study had addressed this principle.

You all know that there are dozens and dozens of randomized clinical trials on antibiotics in periodontal treatment. But the usual protocol in these studies is to take untreated patients directly from the street, classify them as either chronic or aggressive periodontitis, and enroll them in a treatment program supplemented with antibiotics or placebo.

That’s actually true. So, when searching for studies in which patients with excellent oral hygiene who had not responded as expected despite properly conducted conventional periodontal therapy consisting of scaling and root planing as well as surgical treatment for better access, there will be none. This is actually an interesting approach. Similarly restrictive search questions could have been applied for, say, adjunctive topical antibiotics, anti-inflammatory treatments, or laser or photodynamic therapy. Most of the available studies had shown some, albeit generally little, additional benefits over scaling and root planing alone, and many are thus promoted by companies and researchers supported by respective companies. There is often plenty of evidence albeit positive effects are small and must be regarded clinically irrelevant.

If respective adjunctive treatments were only to be tested after conventional treatments had failed, then there would currently be hardly any evidence in favor of these adjuncts at all. To conduct these studies would be difficult as well-complying patients with a high standard of oral hygiene could only be randomized after frustrating experiences of recurrent disease despite considerable efforts of the therapist. Note that some adjuncts had rather been marketed as alternative treatments with remarkable extra costs.

Anyway, the growing problem of antimicrobial resistances and a serious innovation gap since 1990 demand that dentists and in particular periodontists do not use antibiotics in the vast majority of their patients at all. Prescription must be restricted to serious and life-threatening conditions. Reducing the need for periodontal surgery (for proper debridement of infrabony and furcational lesions) must not be an indication for adjunct antibiotics. Periodontal surgery remains the treatment of choice for access problems.

Van winkelhoff cocktail

Affiliation: Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Hannover, Germany

Sascha N. Stumpp

Affiliation: Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Hannover, Germany

Mathias Müsken

Affiliations: Institute of Molecular Bacteriology, TWINCORE, Centre of Experimental and Clinical Infection Research, Hannover, Germany, Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany

Nina Ehlert

Affiliation: Institute for Inorganic Chemistry, Leibniz University of Hannover, Hannover, Germany

Andreas Winkel

Affiliation: Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Hannover, Germany

Susanne Häussler

Peter Behrens

Affiliation: Institute for Inorganic Chemistry, Leibniz University of Hannover, Hannover, Germany

Falk F. R. Buettner

Affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

Meike Stiesch

Affiliation: Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Hannover, Germany

Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript.

Citation: Kommerein N, Stumpp SN, Müsken M, Ehlert N, Winkel A, Häussler S, et al. (2017) An oral multispecies biofilm model for high content screening applications. PLoS ONE 12(3): e0173973. doi:10.1371/journal.pone.0173973

Editor: Jens Kreth, Oregon Health & Science University, UNITED STATES

Received: November 28, 2016; Accepted: March 1, 2017; Published: March 15, 2017

Copyright: © 2017 Kommerein et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by funding from the Ministry of Lower Saxony and VolkswagenStiftung (both BIOFABRICATION FOR NIFE: VWZN2860). F.F.R. Buettner was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy, EXC 62/2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Dental implants play an important role in maintaining full oral function after tooth loss [1]. However, dental implant treatment is not without risks: Early implant failure due to biofilm-associated infections can occur before osseointegration is complete. This early failure rate can be up to 4% [2–7]. Furthermore, even after successful osseointegration of the implant, peri-implant mucositis—bacteria-induced inflammation of the soft tissue around the implant—can occur. If left untreated, this may develop into peri-implantitis. While peri-implant mucositis is defined as a marginal and reversible inflammation, peri-implantitis can lead to destruction of supporting bone and therefore to late implant failure [8–12]. In their review “The epidemiology of peri-implantitis”, Mombelli et al. (2012) reported that 5–10 years after implant placement, 20% of the patients and 10% of the implants developed these infections [13]. Previous studies have shown that peri-implantitis is caused by polymicrobial communities [14, 15], which grow as sessile microbial communities, so-called biofilms, on dental implant surfaces. Within these biofilms, different bacterial species coexist synergistically, embedded in a self-secreted, highly structured extracellular matrix [16–18].

Typical early colonizers in the initial biofilm are streptococci, veillonellae and actinomyces [19–21]. Streptococci and actinomyces species are able to co-aggregate and provide attachment sites and growth support to further bacteria, such as veillonellae, which form metabolic relationships with streptococci [22]. Veillonella species in turn, are also able to develop mixed communities with different “late colonizers” [23]. The presence of Porphyromonas gingivalis, a so-called middle colonizer [21], is associated with periodontitis and these bacteria are frequently found at sites of peri-implantitis [24–26].

A current objective in medical and dental research is to improve implant performance by using new implant materials or surface coatings, in order to prevent or decelerate the formation of biofilms on the implant surface, and to optimize current antimicrobial treatment strategies. For the evaluation of novel oral implant materials or anti-biofilm therapies, appropriate test systems are required, such as multispecies biofilm models, which mimic the in vivo situation.

Existing biofilm models have, for example, been established on saliva-coated hydroxyapatite discs [27–31] or saliva-coated contact lenses [32]. Some models include cultivation in medium supplemented with saliva and/or serum [27–29, 32–34] or use pooled saliva samples to grow an in vitro biofilm [35]. These additives closely mimic the natural habitat, but are often directly collected and then pooled from human sources (volunteers) and thus do not comply with uniform quality standards. Furthermore, the biofilms were sometimes grown in flow cells [33, 34] or in culture plates with 24 or fewer wells [27–32, 35]. Few studies have investigated the reproducibility of the biofilm structure and the species distribution. For these reasons, these models allow investigations of interspecies interactions, but are less suited for high throughput screening applications.

Research in this area should aim at developing a multispecies biofilm with a reproducible biofilm structure and bacterial composition, grown in a standardized medium, and which can be used in 96-well plate formats for high content screening. The model should be robust, and easy to handle and should provide precise time-resolved information about the bacterial composition and the spatial species distribution within the biofilms.

The aim of the present study was therefore to establish a four-species biofilm model in 96-well plate format, consisting of the four early and middle colonizers Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis.

The anaerobically grown four-species biofilms were initially analyzed by confocal laser scanning microscopy (CLSM), with respect to biofilm height, biovolume, live/dead distribution and spatio-temporal spreading of the individual species. For this purpose, live/dead staining and simultaneous fluorescence in situ hybridization (FISH) were performed. The use of scanning electron microscopy (SEM) enabled a detailed insight at the morphology of the bacteria within the multispecies biofilm. To estimate the reproducibility of the individual species distribution, each bacterial species within the biofilms was analyzed by (PMA-) qPCR with respect to total and viable cell numbers. The four species biofilm model was used to determine the impact of antibiotics on biofilm formation either as solved additive or embedded in a bioactive coating intended to be used for medical implants functionalization.

Materials & methods

Bacterial strains and culture conditions

Streptococcus oralis ATCC ® 9811 ™ , Actinomyces naeslundii DSM 43013, Veillonella dispar DSM 20735 and Porphyromonas gingivalis DSM 20709 were acquired from the German Collection of Microorganisms and Cell Cultures (DSMZ) and the American Type Culture Collection (ATCC). The bacteria were routinely cultured in brain heart infusion medium (BHl; Oxoid, Wesel, Germany), supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂) at 37°C.

Co-culture and biofilm formation

The optical densities (OD₆₀₀) of bacterial cultures were measured (BioPhotometer, Eppendorf, Hamburg, Germany), adjusted to 0.1 and subsequently diluted in fresh BHI/vitamin K medium to OD₆₀₀ 0.01 for each bacterial species. The colony forming units (CFUs) of the cultures were determined by plating 100 μL of serially diluted suspensions on BHI agar supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany). The plates were incubated for at least 48 hours at 37°C under anaerobic conditions before counting bacterial colonies.

