International Journal of Antimicrobial Agents 27 (2006) 263–266

Interference of antibiotics in the growth curves of oral streptococci

Ana Castilloa,∗, Jose Liebanaa, Encarnacion Lopezb, Pilar Bacab,Jose M. Liebanaa, Marıa J. Liebanaa, Francisca Castillob

a Department of Microbiology, School of Medicine and Dentistry, University of Granada, Spainb Department of Stomatology, School of Dentistry, University of Granada, Spain

Received 25 July 2005; accepted 30 October 2005

Abstract

The growth curve interference (GCI) induced by different antibiotics was studied using reference strains of oral streptococci. This parametermay prove to be useful in preventing subacute endocarditis of odontological origin in high-risk patients. The growth curves using differentconcentrations of antibiotics and with an initial optical density (OD) of 0.05± 0.01 were observed until an OD of 0.9–1.0 was reached. TheGCI was defined as the lowest concentration of antibiotic that modified the growth curve with respect to a control without antibiotic. The GCIvalues were then compared with the minimum inhibitory concentration (MIC) values. In all cases, interference in the growth curves was atl©

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eywords: Antibiotic susceptibility; MIC; Oral streptococci

. Introduction

The viridans group streptococci comprise a heteroge-eous group of microorganisms that are an important partf the normal flora of the oral cavity, and for this reason arelso known as oral streptococci. They may also be isolated

rom areas such as the pharynx, skin, vagina or intestine,here they are not usually considered pathogenic. How-ver, viridans group streptococci may be involved in sepsisnd pneumonia in subjects who are immunodepressed[1–3],

n sepsis and meningitis in neonates[4] and, particularly,n subacute endocarditis, often associated with bacteraemiafter dental procedures[5]. They have also been isolated

rom invasive pyogenic infections in immunocompetent hosts6–8].

These microorganisms were traditionally considered to beniformly sensitive to a wide range of antibiotics[9]. Recenttudies have come to refute this assumption, with reportsf strains with a high resistance to aminoglycosides[10,11]

∗ Corresponding author at: Dpto. de Microbiologıa, Facultad de Medicina,vda. de Madrid, 11, 18012 Granada, Spain.

and penicillin[12–14]and of strains tolerant with respectpenicillin [10] or resistant to macrolides[14,15] and otheantibiotics[16].

Not only does resistance create problems in treatininfections produced by these microorganisms, it isimportant because resistance can be transmitted to otheteria, for example toStreptococcus pneumoniae in the case openicillin [13,14,16]. Increased bacterial resistance has brelated to the use of antibiotics in dental treatment[17,18].Other studies identify an in vitro relationship betweenproduction of slime, certain biotypes and the efficacyantibiotics[10,19].

The objective of the present study was to evaluateinterference in the growth curves of oral streptococciduced by certain antibiotics and to relate this interferentheir minimum inhibitory concentration (MIC) value.

2. Materials and methods

2.1. Assayed strains

el.: +34 958 24 35 49; fax: +34 958 24 61 19.E-mail address: anamc@ugr.es (A. Castillo).

The following streptococcal reference strains were used:Streptococcus mutans Ingbritt, Streptococcus sanguis DSMZ

924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

oi:10.1016/j.ijantimicag.2005.10.017

264 A. Castillo et al. / International Journal of Antimicrobial Agents 27 (2006) 263–266

Table 1Minimum inhibitory concentrations (MICs) of the antibiotics tested against the streptococcal bacterial strains

Antimicrobial agent MIC (mg/L)

S. mutans Ingbritt S. sanguis DSMZ 6777 S. gordonii NCTC 3165 S. oralis NCTC 11427 S. salivarius HHT

Penicillin 0.07 0.07 0.015 0.07 0.015Amoxicillin 0.03 0.12 0.12 0.07 0.03Cefuroxime 0.015 0.07 0.07 0.12 0.015Imipenem 0.003 0.015 0.015 0.003 0.003Erythromycin 0.03 0.015 0.25 0.03 0.03Spiramycin 0.5 0.5 0.5 1 0.5Josamycin 0.12 0.12 0.12 0.12 0.07Roxithromycin 0.25 0.07 0.07 0.12 0.07Clindamycin 0.015 0.03 0.015 0.07 0.015

6777, Streptococcus gordonii NCTC 3165, Streptococ-cus oralis NCTC 11427 andStreptococcus salivariusHHT.

2.2. Determination of the MIC

Antibiotics were tested at concentrations ranging from0.00007 mg/L to 16 mg/L. The MIC was determined using anagar dilution method, a Steer’s replicator and Wilkins Chal-grens agar (Difco Laboratories, Madrid, Spain). An inoculumof 105–106 colony-forming units/mL was used. Plates wereincubated at 36± 1◦C in an anaerobic atmosphere containing85% N2, 10% H2 and 5% CO2 and readings were taken after48 h [9]. The MIC was defined as the lowest concentrationof antibiotic that visibly inhibited growth of the microorgan-isms.

2.3. Growth curve interference (GCI)

The tested strains, taken from a 24 h culture in WilkinsChalgrens broth (Difco Laboratories), were inoculated in9 mL of the same medium until reaching an optical density(OD) of 0.05± 0.01 at a wavelength of 600 nm in a spec-trophotometer (Zuzi series 4200; Zuzi, Beijing, China). Foreach strain, a series of tubes was prepared with the afore-

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3. Results

The MICs of the antibiotics tested are given inTable 1.Table 2 shows the GCI of the 45 tests in which the ODof 0.9–1.0 was reached with the minimum concentration ofantibiotic. In 25 cases (55.6%) this OD was obtained withthe concentration immediately below the MIC (Fig. 1). This

Fig. 1. Growth curves ofStreptococcus mutans Ingbritt under the actionof penicillin: an example of interference (0.03 mg/L) at one concentrationbelow the minimum inhibitory concentration (0.07 mg/L). OD, optical den-sity.

