Copyright© 1988, American Society for Microbiology
Semiquantitative
Culture
Results
and Pathogenic Significance
of
Obligate
Anaerobes in Peritonsillar Abscesses
ANSSI M. M.
JOKIPII,'t*
LIISAJOKIPII,' PEKKA SIPILA,2 AND KALEVI JOKINEN2Departmentof Serology and Bacteriology, University of Helsinki, Helsinki,' andDepartmentof Otolaryngology, University of Oulu, Oulu,2 Finland
Received 22October 1986/Accepted 25 January 1988
Westudied the bacteriain consecutive peritonsillarabscesses using semiquantitation of the primaryculture findings and correlated the resultstoclinicalparameters.Puncture-aspiratedpusfrom 42 abscesses yielded 133
isolates. Group Astreptococciwereisolated 10 times and, unlike other bacteria,wereisolated 4 timesinpure
culture; other beta-hemolytic streptococci werefound in 8 abscesses, and anaerobeswere found in 28. The
infections were polymicrobial, with two to seven bacteria in 83%. Anaerobes were more abundant than
nonanaerobes; members ofthegeneraStreptococcus,Bacteroides, Peptostreptococcus, and Fusobacterium were
the most important quantitatively, considering both frequency and abundance. In patients with ongoing antibiotic treatment, nonanaerobes (butnot anaerobes)were less abundant than in untreated patients. The
abundance ofobligate anaerobes (specifically cocci and gram-positive rods)correlatedtotheseverity ofillness as defined by fever and short duration before hospitalization. With other groups of bacteria, no such
correlationwasfound. The correlationwasnotexplained byadifferencebetween theantibiotic-treatedand the
untreated patients. Theresultsindicatethe value of the semiquantitation of culture data and the frequency and pathogenic significance of obligateanaerobesin peritonsillarabscesses.
Peritonsillar abscess isapotentially life-threatening
com-plication ofacutetonsillitis caused bygroupAstreptococci
and, according to a recent observation (10), of infectious mononucleosis. It requires immediatetreatment,which in its generally accepted form consists of drainage, antibiotics, andtonsillectomy; alternatively, needle aspiration and anti-bioticsmaysuffice (6, 7, 11). Propertreatmentthus relieson
microbiological statistics and shouldcoveressentially all the
bacteria thataresignificant causativeagents.Besides
Strep-tococcus pyogenes, a variety of organisms, notably
an-aerobes, commonly found in the normal pharyngeal flora havebeen culturedfrom peritonsillar abscesses (1, 4, 5, 12, 13). Owingtothe method of sample collection, such culture resultsincludeanunknown proportion of contaminants and
theclinical significance of the remaining organismsas
caus-ative agentsmay vary.
Thepurposeof thisstudywastorecord the bacteria found inaseries of 42peritonsillar abscesses and toestimate their relative significance by semiquantitating culture results and correlating bacteriological data with clinical parameters.
MATERIALSANDMETHODS
Patients. We studied 42 consecutive patients admittedto theDepartment of Otolaryngology, UniversityCentral Hos-pital, Oulu, Finland, and scheduled for tonsillectomy
be-causeofperitonsillarabscess. In additiontocommon demo-graphic information, the history of the present illness and previous episodes of tonsillitis,antimicrobialtreatment,and axillary temperature with an accuracy of 0.1°C were
re-corded (the standard procedure of the outpatient depart-ment).
* Correspondingauthor.
t Presentaddress:DepartmentofMedicalMicrobiology,
Univer-sityofTurku, Kiinamyllynkatu 13,20520Turku,Finland.
Samples. Standard culture media (3) were used, unless
stated otherwise, and all media for anaerobic incubation
wereprereduced in the GasPak system(BBLMicrobiology Systems, Cockeysville, Md.) for 24 horlonger.
Each abscess was punctured, andpus (>1 ml) was
aspi-rated intoasterile syringe. Daytime specimenswere
imme-diately (about 15 s) plated (0.1to0.2 ml)on chocolate agar
andnonselective enriched anaerobic bloodagar, and
night-time specimens stayed in the syringe, which was stored
anaerobically inaGasPakjaratroomtemperature,andwere
plated similarly in the morning. Anaerobic plates were
exposedtoaironly afew seconds before seeding andwere
immediately returned to anaerobic conditions at 37°C for incubation. Aerobic incubationtook place inacandle jarat 370C.