To obtain a multispecies biofilm, suspension cultures of OD₆₀₀ = 0.1 of the four individual species, were mixed equally and fresh BHI/vitamin K medium was added to achieve a final OD₆₀₀ of 0.01. 150 μL of the mixed suspension was applied to individual wells of a 96-well glass bottom plate (Sensoplate; Greiner, Frickenhausen, Germany) and grown as described above. Each experiment was run in triplicate. Additionally, 2.5 mL of the four-species mixtures were cultured for 24 hours at 37°C on glass discs (10 mm) in Petri dishes (35x10 mm; Sarstedt, Nümbrecht, Germany) for subsequent scanning electron microscopy. Moreover, the pH of the culture medium was measured. Therefore, 4 mL of the four-species suspension was cultured in a cell culture multiwell plate (9.6 cm 2 ; Greiner Bio-One, Frickenhausen, Germany) in triplicate. At various time points during biofilm growth (0, 2, 4, 6, 22, 27 and 45 hours), 0.5 mL of the culture medium was sampled and the pH was measured with pH indicator strips (MColorplast ™ ; Merck Millipore, Darmstadt, Germany).

Quantitative and qualitative biofilm analysis

After 24 and 48 hours of growth, the biofilms were stained within the 96-well glass bottom plates by adding SYTO ® 9 and propidium iodide (LIVE/DEAD ® BacLight ™ Bacterial Viability Kit, Life Technologies, Carlsbad, California, USA) to final concentrations of 3.32 μM and 20 μM, respectively. Biofilm image stacks were acquired with an automated confocal laser scanning microscope (CLSM) (SP-8, Leica Microsystems, Wetzlar, Germany), as described elsewhere [36]. In brief, SYTO ® 9 signals were detected using a multi-wavelength argon laser (excitation wavelength 488 nm) and an emission range of 500–550 nm. Propidium iodide was measured with a 561 nm laser and an emission range of 675–750 nm. Image stacks were acquired using a 1024 x 1024 pixel area with a total height of 23 μm and a z-step size of 1 μm. Image data were processed using the Developer XD Software (Definiens, Munich, Germany), with respect to quality (live/dead ratio) and quantity (total and relative biofilm volumes). The IMARIS software (version 7.6, Bitplane, Zurich, Switzerland) was used for 3D reconstruction of biofilms.

Fluorescence in situ hybridization

Biofilms were washed once with Dulbecco’s Phosphate Buffered Saline (PBS; Biochrom GmbH, Berlin, Germany) and fixed using 50% ethanol. After drying the fixed biofilms, the cells were permeabilized with 40 μL of 1 μg/μL lysozyme for 15 min at 37°C. After stopping the lysis by adding 200 μL absolute ethanol for 3 min, the samples were air dried. Fluorescence in situ hybridization (FISH) was modified from Lawson et al. 2012 [37]: 50 μL urea-NaCl buffer [1 M urea, 0.9 M NaCl, 20 μM Tris-HCl (pH 7.0)] together with 2 μL of 100 μM probe were spotted onto the biofilms. The applied probes, their sequences and references are listed in S1 Table in the supporting information. Hybridization was performed for 25 min at 46°C in a Mini-Incubator 4010 (GFL, Burgwedel, Germany). The biofilms were washed twice with 100 μL of prewarmed urea-NaCl washing buffer [4 M urea, 0.9 M NaCl, 20 μM Tris-HCl (pH 7.0)] and then 100 μL urea-NaCl washing buffer was spotted onto the biofilms and incubated for 5 min at 48°C. After two further washing steps carried out as described above, the biofilms were washed once with aqua bidest., covered with 150 μL PBS and visualized using the sequential imaging mode of the confocal microscope SP8 (Leica Microsystems, Wetzlar, Germany). In the first sequence (see S1a Fig in the supporting information) ALEXA Fluor ® 405 signals were detected with a HyD detector using a 405 nm laser and an emission range of 413–477 nm, together with ALEXA Fluor ® 568 (HyD detector / 561 nm laser / 576–648 nm emission range). In the second sequence (see S1b Fig in the supporting information), ALEXA Fluor ® 488 signals were detected with a PMT detector using a 488 nm laser and an emission range of 509–576 nm, together with ALEXA Fluor ® 647 (PMT detector / 633 nm laser / 648–777 nm emission range). Image stacks were acquired with a z-step size of 1 μm. Image stacks were subsequently processed with the Leica Dye separation tool.

Scanning electron microscopy

After 24 hours, the biofilms were washed twice with PBS (Biochrom GmbH, Berlin, Germany) and fixed for 30 min using 2.5% glutaraldehyde (Roth, Karlsruhe, Germany). After dehydrating the biofilms in an ascending series of ethanol concentrations (25%, 50%, 75%, 90%, 100%; ethanol from J.T. Baker, Phillipsburg, New Jersey, USA) the samples were treated in a Balzer CPD 030 Critical Point Dryer (BAL-TEC, Balzers, Liechtenstein). The dried samples were sputter-coated with gold in an E5400 SEM Coating System (Polaron, Watford, United Kingdom). Scanning electron microscopy was performed with a SEM 505 microscope (Philips, Eindhoven, Netherlands); images were processed with the SEM Software 4.5 [38].

PMA treatment and DNA isolation

The biofilms were detached from the glass surface after 24 and 48 hours by vigorous rinsing with a pipette. Subsequently, the cells were washed once with PBS and resuspended in 100 μL fresh PBS. PMA treatment was used for the examination of the viable parts within the biofilm. For this purpose, 50 μL of the cell suspensions was treated with PMA (Biotum, Hayward, California, USA) prior to DNA extraction. The protocol was modified from Alvarez et al. 2013 [30] as follows: PMA was added to the 50 μL aliquot of the cell suspension at a final concentration of 100 μM. The tubes were incubated for 10 min in the dark at 4°C and the photo-reactive dye was activated by blue light irradiation at 470 nm (3W LED light source) for 20 min. To remove unbound PMA, a final washing step with PBS was performed before total DNA extraction. For total and viable cell amounts, the bacterial DNA was isolated using the FastDNA ™ SPIN Kit for Soil (MP Biomedicals, Irvine, California, USA), following the manufacturer’s instructions. DNA was quantified using a NanoDrop 2000c photometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and stored at -20°C until further processing.

Quantitative real time PCR

Quantitative real time PCR (qRT-PCR) was performed using the iQ5 real time PCR detection system (Bio-Rad, Hercules, California, USA). The primers used in this study are shown in S2 Table in the supporting information. The primers for A. naeslundii and V. dispar were designed using the Primer-BLAST tool from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/tools/primer-blast). The primer pairs were checked for specificity against the three other species (data not shown). Each PCR was performed in a total volume of 25 μL containing 12.5 μL iQ ™ SYBR ® Green Supermix (Bio-Rad, Hercules, California, USA), 0.2 μM forward and reverse primers and 1–40 ng of template DNA, depending on the ratio of the individual bacterial species within the biofilm. The qRT-PCR was carried out with an initial incubation of 3 min at 95°C, followed by 40 cycles of denaturation for 10 s at 95°C, annealing for 20 s (individual temperatures see supplementary S1 Table), amplification for 20 s at 72°C and a melting curve analysis. For each species, a standard curve was generated using defined concentrations of genomic input DNA. All experiments were carried out in triplicate. The genomic DNA amount of a target species in the unknown sample was calculated from the standard curve. The corresponding number of bacterial cells was calculated by dividing the measured amount of DNA by the total genome weight per cell (see S3 Table in the supporting information).

