Effects of Propicillin on Mixed Continuous Cultures of Periodontal Bacteria J

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Effects of Propicillin on Mixed Continuous Cultures of Periodontal Bacteria J. S. VAN DER HOEVEN* AND C. W. A. VAN DEN KIEBOOM Laboratory of Oral Microbiology, Research Group TRIKON, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Received 15 March 1991/Accepted 13 June 1991

Experiments were designed to test the antibiotic (1-phenoxypropyl)penicillin (propicillin) against a complex microflora of periodontal bacteria. This was accomplished by using a continuously growing mixed culture that

was obtained by enrichment of periodontal plaque in human serum. Peptostreptococcus species, Prevotella Downloaded from intermedia, Lactobacillus catenaforme, and Streptococcus species were dominant members of the enrichment culture. None of the strains isolated from the enrichment culture exhibited detectable ,-lactamase activity. MICs of propicillin for the organisms ranged from 0.1 to 1.2 mg/liter. Propicillin was added to the cultures in single doses that were repeated once or twice at 24-h intervals, that is, after 2.4 volume changes of the culture vessel. Analyses done 24 h after the last addition of propicillin revealed that total cell counts of the culture were hardly affected by 1 mg of propicillin per liter, although some changes in the microbial composition occurred. The relative insusceptibility of the culture might be explained by the low growth rate. Higher concentrations (5, 10, and 50 mg/liter) of the antibiotic caused 10- to 20-fold drops in total cell counts. In these cultures P. intermnedia was selectively suppressed to below the detection level, whereas other organisms that were equally

susceptible to propicillin were less affected. It was concluded that mixed continuous cultures are a useful tool http://aac.asm.org/ for studying the effects of antibiotics against the periodontal microbiota.

The use of antibiotics as an adjunct to mechanical therapy susceptibility of bacteria to antimicrobial agents (3, 24). in the treatment of recurrent periodontitis (15) is not undis- Furthermore, continuous cultures, in contrast to batch cul- puted. One reason is that the broad-spectrum antibiotics tures, offer the possibility to study mixed populations of frequently used in periodontal therapy, such as tetracycline microorganisms. Accordingly, mixed continuous cultures and amoxicillin, suppress the autochthonous anaerobic mi- have been used as a model for oral microfloras (4, 19). croflora of the digestive tract and lead to a reduction of the The considerations given above provide a rationale for

colonization resistance (21) of the host. The consequences studying the effects of antibiotics by using continuous cul- on May 29, 2021 by guest thereof may be illustrated by the recently reported (12, 14) tures of mixed periodontal microflora. Earlier experiments overgrowth by enteric rods, pseudomonads, and yeasts in have shown that the representativity of these cultures for the the mouths of periodontal patients. Generally, the choice of natural subgingival ecosystem is probably increased when antibiotic for use in patients with periodontitis is hampered serum, as a substitute for gingival tissue exudate, is used as by the fact that a variety of potential pathogenic bacteria, a growth medium (18, 19). The gingival exudate is consid- rather than a single pathogen, is involved. Furthermore, the ered to be the main natural source of nutrients for the in vitro antibacterial activity of an antibiotic does not always subgingival microflora. correlate with therapeutic efficacy (25). The reasons for this In the present study, the effects of a semisynthetic small- discrepancy can differ widely and include phenotypic differ- spectrum penicillin [propicillin; (1-phenoxypropyl)penicillin, ences between bacteria growing in the natural environment potassium salt] were investigated. Compared with classical and in the laboratory, degradation of the antibiotic by penicillin V (phenoxymethyl penicillin), this type of penicil- members of the microflora other than the target organisms, lin is more rapidly resorbed, leading to higher levels of the and interference of the antibiotic with synergistic relation- drug in serum (1). ships in the microbial community, so that relatively insus- ceptible organisms could still be affected. Slow growth rate and restricted availability of nutrients in MATERIALS AND METHODS the natural environment may be major contributors to phe- Cultures. Mixed continuous cultures of periodontal bacte- notypic changes leading to the relative insusceptibility to ria were obtained from the enrichment of subgingival plaque drugs in vivo (2, 3, 20, 23). This may not explain the fact that by using human serum as a growth medium (19). A plaque in the treatment of orofacial abscesses, a good response to sample from a patient's periodontal pocket that showed antibiotic therapy may follow even when resistant patho- bleeding upon probing was removed with sterile paper points genic species are present (8). Possible explanations for this latter phenomenon include the disturbance of synergistic (Johnson and Johnson, Amersfoort, The Netherlands). The was ml of growth of the mixed microbiota in the abscess by the plaque sample immediately transferred into 1 heat-inactivated (20 min, 56°C) human serum and trans- antibiotic. ported to the anaerobic chamber (Braun, Garching, Germa- The chemostat was shown to be a useful tool in studying ny). After vortexing, the whole sample was used to inoculate the effects of growth rate and nutrient limitations on the 9 ml of serum. Following 48 h of incubation at 370C, the culture that was obtained was split into two equal portions of 5 ml that were each transferred to a small (5-ml) continuous- * Corresponding author. culture vessel. These cultures were flushed under stirring