Bacteriology. Theprimary incubation continued for 3 days
or longer, after which the plates (in their anaerobic or
aerobic containers) were transferred to the bacteriological laboratory. For at least 7 days, the primary plates were
examined for distinct colony types to be isolated. The heaviness ofgrowthwasrecordedasthe numberof colonies
per plate: 0; +, 1 to 10; ++, 11 to 50; +++, 51 to 200; ++++, more than 200 or, usually, innumerable. The
or-ganisms were identified by standard procedures (3), the
Phadebact Streptococcus Test (Pharmacia, Uppsala, Swe-den) for grouping ofbeta-hemolytic streptococci, and the API 20A strip (API System S.A., Montalieu-Vercieu, France) and gas chromatography ofpeptone-yeast-glucose (PYG) brothcultureextracts(8)forcharacterizingthe
anae-robes. Wedefined obligate anaerobesas bacteriathatwere
unabletoform colonies duringa48-hcandlejar incubation; thus, microaerophilic streptococci were included in the
viridansgroup.
Statistical treatment. The significance of differences
be-tween frequency distributions was analyzed by the Fisher one-tailedexact test(14).Correlation between the heaviness ofbacterialgrowthand eitheraxillary temperatureor dura-957
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J. CLIN. MICROBIOL. TABLE 1. Organisms isolatedfrom 42 peritonsillarabscesses
Organism
Streptococcuspyogenes,
groupA
Streptococcus sp., beta-hemolytic, non-groupA Streptococcus sp.,viridans
group
Streptococcus faecalis Staphylococcusaureus
Staphylococcus sp., coagu-lase negative
NeisseriaorBranhamellasp. Moraxellasp. Haemophilus influenza Haemophilus sp. Eikenella corrodens Diphtheroidbacillus Bacillus subtilis Candidaalbicans Totalnonanaerobes Peptostreptococcus anaero-bius Peptostreptococcus asaccha-rolyticus Peptostreptococcus sp. Staphylococcus saccharoly-ticus Veillonellaparvula Fusobacterium necrophorum Fusobacterium nucleatum Bacteroidescapillosus Bacteroidesdistasonis Bacteroidesfragilis Bacteroidesmelaninogenicus Bacteroidessp. Propionibacterium sp. Arachnia propionica Bifidobacteriumsp. Lactobacillus sp. Nonsporeforming gram-positive rod Clostridium sp. Total anaerobes
No. ofisolationsa
+ ++ +++ ++++ Total
2 1 1 6 10
2 2 3 1 8
4 4 1 8 17
4 6 3 4 6 1 5 1 7 6 1
2 1 il
1
1 2
1 1
1 3
4 1 12
37 13 12
2 1 2
1
1 1 1
2 1
3
1 1
il 6 5
a Heavinessofgrowth(+ to+ + ++);semiquantitative estimate of numbers
oforganisms, basedonnumbersofcoloniesonprimary plate.
tion ofillness was assessedbySpearman'srank correlation
procedure givingthecoefficientr, andtheone-tailed
proba-bilityvalue (14).
TABLE 2. Multiplicityoforganisms isolated from each of 42
peritonsillarabscesses andfrequenciesof various combinations of anaerobes and nonanaerobes
No. ofabscesses with thefollowingno.of
No. of nonanaerobes:
anaerobes
0 1 2 3 4 5
0 2 5 6 1
1 6 3 3 1 1
2 2 3 2 1
3 2 2
4 i
5
6werenot, and thisinformationhadnotbeen recorded in 18 cases.
Culture results. All
growth
in aerobic and anaerobic cul-turesof punctureaspirateswasrecordedwithoutattempts ata
priori judgement
ofclinical relevance. Amongthe aerobic orfacultativebacteria,
variousstreptococciwerefound mostfrequently, followed by members ofthe family Neisseria-ceae and gram-positive nonsporeforming bacilli (Table
1).
Among theobligate anaerobes, the members of the genera Bacteroides and Péptostreptococcus accounted formost of thefindings.
Of the non-group A beta-hemolyticstrepto-cocci, two were of group C (++ and +++) and the
remaining six were not groupable with the reagents for
groups
A,
B,
C, D,andG. If thebacteriaseenonlyasone orfew colonies (+) are ignored, anaerobes and nonanaerobes wereapproximately equally frequent and the
quantitatively
most
important
were members of the generaStreptococcus
(isolated27times), Bacteroides (16 times),
Peptostreptococ-cus (13 times), and Fusobacterium (5 times) (Table
1).