Statistical analysis

Statistical analysis of the results was implemented using the software package “Statistical Package for the Social Sciences” (SPSS; IBM, Armonk, USA), version 23.0. The univariate Mann-Whitney U test was applied to compare the cell numbers between independent experiments. The level of significance was set at p ≤ 0.05.

Application of the established biofilm model

To demonstrate its applicability, the established multispecies biofilm model was exemplarily tested on a bioactive coating for medical implants with proven antimicrobial characteristics. Therefore, round glass discs with a diameter of 5 mm were functionalized with a mesoporous silica film and loaded with ciprofloxacin as described in Ehlert et al. 2011 [39]. The discs were placed in a cell culture multiwell plate (9.6 cm 2 ; Greiner Bio-One, Frickenhausen, Germany), followed by a 24 h period of biofilm formation. Glassbottom wells, glass discs without coating and glass discs with a mesoporous silica film were used as control.

In addition, the two antibiotics amoxicillin (amoxicillin trihydrate; Dr. Ehrenstorfer, Augsburg, Germany) and metronidazole (Dr. Ehrenstorfer, Augsburg, Germany) were added to the medium (BHI / vitamin K) individually or in combination at two different concentrations (14 μg/mL and 140 μg/mL). The latter is known as the “van Winkelhoff-cocktail” [40] which is a standard treatment procedure for periodontal infections. Multispecies biofilms were prepared and cultivated as described before. After 24 hours of incubation, the biofilms were stained and analyzed (1024 x 1024 pixel area / total height of 30 μm / z-step size of 1 μm) as mentioned above.

Qualitative and quantitative biofilm analysis revealed high reproducibility of the four-species biofilm model

Biofilms, including the four bacterial species S. oralis, A. naeslundii, V. dispar and P. gingivalis, were grown in 96-well plates. Precultures of OD₆₀₀ = 0.1 of the four individual species were mixed, diluted to a final OD₆₀₀ = 0.01 and used for inoculation of the plates. An optical density of 0.01 corresponded to 1.17x10 7 (± 7.81x10 6 ) CFU/mL for S. oralis, 1.63x10 6 (± 1.86x10 5 ) CFU/mL for A. naeslundii, 3.24x10 5 (± 2.30x10 4 ) CFU/mL for V. dispar and 7.38x10 4 (± 5.41x10 4 ) CFU/mL for P. gingivalis. (PMA-) qRT-PCR confirmed the results of the CFU method (see S2 and S3 Figs in the supporting information). Thus, S. oralis dominated the starting mixture amounting to 81.3% of the overall bacteria, followed by A. naeslundii (11.3%), P. ginigvalis (5.1%) and V. dispar (2.2%).

The biofilms were subjected to live/dead staining and subsequently analyzed by CLSM after 24 and 48 hours of biofilm development. Exemplary images of 24 and 48 hour biofilms are depicted in Fig 1. The lower (bottom) parts of the biofilms are shown in Fig 1a (24 hours) and 1c (48 hours); the higher (top) parts of the biofilms in Fig 1b (24 hours) and 1d (48 hours).

Expand Fig 1. CLSM of the four-species biofilms.

Images of the four-species biofilms comprising the bacterial species S. oralis, A. naeslundii, V. dispar and P. gingivalis after (a) 24 hours (bottom of the biofilm), (b) 24 hours (top of the biofilm), (c) 48 hours (bottom of the biofilm) and (d) 48 hours (top of the biofilm) of biofilm growth. Bacteria were live/dead stained with viable cells visualized in green and dead cells appearing red.

In order to quantify biofilm formation, mean biofilm height, total and relative biovolume with respect to the viable parts (green), colocalized parts (orange) and dead parts (red) of the biofilms were calculated from the image stacks.

The mean biofilm height was 6.20 μm (± 0.37) after 24 hours and increased to 6.64 μm (± 0.49) after 48 hours. At both time points, the biofilm height of the three biological replicas was comparable. Moreover, the total biovolume exhibited no significant difference between the 24 or 48 hour replicas. However, the biovolume slightly increased from 4.38x10 5 μm 3 (± 1.37x10 4 ) after 24 hours to 4.71x10 5 μm 3 (± 2.81x10 4 ) after 48 hours (Fig 2a). Analysis of the relative biovolumes revealed that the viable cells dominated the population, accounting for 97.9% (± 0.1) in the 24 hour and 95.2% (± 0.5) in the 48 hour biofilms. The proportion of dead bacteria marginally increased from 24 to 48 hours (Fig 2b).

Expand Fig 2. Quantification of biovolume.

(a) Total biovolume and (b) relative biovolume of the four-species biofilms, including the bacterial species S. oralis, A. naeslundii, V. dispar and P. gingivalis. Each bar shows the mean ± standard error from three different wells of one biofilm growth experiment of the three biological replicates measured after 24 and 48 hours.

Different bacterial species organize in a distinct spatial pattern within four-species biofilms

Localization of the four bacterial species (S. oralis, A. naeslundii, V. dispar, and P. gingivalis) within biofilms was assessed by FISH. Simultaneous staining with specific probes against the different bacterial species enabled a clear distinguishing of the individual species in 24h (Fig 3) and 48h biofilms (Fig 4).

Expand Fig 3. Maximum intensity projection of biofilm image stacks upon species-specific staining of a 24h four-species biofilm by FISH.

(a)–(d) Separate color channels showing the staining of individual bacterial species within the four-species biofilm. (a) S. oralis (MIT-588-Alexa-405; blue), (b) A. naeslundii (ANA-103-Alexa-488; green), (c) V. dispar (VEI-217-Alexa-568; yellow) and (d) P. gingivalis (POGI-Alexa-647; red), (e) Overlay of individual images of the four-species biofilm. Image stacks of 11 single images with a z-step size of 1 μm.

In order to assess the spatial-temporal distribution of the individual bacterial species within the four-species biofilms, a three-dimensional reconstitution of the biofilms at 24h (Fig 5) and 48h (Fig 6) was performed by CLSM. We assessed the fluorescence staining of the four bacterial species in different depths of the biofilm and could show that S. oralis (blue) was by far the dominant species within the whole biofilm building up a hilly structure with areas where these bacteria grew at high densities. This was observed at both time points, after 24 (Fig 5) and 48 hours (Fig 6). V. dispar (yellow) grew in cylindrical microcolonies spanning the entire height of the biofilm longitudinal from the glas surface to the top. Interestingly, V. dispar colonies appeared to grow in craters of the S. oralis layer, clearly separated from the latter one (Figs 5 and 6). Very small numbers of P. gingivalis (red) could be detected, either as single cells or in microcolonies, up to a biofilm height of 7 μm (Figs 5a–5h and 6a–6h). The growth of A. naeslundii (green) could be detected from a biofilm height of 1 μm up to the top of the biofilm (10 μm). A. naeslundii grew in close contact to S. oralis in areas of the biofilm, where S. oralis also grew at high densities (Figs 5c–5k and 6b–6k).

Expand Fig 5. Spatial-temporal distribution of the individual bacterial species within the 24 hours biofilm.

Fluorescence in situ hybridisation of the 24 hour four-species biofilm consisting of the bacterial species S. oralis (MIT-588-Alexa-405; blue), A. naeslundii (ANA-103-Alexa-488; green), V. dispar (VEI-217-Alexa-568; yellow) and P. gingivalis (POGI-Alexa-647; red). (a)–(k) show the 11 images of the biofilm with a z-step size of 1 μm. (l) shows the 3D-reconstruction of the complete biofilm.

Fluorescence in situ hybridisation of the 48 hour four-species biofilm consisting of the bacterial species S. oralis (MIT-588-Alexa-405; blue), A. naeslundii (ANA-103-Alexa-488; green), V. dispar (VEI-217-Alexa-568; yellow) and P. gingivalis (POGI Alexa-647; red). (a)–(k) show the 11 images of the biofilm with a z-step size of 1 μm. (l) shows the 3D-reconstruction of the complete biofilm.