1717 1718 VAN DER HOEVEN AND VAN DEN KIEBOOM ANTIMICROB. AGENTS CHEMOTHER. with 91% N2-4% H2-5% CO2. The pH and redox potential in TABLE 1. Composition of mixed cultures before the addition of the culture, which was measured with a combined electrode propicillin and 24 h after exposure (Applicon, Austin, Tex.), were left at the values attained by Viable counts (10') in the culture. The dilution rate of the cultures was D = 0.1 Mean no. experimental cultures h-1. One of the cultures, the experimental culture, was (range [10'j) of after addition of the exposed to the antibiotic, while the control culture in the Organism viable counts following propicillin in control concn (mg/liter): second vessel served as a source of inoculum for the culturea preparation of subsequent experimental cultures. In this 1 5 10 50 way, several concentrations of propicillin could be tested by using mixed cultures with very similar Peptostreptococcus micros 97 (60-180) 35 19.5 3.2 2.3 microbial composi- Peptostreptococcus 25 (5-70) 89 0.4 2.1 NDb tions. anaerobius Exposure to antibiotic. After stabilization of the culture by Peptostreptococcus species 31 (19-55) ND ND 19.3 13.8 15 volume changes, a single dose of propicillin, which was dissolved in 0.5 ml of water to give a final concentration of 1 Prevotella intermedia 188 (150-220) 107 ND ND ND mg/liter in the vessel, was added to the experimental culture.

