Oral Microinoi Inmmnol 1992: 7: 100-105

Microbiological evaluation of periapical infections in Egypt

M. O. Wasfy\ K. T. McMahon\ G. E. Minah^ W. A. Falkler, Jr.' 'Dental Research Division, tJS Naval Medical Research Unit No. 3, Cairo, Egypt, ^Department of Microbiology, Baltimore College of Dental Surgery, Baltimore, Maryland, USA

Wasfy MO, MeMahon KT, Minah GE, Falkler WA Jr. Microbiological evaluation of periapieal infections iti Egypt, Oral Microbioi Invnwtol 1992: 7: 100-105. This study identifies and correlates proportions of bacteria in periapically involved anterior teeth of 85 adult Egyptian patients. Affected sites were free from caries and periodontal disease but had a history of trauma. The mean number of component bacterial species per specirnen was 3.1. Anaerobic bacteria were the dominant fiora present in specimen cultures, cornprising 73% (190/259) of cultivable bacteria. The most frequently isolated organisms were Eubacterium species (68'^^)), black-pigmented Baeteroides (56"/)), Streptoeoeeus tnorbillorutn (47%) and non-pigmented Bacteroides (37%). These organisms also showed the highest proportional values relative to total cultivable bacteria. The tnean percentages of total viable counts of these isolates were 19.0%, 14.1%, 18.0% and 15.5%, respectively. Statistical analysis of data revealed a significant negative correlation between S. tnorbillorutn and Bacieroides species.

most frequent bacteria encountered in endodontic abscesses are Bacteroides and Fu.sobacteriutn species. The significance of black-pigmented Bacteroides (BPB) in tnixed infections has been discussed by many investigators (2, 14, 24, 32). When BPB are included, avirulent mixtures of oral flora are always pathogenic in animal abscess models (3, 36). Many BPBs have been reported to impair the host itnmune response by the production of extracellular proteases (24, 31, 32). Recently, a new asaccharolytic BPB species named Bacteroides endodontalis was identified from infected root canals (40). This investigation was designed to analyze the microflora associated with presumptive periapical abscesses. Aspects of this microbiological project dida, N e i s s e r i a a n d Veillottella ( 4 , I I , that may differ from previous studies 44). More recent studies have revealed are: 1) use of an indigenous Egyptian the frequent or concomitant presence population as part of the Middle East of anaerobes in mixed oral infections or Africa; 2) establishment of a tnore (29, 30, 35, 41), but no consistent pat- defined pattern of baeteria frequently tern of participant microorganisms has involved in pulpal infections; 3) exploappeared. One study reports a re- ration of proportional count relationlationship between Peptococeus and ships or relative roles of the identified Bacteroides species in 58'yi} of the pa- mierobial commttnities and 4) corretients (2). Oguntebi et al. (28) claim lation or interpretation of the obthat Fusobacterium nueleatutn and tained findings in terms of virulence Streptococcus t7titis were the most fre- factors or elements of mutualism that quent pair in alveolar abscesses. Also, take place in the active endodontic Williatns et al. (42) reported that the disease.

Most abscesses of the maxillofacial region are odontogenic. An endodontic or periapical abscess usually develops when pulpal tissue becomes exposed to oral bacteria following the development of dental caries, traurna or advanced periodontal diseases. However, endodontic infections have been reported without antecedent oral disease (28). Necrotic or infected material frotn pulp tissue or periodontal lesions may extend to the periapical area, causing a relatively rapid destruction of the periapical tissues and abscess fortnation. Such infections have the potential to involve sinuses and other facial spaces of the head and neck. Microorganisms commonly associated with periapical abscesses are viridans streptococci, staphylococci, lactobacilli, enteric bacteria and species of Can-

Key words: oral microflora: periapical abscess; mixed infection Momtaz O. Wasfy, Research Publication Branch, NAMRU-3 FPO New York, NY 09527t600, USA Accepted for publication March 6, 1991