Anaerobes were isolatedasconfluent growth(+++
++)57%(29of51)of thetime,ascomparedwith 24%(20 of 82) for the nonanaerobes. Members ofoneor more of the above four
generaoccurredas+ + + +growthin 27 of the abscesses and
asoneofthemostabundantisolates in 35 of the abscesses. Group A streptococciwere isolatedas a pureculturein4of the 10 cases; besides these, no other organism except one
viridans group streptococcus was seen in pure culture (on theprimary plates).
Anaverage of 3.2 organisms were isolated from 42
peri-tonsillar abscesses, 1.2 anaerobes and 2.0 nonanaerobes. Most or 83% (35 of 42) were mixed infections with up to
seven organisms per abscess (Table 2). In the 28 cases in
which anaerobes were found, an average of 1.8 of them
could be differentiated, and the corresponding number of nonanaerobes in 38 cases was 2.2. The distributions of the
multiplicities of infection with anaerobes versus
nonan-aerobeswerenotnotably different from each other (Table 2).
RESULTS
The study population consisted of29male and 13 female patients withperitonsillar abscesses.Theiragesrangedfrom
15to62yearswitha mean ageof 27.7years.Aretrospective analysis of their clinical records revealedthat 10patients had notexperienced tonsillitis before thepresent occurrence, 6
hadhadoneortwopreviousoccurrences, 11had hadmore
thantwopreviousoccurrences,andtheinformationhad not been recorded in 15cases. During the present episode, the tonsilsof18patientswerecovered with exudation, thoseof
TABLE 3. Heaviness ofgrowthofanaerobes and nonanaerobes invariouscombinations isolatedfrom 42peritonsillar abscesses
No.ofabscesses according to heaviness
Anaerobes ofgrowth of nonanaerobes:
(heaviness ofgrowth)
O + ++ +±+ ++++
0 2 2 2 8
+ 1 2
++ 2 2 i
+++ 1
+ ++ + 2 5 3 2 7
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TABLE 4. Comparison of bacterial findings from peritonsillar abscesses in 21 patients with and
withoutongoing antibiotic treatment
No.of abscesseswith
bacterialgrowth:
Bacteriological With With no
classification antibiotics antibiotics
+or ++or + or ++ or none heavier none heavier
Nonanaerobes 13 8 1 20 <0.0001
Streptococci 14 7 5 16 0.0061
Staphylococci 20 1 20 1 NS
Neisseriaceae 20 1 17 4 NS
Gram-negative rods 19 2 19 2 NS
Gram-positive rods 21 15 6 0.0103
Anaerobes 8 13 9 12 NS
Cocci 15 6 16 5 NS
Gram-negative rods 12 9 12 9 NS
Gram-positive rods 19 2 18 3 NS
Total growth 5 16 21 0.0239
a The Fisher exacttest;NS,P>0.05, onetailed.
Table 3describes the semiquantitative approach appliedto the 42culture results ignoring the taxonomicposition of the isolated organisms. Inall, + + + + growthwasfound in 71%
(30 of 42) of the abscesses and + + or heavier growth was
found in88%(37 of 42). Ifscantygrowth (+) isignored, the anaerobic and nonanaerobicfindingsperabscesswererather
similar with regard to both frequency and heaviness of growth (Table 3). The semiquantitative culture results
re-vealed no obvious dependence (antagonism or synergism)
betweenanaerobes and other bacteria.
Antibiotictreatment.No antimicrobialtreatmenthad been administered during the present illness of 21 patients, 17
were receiving penicillin, 3 were receiving erythromycin,
and 1wastreated withampicillin. In the latter21 cases,the treatment had continued for 1 to 10 days (mean, 3.6 days) before admission. In the treated group, bacterial growth from the abscesses was less heavy (Table 4). There was a
difference between anaerobes and other bacteria. Total nonanaerobic growth was less heavy in the antibiotic-re-ceiving group as compared with the untreated group, and thisfinding wasdue tothe relative lack ofstreptococci and diphtheroids; the amount of anaerobes showed no such correlation with ongoing antibiotic treatment (Table 4). Of the 10group Astreptococci, 5, including 1of thosein pure
culture, were found in patients receiving penicillin and 5, including 3 of those in pure culture, were found in the untreated group.