Two subsequent doses of 1 mg/liter were given at 24-h Lactobacillus catenaforme 40 (9-130) 35 1.7 ND ND Downloaded from intervals, that is, after 2.4 volume changes, during which the concentration of propicillin in the vessel dropped by dilution Streptococcus intermedius 7 (1-16) 71 2.3 3.2 3.8 until it was approximately 10o ofthe original value. One day Streptococcus morbillorum 20 (8-38) ND 8.2 ND ND (24 h) after the last addition, a sample was taken from the Other culture for microbiological analyses. Higher concentrations 18 (1-50) 160C 5 2.1 ND of propicillin were tested in experimental cultures that were Total CFU 426 (370-726) 497 36.7 29.9 18.9 obtained by transferring 2.5 ml from the control culture to a new vessel and a Before the addition of propicillin. then adding serum to a final volume of 5 ml. b ND, not detected. After batch growth for 24 h, the culture was switched to ' Mainly Eubacterium lentum. continuous growth, and after stabilization for 5 to 10 volume http://aac.asm.org/ changes, the culture was exposed to 5, 10, or 50 mg of propicillin per liter added in a single dose. The addition of antibiotic was repeated after 24 h. Samples for microbiolog- propicillin. The tests were read automatically after 24, 48, ical analyses were taken 24 h after the second addition of and 72 h of incubation, anaerobically, and at 37°C. propicillin. ,-Lactamase activity was tested by a microiodometric Microbiological analyses. All microbiological analysis pro- assay (13). The strains were precultured in Iso-Sensitest cedures were carried out anaerobically. Direct samples (0.1 broth for 24 h. Incubations with propicillin were carried out ml) were drawn from the cultures with a syringe and added in an anaerobic environment. to 9.9 ml of saline (0.9% sodium chloride). After vortexing with glass beads (diameter, 0.1 mm), samples were screened RESULTS on May 29, 2021 by guest microscopically for the presence of spirochetes and motile bacteria. Suitable dilutions of the samples were plated onto The bacteria found in the control and experimental cul- enriched cysteine-blood agar plates (19) that were incubated tures before the addition of propicillin are presented in Table for 7 days at 37°C. The dominant flora on the plates was 1. Since these cultures were derived from the same inoculum estimated by picking 25 to 30 colonies from a section of a and the species listed in Table 1 were consistently found in plate not containing more than 100 colonies. Colonies were these cultures, the presence of each organism was expressed subcultured and stored in skim milk (Difco) at -70°C prior to as the mean number of cfu per milliliter of culture and as the further identification. Also, differential counts based on range of the values observed in the different cultures. colonial morphology were done. As a first step, all isolates Dominant organisms in the cultures were Peptostreptococ- were characterized by Gram stain and cellular morphology. cus micros, Peptostreptococcus anaerobius, a Peptostrep- Gram-positive and gram-negative rods were characterized tococcus species that has not yet been further characterized, by using the API 20A system (API S.A., Montalieu-Vercieu, Prevotella intermedia (previously Bacteroides intermedius), France). Streptococci were identified by using the API 20 Lactobacillus catenaforme, Streptococcus intermedius, and Strep system. Weakly gram-positive peptostreptococci, Streptococcus morbillorum. Spirochetes were detected only gram-negative cocci, and some gram-negative rods were occasionally and were always below 0.1% of the total identified by using the API An-Ident system (Analytab counts; motile bacteria were not observed. The pH in the Products, Plainview, N.Y.). If necessary, additional tests cultures was 7.6 ± 0.1 (standard deviation) throughout the were performed, including aerobic growth on blood agar and experiment, while the redox potential was maintained by the determination of acidic fermentation products on PYG broth cultures at a nearly constant level of -300 mV with a (6). standard deviation of 15 mV. Antibiotic susceptibility and 13-lactamase activity. Repre- Repeated exposure of the mixed culture to propicillin at a sentative isolates were tested for the susceptibilities of their concentration of 1 mg/liter caused no drop in the total viable growth to propicillin. This was done by a microdilution broth numbers of cells at 24 h after the third addition of the method (7) in Iso-Sensitest broth (23.4 g/liter; Oxoid) sup- antibiotic (Table 1). Some changes in the composition of the plemented with vitamin K3 (0.05%), hemin (0.1%), Tris culture were observed, however. The increase in the number hydrochloride (3 g/liter), and sodium pyruvate (1 g/liter). of other organisms was mainly accounted for by Eubacte- Propicillin was added in twofold dilution steps to obtain rium lentum, an organism that was found only occasionally concentrations from 0.1 to 12.8 mg/liter. The starting optical in low numbers in other cultures and therefore was not densities in the test were approximately 20% of the value presented as a separate category. In contrast, marked attained after 72 h of incubation in the medium without changes were observed after exposure of the culture to 5 mg VOL. 35, 1991 EFFECTS OF PROPICILLIN ON PERIODONTAL BACTERIA 1719 TABLE 2. MICs of propicillin for bacteria isolated from mixed findings (19), relatively stable consortia of dominant period- cultures before and after the addition of propicillin ontal bacteria can thus be obtained. The stability of the MIC (mg/liter)' mixed continuous cultures was reflected by the nearly constant pH and redox potential values that were attained After propicillin during cultivation. Such pseudo-steady states of complex Organism Before addition at: propicillin bacterial cultures were also reported elsewhere (4, 5, 10), but 1 mg/liter 10 mg/liter they are, theoretically, still difficult to analyze (5, 16). The absence of strong effects of 1 mg of propicillin per liter Peptostreptococcus micros 0.5 (0.3) 0.5 (0.6) 1.4 (1.1) on the mixed culture seems to be in contrast to the Peptostreptococcus 1.2 (0.1) 0.4 (0.2) 1.5 (1.0) suscep- anaerobius tibilities of the organisms to the antibiotic. One explanation Peptostreptococcus species 0.6 (0.5) b 3.6 (2.1) for this discrepancy was the relatively low growth rate in the continuous culture that rendered the organisms less suscep- Prevotella intermedia 0.7 (0.4) 0.2 (0.1) tible (3, 20, 23) compared with those of organisms grown in batches, at their maximal growth rate, in Iso-Sensitest broth, Lactobacillus catenaforme 0.1 (0.1) 0.1 (0.1) which is used in susceptibility testing.