The three-dimensional biofilm structure was further assessed by scanning electron microscopy (SEM) at a higher resolution than what was possible by CLSM. SEM of single-species biofilms (Fig 7a–7d) enabled identification of individual species within a mixed four-species biofilm (Fig 7e). In the mixed biofilm, the species A. naeslundii and V. dispar can be clearly distinguished. Separation of P. gingivalis and S. oralis is more difficult, but still possible due to the typical long chains of S. oralis cocci. This method confirmed the dominance of S. oralis and lower numbers of the three other species within the multispecies biofilm.

Expand Fig 7. Scanning Electron Micrograph (SEM) of 24 hours old biofilms.

(a) S. oralis, (b) A. naeslundii, (c) V. dispar, (d) P. gingivalis and (e) a four-species biofilm. In the mixed community, the individual species are exemplarily highlighted by arrows: S. oralis (a), A. naeslundii (b), V. dispar (c), and P. gingivalis (d).

Different bacterial cell numbers in the four-species biofilm model are stable over time

Total and the viable cell numbers of each individual bacterial species were analyzed after 24 and 48 hours within the biofilm cultures by qPCR (Fig 8). Each experimental setting included three technical replicates. The bacterial compositions of the biofilms were highly reproducible at both time points and confirmed that S. oralis dominated the biofilms after both 24 and 48 hours. V. dispar and A. naeslundii were less frequent and the lowest cell numbers were found for P. gingivalis. The relative frequency proved to be constant in all three experiments at both time points. The mean percentage distributions (total cell amount) of the four bacterial species were calculated for the 24 hour biofilm to be: 96.3% (± 1.5) for S. oralis, 2.3% (± 0.8) for V. dispar, 1.4% (± 0.9) for A. naeslundii and 0.0024% (± 0.0008) for P. gingivalis (Fig 8a). If only the viable cell numbers were considered, the percentage of S. oralis decreased to 80.2% (± 3.1), the other three species increased to 15.1% (± 3.3)–for V. dispar, 4.8% (± 1.2) for A. naeslundii and 0.0043% (± 0.0017) for P. gingivalis (Fig 8b). Comparing the averaged percentage distribution of the 48 hour old biofilms to the values at 24 hours (total cell amount), the percentage of S. oralis decreased at 48 hours to 86.5% (± 6.8), V. dispar increased to 10.9% (± 5.7), A. naeslundii increased to 2.6% (± 1.3) and P. gingivalis increased to 0.012% (± 0.007) (Fig 8c). If only the viable cell numbers were taken into account and compared to the 24 hours viable cell numbers, S. oralis decreased to 69.7% (± 12.9), V. dispar increased to 25.1% (± 12.1), A. naeslundii increased to 5.1% (± 1.4) and P. gingivalis increased to 0.02% (± 0.0315) (Fig 8d).

Expand Fig 8. qRT-PCR analysis representing the relative species distribution within the biofilms.

These consisted of the bacterial species S. oralis (blue), A. naeslundii (green), V. dispar (yellow) and P. gingivalis (red) and were incubated anaerobically for 24 and 48 hours. Each independent biofilm approach (1–3) included three technical replicates (three wells); qRT-PCR was run in triplicate for each biofilm sample. (a) Percentage distribution based on the total cell numbers after 24 hours, (b) percentage distribution based on the viable cell numbers after 24 hours, (c) percentage distribution based on the total cell numbers after 48 hours, (d) percentage distribution based on the viable cell numbers after 48 hours.

pH of medium decreased upon prolonged biofilm growth

The pH of the biofilm medium was measured after 0, 2, 4, 6, 22, 27, 45 hours of biofilm development. The pH was stable within the first 4 hours of biofilm growth at 7.0–7.5. After 22 hours, the pH decreased to 5.0–5.5. Within the following 23 hours, it decreased to 4.5–5.0 (Fig 9).

Expand Fig 9. pH value curve.

pH measurement of the biofilm medium over 45 hours of biofilm growth.

Application of the established biofilm model

The setup of the established biofilm model was tested to demonstrate the effect of antibiotics (embedded or in solution) on biofilm formation: i) a bioactive coating for medical implants with proven antimicrobial characteristics (mesoporous silica film loaded with ciprofloxacin) and ii) amoxicillin and metronidazole solved in growth medium (BHI / vitamin K) individually or in combination (“van Winkelhoff-cocktail”). In order to evaluate the antimicrobial effect, we calculated the relative amounts of the total biovolume with respect to the viable (green), colocalized (orange) and the dead part (red) of the biofilms from the image stacks for i) bioactive coating (Fig 10a) and ii) antibiotics dissolved in the culture medium (Fig 11a). In addition, maximum intensity projections of the different samples were included (Figs 10b–10e and 11b–11h).

Expand Fig 10. Biovolume quantification and maximum intensity projections of four-species biofilms grown on (coated) glass discs.

(a) Relative proportion of the total biovolume of biofilms grown on different surfaces as indicated in the figure. (b-e) Images of 24 hour-old four-species biofilms established in glassbottom wells (b), on glass discs (c), on mesoporous silica coated glass discs (d) and on mesoporous silica coated glass discs containing ciprofloxacin (e). Bacteria were stained live/dead (viable cells: green; dead cells: red).

(a) Determination of total biovolumes of 24 hour-old four-species biofilms grown under conditions as indicated in the figure. (b-h) Images of biofilms grown in glassbottom wells without supplementation of antibiotic (b), with 14 μg/mL amoxicillin (c), with 140 μg/mL amoxicillin (d), with 14 μg/mL metronidazole (e), with 140 μg/mL metronidazole (f), with 14 μg/mL amoxicillin and 14 μg/mL metronidazole (g) and with 140 μg/mL amoxicillin and 140 μg/mL metronidazole (h). Antibiotics were added to BHI / vitamin K medium. Bacteria were stained live/dead (viable cells: green; dead cells: red).

Biofilm formation on glass discs (control) was augmented in comparison to glass wells and resulted in a doubling of biovolume (Fig 10). The biovolume on glass discs coated with mesoporous silica was slightly decreased in comparison to the uncoated control. On glass discs coated with mesoporous silica and ciprofloxacin the biovolume did not alter considerably compared to the control without antibiotic but the proportion of dead cells massively increased (up to 70%; Fig 10a and 10e).

Supplementation of culture media with 14 μg/mL amoxicillin, 14 μg/mL metronidazole or a combination of both did not affect the total biovolume (Fig 11a). A reduction of the total biovolume to less than 20% of the control and a major increase up to 50–70% in the dead proportion was observed for amoxicillin (140 μg/ mL) alone and in combination with metronidazole (140 μg/mL).

Discussion

Bacterial adhesion and biofilm formation on dental implants often cause peri-implantitits that can finally lead to implant loss. Methods for testing of implant surfaces concerning bacterial adhesiveness or antimicrobial properties are highly demanded and thus in the present study, we have established a multispecies biofilm model. Using an array of different methods, including CLSM, SEM, qRT-PCR and FISH, we unambiguously demonstrated that this biofilm model is robust and highly reproducible, which is a prerequisite for envisioned high throughput analyses of e.g. anti-biofilm activity of implant surfaces. Our focus was on developing an easy-to-use biofilm model without the need for nutritional supplements that are not manufactured according to uniform quality standards and are of limited availability, such as human saliva and to apply this test system to high content screening.