Material and methods Patients and samples

Eighty-five patients aged 15-45 years presenting for treatment at the Faculty of Oral and Dental Medicine, Cairo University, were selected for this study. None had systemic diseases or had received antibiotic therapy for a tninitnum of one month before sample collection. The patients complained of symptoms of acute infiatnmation that involved variable degrees of pain and swelling and periapical radiolticency at one of the upper or lower anterior permatient teeth. Employed clinical criteria were discussed in detail by Matusow (21, 22) and Sundqvist (35). The teeth in question were intact with no evidence of caries or periodontal disease. The pulps were nonvital atid necrotic as determined by electrical and thermal testitig. All patients had the history of an earlier trauma, 3-4 months ago, and no itntnediate or subsequent abscess fortnation had occttrred. To approach the periapical tegion, involved teeth were routinely scaled, polished, isolated with a rubber datn and thotoughly swabbed with betadine (Purdue Frederick, Norwalk, CT). Access to root canals was established through the pulp chamber. Pus in periapical area was collected by the insertion of a sterile paper point into the canal for 1 tnin (18) under nitrogen

Microbiology of root canal ittfections

gas flow and then placing into 1 tnl of reduced transport fiuid (RTF) (37) in a screw capped vial still under a nitrogen gas flow. Vials were tightly capped and transported within 45-60 tnin to the Dental Research Division at the US Naval Medical Research Unit No. 3, Cairo, Egypt for tnicrobiological assay. The samples were itntnediately introduced into an anaerobic chamber (Coy Manufacturing, Ann Arbor, MI) for processing in an atmosphere of 85% nitrogen, 10% carbon dioxide and 5% hydrogen.

101

Tabie I. Relationship between the number of microorganistns* associated with periapical infections and their frequency oi' occurrence No. of component mtcroorgantstiis

Frequency in 85 specimens

0 1 2 3 4 5 6

29 13 11 4

Total

85

7 1

20

% of specimens

No. of isolates

8.2 1.2 23,5 34.1 15.3 12.9 4.7

0 1 40 87 52 55 24 259

• The mean nutnber of component bacteria per cultivable specimen is 3.1.

Cultivation, isolation and identification

Each sample was dispersed by low-energy sonication (Knotes Ultrasonic Cell Disruptor, Electro-Mechanical Instrument, Perkasie, PA) for 10 s and then serially diluted 10-fold in RTF. Appropriate dilutions were inoculated (0.05 ml quantities) onto duplicate plates of reduced Brueella blood agar supplemented with hemin and menadione (BRBA) (10). These were incubated at 37°C for 6-8 d (14). Plates containing 30-300 colonies were selected for isolation and identification procedures that included the detertnination of colonial morphology, gratn-stain reaction, catalase activity and ability to grow aerobically. The nutnber of colonies recovered were detertnined using a dissecting tnicroscope (Bausch & Lotnb, Rochester, NY) and expressed as percentages of total viable counts (TVC). Pure cultures from each tnorphological category were obtained by triple streaking of the various colonial morphologies onto BRBA with appropriate incubation. Identification was perfonned using suitable API rapid identification kits (Analytab Products, Plainview, NY) following the recotnmendations of the manufacturet. Bacteria not definitively identified by these techniques were identified using conventional tube tnethods as described by Holdetnan et al. (17). Statistical analysis of microbiological data was perfortned to study the correlation between bacterial cotnbinations in specimens where cultures yielded more than otie microorganism (13).