Clinical correlations. Measurements of axillary
tempera-ture atthe time of admissionwereavailablein the records of
36 patients. Fever correlated significantly with the semi-quantitatively assessed bacterialgrowth (Table 5). Thiswas
found withtotal anaerobic growth and withthe heaviness of growth of anaerobic cocci, but not with nonanaerobes or
with the othermorphologic classes of anaerobes.
Totalbacterial growthperabscesswasheavier the shorter
the duration of illness before admission (Table 6). More specifically, this correlation was found with the anaerobes
and among them with the cocci and gram-positive rods.
Similar correlations were found within the group of 21
antibiotic-treated patients: total growth,r, =0.403, P<0.05 (Spearman's rankcorrelation); total anaerobes,r, =0.524, P
< 0.01; anaerobic cocci, r, = 0.504, P < 0.025; anaerobic
gram-positive rods,r, = 0.294, 0.05 < P< 0.10.
DISCUSSION
The adoption of anaerobic technology and the ensuing bacteriological reevaluations have alteredourviews ofmany
infectious processes. That infections arederived from
nor-malflora has becomeanaccepted principle,and peritonsillar
abscess is anexample. Increased work load in the
microbi-ological diagnosis andambiguityofinterpretation whenever contamination from the normal floracannotbe excludedare
unfortunate by-products of theprogress. Thepresent inves-tigation showed thatsemiquantitative datamay be useful in themicrobiological work and that clinical relevancemay be
attributedtolaboratory findings inadifferentialmanner.
Nighttime specimens were kept in syringes in anaerobic
jars until morning to ensure anaerobiosis without
compro-mising the series of consecutive patients. The significance of the procedure was not investigated. There were no gross
differences between daytime and nighttime specimens as
regards the culture results, but we did not analyze subtle differences, as this would be unjustified in the absence of
parallel experiments with divided specimens.
Group A streptococci are implicated as pathogens in
peritonsillar abscesses, because thecomplication is usually
preceded by tonsillitis. Previous studies have revealed S.
pyogenesinnotmorethan39.3%or35 of 89cases(1, 4, 12,
13), and the present finding was even less (23.8%).
Non-group A beta-hemolytic streptococci were isolated twice (2.2%)by the previousgroups,whilewefoundeight (19.0%). Thismayreflectamoregeneralswitch fromgroupA toother
groups, notably C, which we have noticed in several other
infections recently (unpublished data), and the total
fre-quency of beta-hemolytic streptococci in peritonsillar
ab-scesses in both our series and the previous ones is very similar or about 40%. Finally, ourresults confirm that itis characteristic of S. pyogenes to occur in pure culture;
TABLE 5. Correlation betweenaxillarytemperature and heaviness of bacterialgrowthfromperitonsillarabscesses of 36 patients
Bacteriological Mean temp(°C) +SD (no. of patients)
classification________________________________r.j
classification + growth or none + +growthor heavier
Totalgrowth 37.5 ± 0.62(5) 37.6 ± 0.63 (31) 0.381 <0.025
Nonanaerobes 37.5 ± 0.56(13) 37.7 ± 0.67 (23) 0.168 NS
Anaerobes 37.5 ± 0.64(16) 37.7± 0.62 (20) 0.356 <0.025
Cocci 37.5 ± 0.60(27) 38.0± 0.63 (9) 0.351 <0.025
Gram-negativerods 37.6 ±0.64(22) 37.7± 0.63 (14) 0.239 NS
Gram-positive rods 37.6 ±0.63 (32) 37.7 ±0.70 (4) 0.225 NS
aSpearman'srankcorrelationcoefficient,usingthewholerangeofsemiquantitative growthreadingsfromOto++++;NS,P>0.05,onetailed.
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960 JOKIPII ET AL.
TABLE 6. Correlationbetweenacutenessof illness and heaviness of bacterialgrowthfromperitonsillarabscesses of 42 patients
Bacteriological Mean date of onset ofsymptoms+ SD (no. of patients) rb pb
classification +growthor none + +growthorheavier
Totalgrowth -7.0 ± 1.41 (5) -4.3 ± 2.93 (37) 0.265 <0.05
Nonanaerobes -5.6 ± 3.08(14) -4.1 ± 2.75 (28) 0.203 NS
Anaerobes -5.8 ± 3.11 (17) -3.8± 2.55(25) 0.381 <0.01
Cocci -5.1 ± 3.22(31) -3.3 ± 1.10 (11) 0.321 <0.025
Gram-negativerods -4.9 ± 3.01(24) -4.2± 2.84 (18) 0.204 NS
Gram-positiverods -4.8 ± 3.01(37) -3.0± 1.58 (5) 0.325 <0.025
a Date of admittance isday0.
b Spearman's rankcorrelationcoefficient,usingthe wholerangeofsemiquantitative growth readingsfrom to++++ +;NS,P>0.05,onetailed.
previous studies recorded this 17 times in 89patients (1,4, 12, 13).
