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Interference by Aerobic and Anaerobic Bacteria in Children With Recurrent Group A  -Hemolytic Streptococcal Tonsillitis
Itzhak Brook, MD, MSc;
Alan E. Gober, MD
Arch Otolaryngol Head Neck Surg. 1999;125:552-554.
ABSTRACT
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Objective To compare the frequency of recovery of aerobic and anaerobic bacteria with interfering capability of group A  -hemolytic streptococci (GABHS) in the tonsils of children with and without a history of recurrent GABHS pharyngotonsillitis.
Patients and Methods Tonsillar cultures were taken from a group of 20 children with and 20 without history of recurrent GABHS pharyngotonsillitis.
Results Eleven aerobic and anaerobic isolates with interfering capability with GABHS were recovered from 6 (30%) of the 20 children with recurrent GABHS, and 40 such organisms were isolated from 17 (85%) of the 20 without recurrences (P<.01). The interfering organisms included aerobic ( -hemolytic and nonhemolytic streptococci) and anaerobic organisms (Prevotella and Peptostreptococcus species).
Conclusions The tonsils of children with a history of recurrent GABHS infection contain fewer aerobic and anaerobic bacteria with interfering capability of GABHS than those without the history of recurrent GABHS infection. The presence of these interfering bacteria may play a role in preventing GABHS infection.
INTRODUCTION
PENICILLIN'S failure to eradicate group A -hemolytic streptococcal (GABHS) tonsillitis is of great clinical concern.1 Various theories have been suggested to explain this phenomenon. One explanation is that -lactamase–producing bacteria protect GABHS by inactivating penicillin.2 Another theory is that -hemolytic streptococci (AHS) that are part of the normal oral flora may play an important role in the eradication of GABHS. Some of the AHS isolates have been shown in vitro to compete and thus interfere with GABHS growth.3-4 The oropharynx is known also to be colonized by other organisms (nonhemolytic streptococci and Neisseria species) that are also capable of interfering with GABHS.3 However, the role of these organisms, as well as anaerobic bacteria in recurrent GABHS tonsillitis, has not been studied before.
This study was designed to compare the recovery of all interfering aerobic and anaerobic bacteria from the tonsils of children with recurrent GABHS tonsillitis and those without such a history.
PATIENTS AND METHODS
PATIENTS
Children seen sequentially for their annual physical examination by us between June 1, 1992, and June 30, 1996, were included in the study. We excluded those who received antimicrobial therapy or had GABHS tonsillitis in the previous 2 months. Only 1 child per household was enrolled in the study. Included in the study were 40 children (29 boys), with a mean age of 8.2 years (age range, 5-14 years).
Twenty of the children had a history of recurrent GABHS tonsillitis, which was defined as having at least 3 episodes of microbiologically documented GABHS infection within the past 6 months, that was associated in each instance with tonsillar and/or pharyngeal erythema and 1 or more of the following: sore throat, tonsillar exudate, fever (body temperature >37.5°C), and tender cervical lymphadenitis. Another group of 20 children had no history of GABHS infection within the past 2 years. No difference was noted in the ages and sex of the 2 groups.
MICROBIOLOGY
The tonsillar and pharyngeal areas of the patients were swabbed with 2 sterile cotton swabs. Aerobic cultures were obtained by placing a swab in modified Stuart bacterial transport system (Baltimore Biological Laboratories, Cockeysville, Md). Cultures for anaerobes were obtained by introducing a swab into the anaerobic transport media (Port-A-Cul; Baltimore Biological Laboratories; and Becton Dickinson Labware; Microbiology Systems, Cockeysville). The specimens were inoculated within 3 hours of collection.
Sheep blood (5%), chocolate, and MacConkey agar plates (Baltimore Biological Laboratories) were inoculated to isolate aerobic organisms. The plates were incubated at 37°C aerobically (MacConkey agar) and under 5% carbon dioxide (blood and chocolate agars) and examined at 24 to 48 hours. To isolate the anaerobes, the specimens were plated on prereduced Brucella agar (Baltimore Biological Laboratories) supplemented with vitamin K1 and sheep blood, an anaerobic blood agar plate containing kanamycin sulfate and vancomycin hydrochloride, an anaerobic blood plate containing phenylethyl alcohol, and placed in enriched thioglycollate broth.5 These media were incubated in anaerobic jars at 37°C and examined at 48 and 96 hours. Bacteria were identified using conventional methods.5-6 GABHS and AHS were preliminarily identified by colonial morphology and type of hemolysis. Characterization of GABHS was done by bacitracin-disk sensitivity and by serologic grouping using latex-agglutination techniques. All microbiologic studies were done without knowledge of the patient group.
METHODS OF TESTING FOR INTERFERENCE
The inhibitory activity of 5 separate colonies of all aerobic and anaerobic bacterial strains recovered from each patient was individually tested against a single recent clinical isolate of GABHS (Streptococcus pyogenes), as previously described.7 In brief, minidrops of log-phase broth cultures of the isolates were transferred with a Steer steel pin replicator to blood agar plates and allowed to dry for 15 minutes at room temperature. A sample of a log-phase broth culture of the target GABHS strain was applied adjacent to each of the tested isolates, and the plates were incubated in 5% carbon dioxide at 37°C for 24 hours. Inhibition of the GABHS or the other strains was registered as previously described.4 Statistical significance was calculated using the  2 test.8
RESULTS
GABHS was recovered from 3 of the group (15%) with history of recurrent GABHS tonsillitis and from 2 of the group (10%) without such history. Organisms with interference capability were not recovered from any of the 3 children with history of recurrent GABHS tonsillitis that were colonized with GABHS. These organisms were isolated from 1 of the 2 patients of the group without such history that was colonized with GABHS.