Different multispecies biofilm models have already been described [27–35, 41]. However, they mainly focus on biological aspects of bacterial interactions or biofilm development and they are less suitable for high content screening applications, due to (I) the use of human saliva and/or serum, which makes the growth medium less reproducible [27–30, 33, 34] and/or (II) the use of culture plates with 24 or fewer wells [27–33, 35]. For example a three-species biofilm model, including S. oralis, A. naeslundii and F. nucleatum, and a four-species biofilm model, consisting of S. gordonii, A. naeslundii, V. atypica and F. nucleatum, were established in saliva-containing medium [33, 34]. Similarly, a five-species model (S. oralis, S. sobrinus, A. naeslundii, V. dispar, F. nucleatum) and a six-species biofilm model (S. oralis, A. naeslundii, V. parvula, F. nucleatum, P. gingivalis, A. actinomycetemcomitans) were cultivated on saliva-coated discs [27, 28]. The 10-species subgingival Zurich Biofilm model [29] is very complex and close to in vivo situations. The bacterial composition and biofilm stability were evaluated for three different growth media that, in part, contained saliva and/or human serum. The results revealed that different growth media affect biofilm stability, development and bacterial composition. Another in vitro biofilm model system has been developed without saliva, on rigid gas-permeable hard contact lenses (RGPLs), in order to study the interactions of the bacteria with epithelial cells [32]. The authors used commercially available fetal bovine serum (FBS) for RGPL-coating, which is much more reproducible than collected and pooled saliva.

In the present study we demonstrated that biofilm height, as well as the total and relative biovolumes of the four-species biofilm, were highly reproducible after 24 and 48 hours. Furthermore, the individual cell distributions of the four bacterial species, as determined by (PMA-) qRT-PCR, were also highly reproducible and did not change over time. When comparing the total and viable parts of a biofilm, there may be fluctuations in the percentage distribution. It is therefore important to determine both the total and the viable fractions of the biofilm.

In vivo studies on plaque development have demonstrated that streptococci are the predominant colonizers at early time points: They made up to 60% of the total after 4 hours and up to 90% after 8 hours of plaque formation [19]. Moreover, 16S rRNA gene sequencing studies with a retrievable enamel model have found that about 66% of all bacteria were streptococci after 4 hours of enamel colonization and 80% after 8 hours [21]. In the same study, veillonella made up to 10% and actinomyces up to 7.8% of the total after 4 hours of plaque development. Porphyromonas were present at a maximum of only 1.5% [21]. These findings therefore resemble those in our in vitro biofilm model: Assessing the viable part of our biofilm model, the distributions of the four species S. oralis [24 hours: 80.2% (± 3.1); 48 hours: 69.7% (± 12.9)], V. dispar [24 hours: 15.1% (± 3.3); 48 hours: 25.1% (± 12.1)], A. naeslundii [24 hours: 4.8% (± 1.2); 48 hours: 5.1% (± 1.4)] and P. gingivalis [24 hours: 0.0043% (± 0.0017); 48 hours: 0.019% (± 0.0315)] were very similar to the native situation in early plaque.

The urea-NaCl-FISH assay was first described for Staphylococcus aureus [37]. We have already used this method in a study on the simultaneous staining of three species [42]. Our results show that urea-NaCl-FISH assay is also suitable for simultaneous FISH analysis of four different gram-positive and gram-negative bacterial species embedded in a biofilm. The FISH results not only confirmed the CSLM and qRT-PCR data but provided additional information on biofilm architecture and species distribution. S. oralis built up the main structure of our in vitro biofilm model, similarly described in in vivo biofilm studies [19–21]. A. naeslundii grew closely together with S. oralis. This direct contact between streptococci and actinomyces has already been described in literature and was first discovered in 1970 [43]. Later studies revealed that coaggregation is, inter alia, mediated through the quorum-sensing regulatory molecule AI-2 [44]. Furthermore, actinomyces are able to recognize receptor polysaccharides (RPS) on streptococci through their type 2 fimbriae. The subsequent coaggregation is highly specific [22, 45]. Veillonella also showed metabolic interactions with streptococci in previous studies and some Veillonella species are able to use lactic acid, which can be produced by streptococci, as fermentation substrate [46–48]. Interestingly, in our biofilm model colonies of V. dispar were spatially clearly separated from S. oralis. It would be very interesting to analyze interspecific interactions and their influence on biofilm formation within the described multispecies biofilm model in further studies.

The increase in A. naeslundii and V. dispar after 48 hours, which could be detected by qPCR, could also be confirmed by FISH. No spatial change in the individual bacterial species within the biofilm was identified.

The pH measurements revealed that the pH of the biofilm medium decreased from pH 7.0–7.5 to pH 4.5–5.0 after 45 hours. In a study by Takahashi et al. (1990), P. gingivalis could only grow at pH from 6.5 or higher, whereby the optimum pH for proteolytic activity was detected at pH 7.5 to 8.0 [49]. Because P. gingivalis is very sensitive to pH, this may be the reason for the low numbers of this species in our biofilm. The decrease in pH can be attributed to the species S. oralis, A. naeslundii and V. dispar. S. oralis is known to be acid tolerant [50] and due to its ability to metabolize sugar, it can even produce acids independently [51]. Veillonella species are also known to produce acids (hydrogen sulfide; H₂S) under appropriate conditions [52, 53]. Even A. naeslundii is able to produce acids from glucose, best at pH of 7.0 [54]. These characteristics might explain why A. naeslundii and V. dispar have no problems growing in medium with a lower pH and increase in cell number.

It is important to note that we have measured the pH of the medium which does not exactly reflect the pH within the biofilm. Within multispecies biofilms the pH profiles can be heterogeneous depending on the location and the local microbial composition. Therefore, Hwang et al. (2016) established a method for simultaneous spatio-temporal analysis of pH microenvironments [55]

It would be interesting to use such a method to analyze the pH within our biofilm model in response to different culture conditions and the effect of the pH changes within the biofilm on biofilm formation and composition in further studies. Since diverse bacterial species response differently to pH shifts and are even able to produce acids themselves, antibiotic-treatment may take an additional influence on the biofilm: Next to the primary effect of killing, it could secondary influence the bacterial population due to pH shift (by eliminating acid producing bacteria). This is another interesting aspect which we would like to investigate with our model. The experiments shown in Figs 10 and 11 demonstrate the application of our biofilm model which can be used for both, the testing of bioactive and antimicrobial surfaces and for the testing of antimicrobials / antibiotics in solution.

In conclusion, a new four-species biofilm model was established that mimics the native situation, and is robust and highly reproducible. It is thus perfectly suited for the investigation of protective effects of novel antimicrobial surfaces or alternative antimicrobial treatment strategies. While live/dead staining can be used to determine overall viability, more labor-intensive methods—(PMA-) qRT-PCR and FISH—are capable of resolving species-specific viability, in addition to the spatial distribution of biofilms on selected, promising materials and thus, allow a detailed understanding of bacteria-surface interactions.

Supporting information

S1 Table. Species-specific 16S rRNA probes for fluorescence in situ hybridization.

S2 Table. Species-specific primer pairs used in qRT-PCR to identify the four different bacterial species within the biofilm.

S3 Table. Genome size, corresponding accession number and the calculated genome weight used for quantification of the individual species.

S1 Fig. Sequential scan of the FISH stained biofilms.

(a) In the first sequence, ALEXA Fluor ® 405 signals were detected with a HyD detector using a 405 nm laser and the emission range of 413–477 nm, together with ALEXA Fluor ® 568 (HyD detector / 561 nm laser / 574–648 nm). (b) In the second sequence, ALEXA Fluor ® 488 signals were detected with a PMT detector using a 488 nm laser and a range of 509–579 nm, together with ALEXA Fluor ® 647 (PMT detector / 633 nm laser / 648–777 nm).