Fig. 2. Growth curves ofStreptococcus gordonii NCTC 3165 under theaction of spiramycin: an example of interference (0.12 mg/L) at two con-centrations below the minimum inhibitory concentration (0.5 mg/L). OD,optical density.

mentioned OD value and the nine studied antibiotics wadded in a scaled series of seven concentrations greateequal to and below the MIC, giving a total of 315 expments. As the growth control for each assay, a tube withsame medium and inoculum but with no antibiotic was uThe tubes were incubated in a bain-marie at 36± 1◦C. Spectrophotometry readings of the OD were taken every hour.experiment was considered complete when an OD of 0.9was reached with the lowest concentration of antibioticmodified the growth curve with respect to the control. Tvalue is referred to as the GCI.

The following antibiotics were used: penicillin, amocillin, cefuroxime, erythromycin, spiramycin, roxithromycand clindamycin (Sigma Chemical Co., St Louis, Mimipenem (Merck Sharp Dohme, Madrid, Spain)josamycin (Ferrer Grupo, Barcelona, Spain).

,

A. Castillo et al. / International Journal of Antimicrobial Agents 27 (2006) 263–266 265

Table 2Concentration of antimicrobial and time corresponding to optical density values of 0.9–1 for thestreptococcal bacterial strains tested

finding was consistent for cefuroxime with all the strains,and it was nearly always the case for amoxicillin (with oneexception forS. salivarius HHT). In nine cases (20%) the ODof 0.9–1.0 was obtained with concentrations two under theMIC value (Fig. 2), and in 11 instances (24.4%) the concen-trations were clearly lower than the MIC (Fig. 3). This cleardifference between the MIC and the GCI was not observedat all for S. sanguis or S. gordonii, nor was it associated inany case with amoxicillin or cefuroxime. It was more fre-quent forS. mutans Ingbritt andS. oralis NCTC 11427, andwith the antibiotics imipenem, spiramycin, josamycin androxithromycin. The OD 0.9–1.0 values were reached in vary-ing periods of time. In seven cases (15.6%) it took 4 h. It is

F no entra-t ticald

noteworthy thatS. mutans Ingbritt, S. gordonii NCTC 3165andS. salivarius HHT did not reach the established OD in4 h. It took 5 h in 14 cases (31.1%) and 6 h in the remainderof the experiments (53.3%).

4. Discussion

Viridans group streptococci, also known as oral strepto-cocci, are increasingly involved in human infections, mostnotably perhaps in subacute endocarditis. These infectionsoften take place after some sort of dental procedure. For thisreason, preventive clinical trials have been developed usingantibiotics, especially for high-risk patients. Depending onthe patient, such measures may involve amoxicillin, clin-damycin or erythromycin given orally, or parenteral adminis-tration of ampicillin and vancomycin[20]. Preventive inter-ventions are also used when there is a risk of bacteraemia ofoncohaematological origin[21]. As far as the treatment ofviridans streptococcal endocarditis is concerned, combina-tions of antibiotics such as penicillin and aminoglycosidesor vancomycin with aminoglycosides may be used, withthe duration of therapy depending on the patient’s response.Alternative therapeutic or prophylactic regimens have beenproposed by various authors[22,23].

The different patterns of susceptibility of different speciest uchr wl-e

t pro-v he

ig. 3. Growth curves ofStreptococcus oralis NCTC 11427 under the actiof imipenem: an example of interference (0.0003 mg/L) at three conc

ions below the minimum inhibitory concentration (0.003 mg/L). OD, opensity.

o a number of antibiotics and the risk of transmission of sesistance[11,16] make it necessary to increase our knodge of the activity of antibiotics on oral streptococci.

A number of authors have described parameters thaide a dynamic view of antibiotic activity in vitro. Such is t

266 A. Castillo et al. / International Journal of Antimicrobial Agents 27 (2006) 263–266

case of the post-antibiotic effect, the post-antibiotic sub-MICand the sub-MIC effect for some antibiotics onS. gordoniiandS. sanguis [24], as well as the MAC described by Gem-mel and Lorian[25], which is the minimal drug concentrationproducing a one-log decrease in cell numbers with respect tothe control. The parameter defined in our study as the GCI,or the lowest concentration of antibiotic that modifies thegrowth curves of the bacterial strains tested, also offers a per-spective on the activity of antibiotics on oral streptococci andcould be considered of a utility similar to that of the MAC.

In conclusion, the most important finding of this study isthat the antibacterial activity of the tested antibiotics is main-tained from 4–6 h, not only with sub-MIC concentrations buteven with concentrations two, three or more times below theMIC value. These results point to the in vitro efficacy of thetested antibiotics at subinhibitory concentrations and suggest,in turn, the potential utility of establishing preventive mea-sures for those cases of subacute endocarditis stemming fromdental interventions in patients at high risk.

Acknowledgments

This study was supported in part by the AndalusianRegional Government through the research project ‘Microbi-ology, Immunology and Epidemiology of Oral Diseases’, andb peanR

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[17] Erickson PR, Herzberg MC. Emergence of antibiotic resistantStrep-tococcus sanguis in dental plaque of children after frequent antibiotic

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