Eleven aerobic and anaerobic isolates with interfering capability vs GABHS were recovered from 6 (30%) of the 20 children with recurrent GABHS, and 40 such organisms were isolated from 17 (85%) of the 20 without recurrences (P<.01) (Table 1). The interfering organisms included aerobic (-hemolytic and nonhemolytic streptococci) and anaerobic bacterias (Prevotella and Peptostreptococcus species).
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Isolates With Interference Capability in Children With and Without a History of Recurrent GABHS Tonsillitis*
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COMMENT
This study compared the recovery rate of all aerobic and anaerobic bacteria capable of interfering with GABHS in children with or without a history of recurrent GABHS tonsillitis. This study shows, as was previously described,3-4,7, 9-10 that AHS isolates are less often recovered in children with recurrent GABHS tonsillitis than in those without such history. Interfering AHS isolates were previously recovered in up to a third of the patients who did not develop pharyngotonsillitis,4, 7 or those with tonsillitis that responded to penicillin therapy.9-10 However, when we also studied all other aerobic and anaerobic organisms capable of interfering with GABHS, their presence was detected in 85% of the patients without a history of recurrent GABHS tonsillitis. Therefore, presence of organisms with interfering potential may play a greater role than was previously known in the prevention of GABHS tonsillitis.
We were able to show for the first time that organisms other than AHS can also interfere with GABHS in vitro growth. This includes aerobic nonhemolytic streptococci as well as the anaerobic bacteria Peptostreptococcus and Prevotella species. The potential in vitro inhibitory activity of these 2 species of anaerobic bacteria against other organisms was previously reported.11
Reestablishment of colonization of the oropharynx by AHS with interfering capabilities can be used to prevent recurrent GABHS tonsillitis. Roos et al12 recently treated children with recurrent tonsillitis by colonizing their tonsils with interfering AHS or placebo. Clinical recurrences occurred in 2% (1 of 51 children) of the AHS group and 23% (14 of 61) of the placebo-treated group.
It is possible that maintenance of the normal oropharyngeal flora that possess inhibitory potential of pathogens could contribute to the reduction of recurrent tonsillar infection. It is yet to be determined if using antibiotics with a wide-spectrum efficacy against members of the oral flora may enhance colonization with potential pathogens, such as GABHS. Further studies are warranted to investigate whether maintaining a normal flora in the oropharynx could be beneficial in preventing GABHS tonsillitis.
AUTHOR INFORMATION
Accepted for publication November 20, 1998.
Corresponding author: Itzhak Brook, MD, MSc, PO Box 70412, Chevy Chase, MD 20813-0412 (e-mail: Brook{at}mx.afrri.usuhs.mil).
From the Department of Pediatrics, Georgetown University, School of Medicine, Washington, DC.
REFERENCES
1. Kaplan EL, Johnson DR. Evaluation of group A streptococcus from the upper respiratory tract by amoxicillin with clavulanate after oral penicillin treatment failure. J Pediatr. 1988;113:400-403.
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2. Brook I. The role of a beta-lactamase–producing bacteria in the persistence of streptococcal tonsillar infection. Rev Infect Dis. 1984;6:601-607.
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3. Crowe CC, Sanders E, Longley S. Bacterial interference, II: the role of the normal throat flora in prevention of colonization by group A streptococcus. J Infect Dis. 1973;128:527-532.
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4. Grahn E, Holm SE. Bacterial interference in the throat flora during a streptococcal tonsillitis outbreak in an apartment house area. Zentralbl Bakteriol Mikrobiol Hyg [A]. 1983;256:72-79.
5. Summanen P, Baron EJ, Citron DM, Strong CA, Wexler HM, Finegold SM. Wadsworth Anaerobic Bacteriology Manual. 5th ed. Belmonth, Calif: Star Publishing Co; 1995.
6. Murray PR, Baron EJ, Pfaller MA, Trenover PC, Yolken RH. Manual of Clinical Microbiology. 5th ed. Washington, DC: American Society for Microbiology; 1993.
7. Grahn E, Holm SE, Roos K, Ekedahl C. Interference of alpha-hemolytic streptococci isolated from tonsillar surface, on hemolytic streptococci, Streptococcus pyogenes: a methodological study. Zentralbl Bakteriol Mikrobiol Hyg [A]. 1983;254:459-468.
8. Converse WJ. Practical Nonparametric Statistics. 2nd ed. New York, NY: John Wiley & Sons Inc; 1980.
9. Roos K, Grahn E, Holm SE. Evaluation of beta-lactamase activity and microbial interference in treatment failures of acute streptococci tonsillitis. Scand J Infect Dis. 1986;18:313-319.
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10. Brook I, Gober AE. Role of bacterial interference and beta-lactamase–producing bacteria in the failure of penicillin to eradicate group A streptococcal pharyngotonsillitis. Arch Otolaryngol Head Neck Surg. 1995;121:1405-1409.
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11. Murray PR, Rosenblatt JE. Bacterial interference by oropharyngeal and clinical isolates of anaerobic bacteria. J Infect Dis. 1976;134:281-285.
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12. Roos K, Holm E, Grahn-Hakansson E, Lagergren L. Recolonization with selected alpha-streptococci for prophylaxis of recurrent streptococcal pharyngotonsillitis: a randomized placebo-controlled multicenter study. Scand J Infect Dis. 1996;28:459-462.
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