Results

Of the selected teeth, 91.8% were positive for tnicroorganistns upon culture, and 7 did not yield any bacterial growth. As shown in Table 1, 62.8% (49/78) of the cultivable periapical specitnens contained a mixture of 2-3 organisms, and

the retnainder of specimens had up to 6 species present. One specimen harbored a single mierobial species. Streptococcus tnorbilloruni. There was an average recovery of 3.1 bacterial species per cultivable specitnen. The detailed bacteriological analyses of this fiora and the relative prevalence of individual species in endodontic specitnens is outlined in Table 2. Of 259 isolates from 28 representative species, anaerobes (190 iso-

lates) were the predominant microorganisms isolated (73.4%). Specimens frotn which only anaerobes were recovered represented 30.8% of the total (24/ 78). The tnost frequently recovered anaerobic isolates in this population were Eitbaeterium lentum (51.3%)> S. tnorbillorutn (47.4%), Baeteroides intermedius (39.7'yii) and Bacteroides ureolyticus-Uke bacteria (28.2%). The individual bacterial species were also grouped accord-

Tai>le 2. Ptevalence of bacterial species isolated from 78 culture-positive root canals* Frequency in 78 specimens

% of specimens

"A Total

Microorganisms

Species

luihacterium

E. lentum E. iimosum

40 13

51.3 16.7

68.0

Blackpigmented Bacteroides

B. B. B. B. B.

31 5 3 3 2

39.7 6.4 3.9 3.9 2.6

56.5

intermedius etidodontalis dentieola melatiinogenicus iocschii

37

47.4

47.4

22 4 3

28.2 5.1 3.9

37.2

sanguis mitis mitieri intertncdiiis

9

30.7

4 4

11.5 9.0 5.1 5.1

viseo.sus odontolyticus tsraeiii meyeri

9 8 4 I

11.5 10.3 5.1 1.3

28.2

F. nucleatutn L. buccalis F. mortiferwn

12 3 1

15.4 3.8 1.3

20.5

Veillonella

V. parvula

14

18.0

18.0

Propionibacteriwn

P. gratnilosum P. acne

5

10.2

• 3

6.4 3.8

Streptococcus morbilloriim Other Bacteroides

B. weolyttcus B. buecae B. oralis

Other Streptococcus

S. S. S. S.

Actinomyces

A. A. A. A.

Fusobacterium

7

•

Peptoslreptococcus

P. micros P. magnus

6 1

7.7 1.3

9.0

Staphylococcus

S. epidermidis

5

6.4

6.4

* Seven satnples showed negative cultures.

102

Wasfy et al.

Table 3. Mean proportional recovery of 259 bacterial isolates (expressed as CFU% I'.v total viable counts at 10" dilution/0.05 ml plate inoculum) Mierobial categories Gram-positive rods: Eubacteriutn spp. Actinomyces spp. Propionit)acterium spp. Gram-negative rods: Black-pigmented Bacteroides Oiher Bacteroides spp. Fusobacteriutn spp. Gram-positive cocci: S. morbiltorum O\\\sr Streptococcus spp. Anaerobic cocci Staphylococcus spp. Gram-negative cocci: Veillonella parvula

Frequency in 78 specimens 53(68.0%) 22(28.2%) 8(10.3%)

Range of CFU% Mean proportion in positive cultures of CFU + SD* 0.00±41.3 0.00 + 95.6 0.00+100.0

19.0 + 28.6 3.5 + 13.1 3.3+15.1

44(56.4%) 29(37.2%) 16(20.5%)

0.00 ±93.5 0.00 ±94.0 0.00±41.3

14.1 ±25.7 15.5 ±27.4 14.1 ±25.7

37(47.4%) 24(30.8%) 7(9.0%) 5 (6.4%i)

0.00-100.0 0.00-100.0 0.00-71.4 0.00-96.0

18.0±30.2 7.6±21.0 2.5±11.0 5.9 ±20.1

14 (1 B.O'VI,)

0.00-90.0

6.1 ± 18.9

kaline phosphatase, a-glucosidase and with a-L-fucosidase in 2 cases). These reactiotis were essentially identical to the ATCC strain of B, endodontalis, 35406. Differentiation of 5. intermedius and B. endodontalis was tnade with the API-zym system. In this assay, B. intertnedius was positive for alpha glueosidase activity and B, etidodontalis was negative. B. endodontalis showed a mean percentage prevalence of less than 1.0% and was isolated from only 6.4% (5/78) of the cultivable specimens (Table 2). Statistical analysis of the data revealed a negative correlation {P