Higher concentrations of propicillin, however, caused a Downloaded from Streptococcus intermedius 0.6 (0.4) 0.6 (0.6) 1.2 (1.0) significant drop of all populations in the culture, in particular Streptococcus morbillorum 0.5 (0.6) that of P. intermedia. The specific response of P. intermedia attracts attention because other organisms in the culture Unidentified NDc 0.1-0.3 0.6-5.0 were equally susceptible to propicillin. Previous experi- a Values are means (standard deviations) of four isolates tested. ments (17) have suggested that mixtures of periodontal b _, not found in the culture. bacteria can grow synergistically in serum, whereby immu- c ND, not determined. noglobulin-cleaving organisms, such as P. intermedia, have an important role. This can be understood from the fact that immunoglobulins appear to be preferentially degraded and of propicillin per liter. P. intermedia dropped to below the utilized as nutrient sources by such consortia (17). It is http://aac.asm.org/ level of detection, while other species, with the exception of tempting to assume that the suppression of P. intermedia by P. micros, dropped to considerably lower levels at 24 h after propicillin resulted in a drop of total counts in the mixed the second addition of the antibiotic. Furthermore, the total culture because of a substrate (immunoglobulin) limitation. viable counts in the culture dropped significantly. Higher This hypothesis remains to be investigated, however. From concentrations of propicillin resulted in the suppression ofP. the results of the present experiment, it might be speculated intermedia and L. catenaforme to below the level of detec- that propicillin could be used to suppress P. intermedia in tion and 10- to 20-fold reductions of total viable counts the periodontal pocket or orofacial abscesses. In these (Table 1). abscesses, the isolation frequency of black-pigmented anaer- The MICs for strains isolated from the mixed cultures are obes, mainly P. intermedia, and other Peptostreptococcus given in Table 2. In the primary culture, MICs ranged from species appears to be remarkably high (9, 11, 22), suggesting on May 29, 2021 by guest 0.1 (L. catenaforme) to 1.2 (P. anaerobius) mg/liter. Higher an analogy between the growth of these organisms in serum MICs were recorded for strains from cultures that were cultures and abscesses. exposed to 10 mg of propicillin per liter. In summary, it can be concluded that the effect of antibi- The 3-lactamase activities of several isolates against prop- otics on complex microfloras cannot sufficiently be predicted icillin were assayed iodometrically and expressed semiquan- from the susceptibilities of the individual organisms. The titatively by using a scale of from 0 to 4. The P-lactamase- mixed continuous culture may offer a model to obtain producing strain Escherichia coli HB101 was used as a additional information, which could be used for predicting reference (score 4). ,B-Lactamase activity was not detected and understanding the effects of antibiotics in the clinical among the strains isolated from the primary enrichment situation. (control) culture or the culture obtained after the addition of 1 mg of propicillin per liter. Some of the isolates from the REFERENCES culture exposed to 10 mg of propicillin per liter exhibited 1. Banfai, P., H. Schirger, A. Wolf, and H. Mager. 1985. Penicillin- ,B-lactamase activity. One of seven different Peptostrep- Wirkspiegel im Serum und im Tonsilgewebe. Therapiewoche tococcus isolates had an activity score of 3, while one of the 35:5816-5818. two isolates of P. micros and a gram-positive coccus that 2. Brown, R. W., and P. Williams. 1985. 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