The results of qPCR (total cells) and PMA-qPCR (viable cells) show the percentage distribution of the four species S. oralis, A. naeslundii, V. dispar and P. ginigvalis in the start-mixture.

S3 Fig. Comparison of the percentage distribution of the viable cells determined by PMA-qPCR with the results determined by CFU analysis.

Acknowledgments

This work was carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative. NIFE is the Lower Saxony Center for Biomedical Engineering, Implant Research and Development, a joint translational research centre of the Hannover Medical School, the Leibniz University Hannover, the University of Veterinary Medicine Hannover and the Laser Zentrum Hannover e. V.

Funding information

The BIOFABRICATION FOR NIFE Initiative is financially supported by the ministry of Lower Saxony and the VolkswagenStiftung (both BIOFABRICATION FOR NIFE: VWZN2860). F.F.R. Buettner was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy, EXC 62/2).

Author Contributions

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Van winkelhoff cocktail

University of Florida/USAID/SADCC

Heartwater Research Project,

Box 8101 Causeway, Harare, Zimbabwe.

Heartwater has an economically significant impact on livestock. The disease is caused by the rickettsial organism Cowdria ruminantium and is transmitted by ticks of the genus Amblyomma. In Africa heartwater is the second major tick-borne disease after theileriosis and its occurence spans most of the sub-Saharan region and the neighbouring islands. In southern Africa it is considered to be the main tick-borne disease. The principal objective of the University of Florida/USAID/SADCC Heartwater Research Project is to develop new vaccines and diagnostic tests to replace the current cumbersome methods of immunisation and diagnosis for heartwater. The Project's goal is to understand the molecular and immunological aspects of heartwater with respect to developing improved vaccines which would contain a repertoire of protective immunodominant antigens Of the approaches being pursued, the first is to develop a tissue culture-derived vaccine, and the second involves cloning of genes encoding antigens to C. ruminantium which are recognised by the immune responses and which may be useful as vaccine candidates. These approaches are discussed in this paper.

The prevalence of heartwater has a significant economic impact on livestock. Caused by the rickettsial organism Cowdria ruminantium and transmitted by ticks of the genus Amblyomma in Africa, heartwater is the second mayor tick-borne disease after theileriosis. Its occurrence spans most of the sub-Saharan region and the neighbouring islands (Uilenberg, 1983). In southern Africa it is considered to be the main tick-borne disease.

Economic losses due to heartwater are a result of the high mortality rate it causes, which ranges between 20 and 90% (Uilenberg, 1987), and the reduced productivity it engenders in both clinically ill and surviving/recovered animals which become chronic carriers of the disease.

Heartwater is routinely controlled by acaricides and artificial immunisation. However, tick control is expensive and ticks build up a resistance to acaricides. Hence complete disease control using this method is not really achievable. In addition, intensive dipping against ticks is difficult to sustain in many African countries.

In the artificial immunisation method, young animals (less than six months old) are inoculated with infected blood to maximise their age-related innate immunity to heartwater. This process has given varied results, as it does not reproducibly induce immunity in the vaccinated animals. In older animals, artificial immunisation is achieved by infection with blood infected with the Ball-3 heartwater strain (Oberem and Bezuidenhout, 1987) followed by treatment with tetracyclines (Uilenberg, 1990; van der Merwe, 1987). The infection-and-treatment method has been used with a degree of success as animals that have recovered from heartwater infections have been shown to be immune to homologous and, in most cases, heterologous challenge (Uilenberg et al, 1983; van Winkelhoff and Uilenberg, 1981). However, the infection-and-treatment method has limitations. The vaccine has to be stored in liquid nitrogen or dry ice up to the time of inoculation of the animals; the vaccine has to be administered intravenously; vaccinated animals have to be monitored clinically and treated with a curative dose of tetracyclines during the febrile reaction; some vaccinated animals may die due to acute reactions to the vaccine despite administration of tetracyclines; other disease-causing organisms may be accidentally transmitted in the blood vaccine; and lastly, overall this method is cumbersome and cannot be applied on a wide scale.

Clinically ill animals in the field may be treated with tetracyclines. However, the course of the disease is rapid and by the time the clinical reaction is detected it is usually too late for antibiotic therapy.

These control methods against heartwater have been used for many years and currently there are no suitable alternatives.

The principal objective of the University of Florida/USAID/SADCC Heartwater Research Project was to develop new vaccines and diagnostic tests to replace the current cumbersome methods of immunisation and diagnosis against heartwater. The project's initial goal was to propagate C. ruminantium organisms in vitro.

This goal has been achieved and efforts are now concentrated towards studying the molecular and immunological aspects of heartwater with respect to developing improved vaccines that contain a repertoire of protective immunodominant antigens.

Two approaches are being pursued. The first is to develop a tissue-culture-derived vaccine. The second involves cloning genes encoding antigens of C. ruminantium that are recognised by the immune system and that may be useful as vaccine candidates.

In the mammalian host, C. ruminantium is found both intracellularly or extracellularly. The intracellular stages are found in macrophages, neutrophils and endothelial cells, where they develop into colonies containing morphologically distinct organisms (Jongejan et al, 1991). This stage eventually gives rise to the extracellular stage, and the organisms are found to be freely circulating in the blood looking to invade new cells. Antigens of intracellular organisms are probably a target of cell-mediated immune responses, whereas antigens of the extracellular organisms are probably a target of antibody-mediated immune responses. In this report the strategy of developing vaccines based on the identification of antigens recognised by the antibody responses will be discussed.

Normal and immune antisera were prepared from the blood of 175 sheep that had been inoculated and had recovered after experimental infection with the Crystal Springs Heartwater strain of Zimbabwe. After recovery the 175 sheep were resistant to further homologous challenge.

Propagation and isolation of Cowdria ruminantium

Cowdria ruminantium cultures were maintained in bovine endothelial cells using the Byrom and Yunker (1990) method. Various strains of Heartwater were propagated for analysis, namely Crystal Springs, Zwimba, Palm River, Nyatsanga, Lemco T3, Highway (of Zimbabwe), Ball-3, Welgevonden (of South Africa) and a strain from Nigeria. The C. ruminantium organisms were isolated from cell culture supernatants after complete lysis of the host cells. The organisms were washed three times in phosphate buffered saline pH 7.4 (PBS) at 30,000 G for 30 minutes each wash and processed as necessary.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting

Analysis of C. ruminantium proteins was achieved by SDS-PAGE and Western blotting (Johnstone and Thorpe, 1988). The electro-phoresed proteins were transferred to nitrocellulose, blocked for 1 hour in 0.25% gelatin, and probed with immune antisera. The reaction of the antisera was developed by incubation of the blots with peroxidase-labelled protein-G and substrate (Kirkegaard and Perry, USA).

The analysis revealed the recognition of several C. ruminantium proteins by the immune antisera and also showed that the 32 kD protein was the most immunodominant. Furthermore, the immunodominant proteins of C. ruminantium from geographically different strains of heartwater cross-react in Western blotting analysis with the Crystal Springs strain of Zimbabwe, indicating antigenic conservation of these proteins. Other investigators have also shown cross-reactions between different strains of heartwater by Western blotting and by cross-protection studies (van Winkelhoff and Uilenberg, 1981; Uilenberg et al, 1983; Rossouw et al, 1990). High-titred immune antisera are being used to screen genomic DNA libraries to isolate genes encoding the immunodominant proteins of C. ruminantium. A gene encoding the 21 kD protein of C. ruminantium has been cloned and a vaccine trial using the recombinant protein as a vaccine candidate is under way. As other genes are cloned, their recombinant proteins will be tested in vaccine trials and, if necessary, used in a cocktail vaccine to optimise the induction of protective immune responses against heart- water.

The approach to develop a vaccine against heartwater using tissue culture derived organisms has so far yielded encouraging results and is being evaluated in greater detail.