Eventhough there isa subgroupofpatientswithgroup A
streptococciinpureculture,mostperitonsillarabscesses are causedbypolymicrobialinfections.Theaveragemultiplicity of culture resultsinearlier serieshas been2.3organismsper abscess (1, 5, 12, 13), and we found 3.2 per abscess.
Likewise, most peritonsillar abscesses are anaerobic infec-tions:66.1% on the average in 242 earlierpatients(1, 4, 5,12, 13) and 66.7% in our series (some authors included micro-aerophilic streptococci, butwe countedonly obligate
anae-robes). The individual variation of the culture results is great, andnearly alltheorganisms arecommon members of
thenormal flora. Sincetreatment
always
includesantibioticsbefore culture results are
available,
the variation creates aproblemofchoice ofdrug. The similarity with normal flora creates aproblemofinterpretation ofculture results. Much of the presenteffortwas directed to thesetwoquestions.
The rationale behind the quantitation of culture results was twofold. Contaminating bacteria originating from the normalfloraatthe siteofpuncture orfromelsewhereduring handling ofthe sample orthe cultures are expected to be fewer innumberontheaverage than bacteria from thepus.
Second, it seems reasonable to suggest that abundant iso-lates are more likely causes of clinical illness than are
bacteria evidenced only by a few colonies. The present
approach was crude, and the semiquantitation was not
intended to refer to exact numbers of bacteria inside
ab-scesses, but to evaluate the possibilities of the normal procedures of a diagnostic laboratory. Dilution and the
calculation of CFU per volume ofpus is too tedious for routine diagnostic work. Furthermore, our method of
pro-viding anaerobiosis for the primary cultures has proved
efficient in revealing anaerobes in otolaryngological
infec-tions (9). We were unable simultaneously to adhere to the
method,tocollectunselected patientsconsecutively,and to carry outquantitative bacteriology. Therefore, our
compro-mise was toreplace the latterby
ils
approximation, colonycounts onprimary plates.With the crude butsimple method
it was possible to reduce the list ofsignificant bacteria to speciesof Streptococcus, Bacteroides, Peptostreptococcus,
and Fusobacterium. One or more species of these four genera wereamongthemostabundant isolatesin83% or 35
of 42 cases. In general terms, the weighted results empha-sized obligate anaerobes. These technical considerations
serve the purposes ofdescription and communcation, and thepossible clinical relevance remainsa suggestion.
Axillary temperatureand thedurationoftheillnessbefore admission were available for the
evaluation
of the clinical severity ofthe condition at the time of the microbiologicalintervention.
These parameters correlated with theabun-dance of anaerobes and more specifically with anaerobic
cocci
(and gram-positive rods)
intheperitonsillar abscesses.Adifference betweentheantibiotic-treatedand theuntreated
groups was excluded as an explanation ofthe finding. The
probable
causality behind the correlation is that anaerobic cocci andgram-positive
rods aggravate the symptoms and are thus to be regarded as clinically significantlaboratory
findings. Conceivably,
numerous other factors also deter-mine theseverity
of symptoms. The lack of sucha clinicalcorrelation in the case of other bacterial groups is no evidence
against
theirsignificance,
which remains to be evaluated infuture studies. While the importanceof anaer-obicinfections as awholeisgenerally accepted, that ofthe numerouscomponentsof normalflora-derivedpolymicrobial
infectionsremainsdebatableinmost cases.Few attempts to sort out
pathogenic
significance in mixed infections have beenreported, and it isinterestingto note that suchimpor-tancehas been ascribedtoanaerobic gram-positive cocci in amurine subcutaneous abscess model(2). The samegroup ofbacteria was emphasized in our work. The present
ap-proach to ascribe pathogenic significance to a component may bewidelyapplicable tomixedinfections.
ACKNOWLEDGMENTS
We thankIrjaJouhtenandMarjaKorvala forexcellenttechnical assistance.
This work was supported by a grant from the Sigrid Jusélius Foundation, Helsinki, Finland.
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