Byrom B and Yunker C E. 1990. Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants. Cytotechnology 4:285 290.

Johnstone A and Thorpe R. 1988. Immunochemistry in Practice. 2nd edition. Blackwell Scientific Publication, UK

Jongejan F. Zandbergen T A, van de Wiel P A, de Groot M and Uilenberg G. 1991. The tick-borne rickettsia Cowdria ruminantium has a Chlamydia-like development cycle. Onderstepoort Journal of Veterinary Research 58:227-237.

Oberem P T and Bezuidenhout J D. 1987. The production of heartwater vaccine. Onderstepoort Journal of Veterinary Research 54:485 488.

Rossouw M, Neitz A W H. De Waal D T. du Plessis J L, Van Gas L and Brett S. 1990. Identification of the antigenic proteins of Cowdria ruminantium. Onderstepoort Journal of Veterinary Research 57:215-221.

Uilenberg G. 1983. Heartwater (Cowdria ruminantium infection): Current status. In: Advances in Veterinary Science and Comparative Medicine. pp. 427-480.

Uilenberg G. Zivkovic D, Dwinger R H. Ter Huurne A A H M and Perie N M. 1983. Cross immunity between strains of Cowdria ruminantium. Research in Veterinary Science 35:200-205.

Uilenberg G. 1990. Present and future possibilities for the control of cowdriosis and anaplasmosis. The Veterinary Quarterly 12:39-45.

van der Merwe L. 1987. The infection and treatment method of vaccination against heartwater. Onderstepoort Journal of Veterinary Research 54:489-491.

van Winkelhoff A J and Uilenberg G. 1981. Heartwater: Cross immunity studies with strains of Cowdria ruminantium isolated in West and South Africa. Tropical Animal Health and Production 13: 160 164.

Aspects of doing business in Flanders (Belgium)

Description

Guy TROUVEROY, H. E. Ambassador of Belgium to the Russian Federation, Belgian Embassy in Moscow

• Expanding into Western Europe: the Flanders' business case

John Verzeele, Director Inward Investment, Flanders Investment and Trade

• Antwerp as a business location: possibilities and peculiarities of doing business

Gerard Van Winkelhoff, CEO/Senior Investment Promotion Manager, Antwerp headquarters

• Flemish Brabant – Gracefully Genius

Thomas Jansen, policy officer internationalization, Province of Flemish Brabant

• Ghent: the best idea is to go where the best ideas are

Brecht Lootens, Investment promotion manager, City of Ghent

Koen Schoors, professor, UGent and Vlerick Business School

12:20-14:20 Session 2 .

Marc De Mil, Ministry of Finance of Belgium

• Investing in the Flemish region. The experience of Russian companies

• Immigration law: visa, work permit and other issues.

Eric Jacquemin, Consul, Belgian Embassy in Moscow

• Image of Flanders

Ksenia Kirichenko, Representative of the Office of Tourism Flanders-Brussels in Russia, the Office of Tourism Flanders and Brussels in Russia

Regarding conference agenda please contact — Elena Zaytseva

Eric Jacquemin

Graduate studies: Master in LAW, Vrije Universiteit Brussel 1985 — 1987

Master in CRIMINOLOGY, V.U.B., 1983 — 1985

Bachelor in LAW, V.U.B., 1980 — 1983

Secondary education: Koninklijk Atheneum St. Pieters Woluwe, 1974 — 1980

Belgian Federal Public Service FOREIGN AFFAIRS :

Consul in Moscow, since 2010

Consul in London, 2006 — 2010

Consul in Beijing, 2002 — 2006

Consul in Dar es Salaam, 1998 — 2002

Vice-Consul in London 1997 — 1998

Trainee 1996 — 1997

Managing Director Glass & Design, Brussels 1995 — 1996

Head of sales, Interior designers, Brussels 1993 — 1995

Head of Administration, Yaoundé office 1991 — 1993

AGIC CAMEROUN, Agence Générale d'Importations Commerciales :

Freelance representative for industrial tools in Yaoundé, Cameroon 1990 -1991

PIEDBOEUF — INTERBREW, Judicial and Commercial Department, Brussels 1988 — 1990

Military Service, 15th Wing Belgian Air Force, Mission Planning, Melsbroek 1987 — 1988

John Verzeele

As Director Inward Investment, he is in charge of attracting and assisting foreign investors to Flanders (Belgium). Vast experience with all types of investment projects.

Travels the world to convince investors to choose Flanders for their European projects and as such he has given numerous presentations to a wide variety of audiences.

Marc De Mil

He informs foreign companies of the attractive tax regimes Belgium has to offer with regard to foreign investments and assist them in their contact with Belgian tax administrations

Brecht Lootens

Brand management and communication, Vlerick Business School, 2012

Executive Master in Public Management, Antwerp Management School, 2011

• Graduated magna cum laude

• The MBA for the public sector" combines general management competences and a

thorough knowledge of the (Belgian / European) public sector

• Dissertation on innovation management in local government

Aanvullende Opleiding Internationale betrekkingen en conflictbeheersing, Katholieke

Universiteit Leuven, 2003

• Graduated magna cum laude

Licentiaat in de Sociale en Culturele Antropologie, Katholieke Universiteit Leuven, 2002

• Graduated cum laude

• Dissertation: Getuigenissen uit't Huis. Naar een beter begrip van de identiteitsbeleving

van niet-begeleide buitenlandse minderjarigen. (promotor: prof. Dr. Marie-Claire Foblets)

Kandidaat in de Geschiedenis, Rijksuniversiteit Gent, 2000

• Graduated cum laude

Latijn-Wetenschappen, Sint-Hendriks en Zusters Maricolen Instituut Deinze, 1998

City of Ghent, Department of Economy, Investment Promotion Manager, November 2011-present

• Responsible for the attraction and development of foreign investment in the city of Ghent

• Economic city marketing and investment promotion strategy

• Relation management for companies interested in investing in Ghent

• Providing ongoing services to companies locating here:

o Providing information on key business sectors and industrial clusters

o Assistance in setting up a business

o Introduction to potential partners in local industry, government, service providers and research institutions

o Advice on real estate solutions and locations

Project leader Sustainable Economy and Eco-Innovation, April 2008 — October 2011

Strategic Policy Advisor, April 2008 — May 2010

Corporate Social Responsibility (CSR), September 2006 — April 2008

Adecco Belgium, CSR department, Social Responsability Advisor, January 2006 — August 2006

Steunpunt Allochtone Meisjes en Vrouwen vzw, Staff member interculturalisation, December 2003 — December 2005

Koen Schoors

Thomas Jansen

He has degrees in Economics and Japanese Studies, as well as an LL.M in International Business Law and an advanced M.Sc in European Politics and Policies. He has studied and lived in Belgium, The Netherlands, Japan and Thailand.

He has been tasked by government to synchronize the global needs of international investors with local opportunities in Flemish Brabant, the knowledge region encircling Brussels.

Ksenia Kirichenko

Ksenia Kirichenko leads travel trade marketing department of TMI Consultancy, a British PR and marketing agency specialised in travel marketing and destination management services. Since 2012 TMI represents Flanders national tourism organisation at the Russian market. Visit Flanders representation in Russia develops Flanders marketing strategy aimed to promote Flanders and Brussels as travel and business destination for Russians and implements this strategy trough communication with the leading Russian touroperators and travel media.

Gerard Van Winkelhoff

Relevant professional experience:

CEO/Senior Investment Promotion Manager.

Antwerp Headquarters is an Investment Promotion Agency, supported by the City and Port of Antwerp, and 21 stakeholders from the private sector.

Main tasks of AHQ are: trade promotion, investment attraction, location services, accountmanagement, full support of international companies setting up shop in Antwerp.

1) Waardetoevoeging door verdere ontwikkeling van de "droge"kant van de haveneconomie (Value Creation by further development of the 'dry' side of the Port Economy)

2) Achterlandverbindingen (corridor Antwerpen-Luik): ontwikkeling infrastructuur, samenwerkingsverbanden, ketenregie (Hinterland connections (corridor Antwerp-Liège): development of infra structure, alliances, chain management

2010 — 2011: Communication- , language- and branding agency Mo, Rotterdam, the Netherlands

Partner and 'cooperating' partner. In this capacity, I was responsible for writing a long-term business plan, expand client base, boost turnover, streamline business processes and hire new emplyees.

2009 – 2010: ArcheoLogic, archeological consultancy for spatial development, archeology and cultural history, Woerden, the Netherlands

General manager, responsible for the overall management of the organisation, further development and market positioning of the company for the long term. Turnover € 35M. 60 employees.

1992 — 2009: OntwikkelingsBedrijf Rotterdam/Rotterdam Development Corporation (OBR, one of the biggest municipal organisations in Rotterdam), Department Economic Development, Rotterdam, the Netherlands

Senior Manager Business Development (focus on cluster Port & Industry)/Senior Brand & Marketing Manager/Policy maker/Project and Strategic Director/ Managing Director Rotterdam Investment Agency. Turnover €350M, Rotterdam, the Netherlands

Set up and managed a new commercial, internationally oriented department for job creation,investment attraction & trade promotion. In combination with Deputy Chief Marketing Officer.

2004 — 2007: Rotterdam Development Corporation, Rotterdam, the Netherlands

Deputy Chief Marketing Officer. Worked on the Rotterdam brand, promoted the brand in the international arena, developed long term marketing/communication programmes.

Managed day-to-day implementation of client engagements and project teams.

1989 — 1992: ComText Belgium, later part of Norske Personal Gruppen, a Norwegian company for (technical) translation, interpreting and software localization, Brussels, Belgium

Founder and General Manager. After the acquisition in 1991 General and Commercial Director for Europe.

Global Market Expansion. Expanded geographic market coverage for the Group by adding a significant number of strategically located offices across Europe, Russia (Moscow), U.S.A. (New York) strategic regions worldwide.

1986 — 1989: Technical translation/text agency TxT, part of a big Belgian holding company, Antwerp, Belgium

General and Commercial Manager.

Number of employees: 25. Turnover: €13M.

University teacher technical translation, librarian sciences, terminology

1981 – 1983: De Nieuwe Gazet/Het Laatste Nieuws (a national newspaper in Belgium), Antwerp, Belgium

Journalist Economic and Cultural department/Editor

1979 — 1981: City of Eindho¬ven, the Netherlands

Senior Information Officer/Head Information Department

1976 – 1979: INK Taalservice, Amsterdam, the Netherlands

Co-founder first translation agency for automation/software localisation in the Netherlands. Languages: Spanish, English, French, German.

- English (very good oral and written skills)

- Spanish (very good oral and written skills)

- French (good oral and written skills)

- German (good oral and written skills)

Title: 'Consolidation of Printing Houses and Reduction of Information, 'with an additional paper 'Book and Information'.

Title: 'Not left, not right, but interesting. Some critical and theoretical observations on verzuiling (denominational segregation), ontzuiling (denominational dissegregation) and vertrossing (Americanisation of the media)'. A media-sociological and media-historical approach towards these phenomenona.

- Courses Political Economy, Antwerp University

- Krauthammer: Leadership and Management, Personal Benefits

- Mediatraining Peter Schuiten Rotterdam

- How to read handwritings in old Dutch and courses Genealogy, Record Office Rotterdam

- Reading the hieroglyphs, Leiden University

- Collecting and study of old and antiquarian books and handwritings

- Collecting and study of old city maps, country maps and nautical charts

- Digital photography, computers

- Archeology and national (cultural) heritage

- Study and writing my doctoral dissertation

Periodontology

A Restrictive Approach to the Use of Antibiotics in Periodontics

Are Biofilm Infections Different?

When having revised a popular textbook on Periodontology for undergraduates for its 3rd edition I slightly updated a table which I had used in teaching for a decade. It was once published in an important article by late Sigmund Socransky and Anne Haffajee [1] where these two most influential authors in Oral Microbiology made their point that biofilm infections are in fact different (and difficult to target). The table is featured on the first page of the article but may easily be overlooked. But it contains important ideas which are still valid. Socransky and Haffajee differentiate acute, chronic delayed and biofilm infections and present respective examples. While many acute infections are fortunately self-limiting and require just supportive treatment [2] chronic infections need antibiotics since the host does not properly cope with the causative agents. I have added as typical examples acute necrotizing ulcerative gingivitis, or ANUG, which in most cases resolves under oral hygiene improvement and cautious scaling (supported by antiseptics), and necrotizing ulcerative periodontitis, or NUP, which is longstanding and indicates that the host, for instance due to immune suppression in the course of HIV infection, elicit an improper, impaired response. Antibiotics are not indicated in the former case while they are in the latter; usually metronidazole is the drug of first choice [3]. If the causative agent in delayed infection is known, therapy involving suitable antibiotics is easy. In the past, that was not the case for example for syphilis (Treponema pallidum) where all kinds of mercury treatment had been applied. The disease could only be cured after the detection of penicillin. The same holds, for example, for gastrointestinal ulcers caused by Helicobacter pylori, which has been proven only in the 1980s. Gastric ulcers are now easily treated by high dosages of antbiotics after the bug had been identified.

пятница, 16 февраля 2018 г.

van_winkelhoff_cocktail

Antibiotikatherapie der Parodontitis mit Metronidazol

Metronidazol ist gegen wichtige Markerkeime der Parodontose wirksam

Van-Winkelhoff-Cocktail deckt das Erregerspektrum ab

Nebenwirkungen von Metronidazol

Empfohlene Dosierung

Leistungen

Van winkelhoff cocktail

Antibiotic treatment/therapy

All services provided by our surgery in Darmstadt

Adress, mail and how to find

Adress, mail and how to find

Release of metronidazole from electrospun poly( l -lactide-co- d / l -lactide) fibers for local periodontitis treatment

Objectives

Significance

Choose an option to locate/access this article:

Topic 3 Antibiotics in Periodontology

ИГРАТЬ

Periodontology

Van winkelhoff cocktail

Sascha N. Stumpp

Mathias Müsken

Nina Ehlert

Andreas Winkel

Susanne Häussler

Peter Behrens

Falk F. R. Buettner

Meike Stiesch

Introduction

Materials & methods

Bacterial strains and culture conditions

Co-culture and biofilm formation

Quantitative and qualitative biofilm analysis

Fluorescence in situ hybridization

Scanning electron microscopy

PMA treatment and DNA isolation

Quantitative real time PCR

Statistical analysis

Application of the established biofilm model

Qualitative and quantitative biofilm analysis revealed high reproducibility of the four-species biofilm model

Different bacterial species organize in a distinct spatial pattern within four-species biofilms

Different bacterial cell numbers in the four-species biofilm model are stable over time

pH of medium decreased upon prolonged biofilm growth

Application of the established biofilm model

Discussion

Supporting information

Acknowledgments

Funding information

Author Contributions

References

Van winkelhoff cocktail

Aspects of doing business in Flanders (Belgium)

Description

Eric Jacquemin

John Verzeele

Marc De Mil

Brecht Lootens

Koen Schoors

Thomas Jansen

Ksenia Kirichenko

Gerard Van Winkelhoff

Periodontology

A Restrictive Approach to the Use of Antibiotics in Periodontics

Are Biofilm Infections Different?

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пятница, 16 февраля 2018 г.