European Journal of Clinical Microbiology & Infectious Diseases (2021) 40:1461–1470

https://doi.org/10.1007/s10096-021-04180-y

ORIGINAL ARTICLE

Microbiology of parapharyngeal abscesses in adults: in search

of the significant pathogens
Tejs Ehlers Klug 1 & Thomas Greve 2 & Camilla Andersen 2 & Pernille Hahn 3 & Christian Danstrup 4 &
Niels Krintel Petersen 1,4 & Mirjana Ninn-Pedersen 4 & Sophie Mikkelsen 4 & Søren Pauli 1 & Simon Fuglsang 1 &
Helle Døssing 5 & Anne-Louise Christensen 6 & Maria Rusan 1 & Anette Kjeldsen 5

Received: 2 December 2020 / Accepted: 1 February 2021 / Published online: 10 February 2021
# The Author(s) 2021

Abstract
We aimed to describe the microbiology of parapharyngeal abscess (PPA) and point out the likely pathogens using the following
principles to suggest pathogenic significance: (1) frequent recovery, (2) abundant growth, (3) growth in relative abundance to
other microorganisms, (4) percentage of the isolates recovered in both absolute and relative abundance, (5) more frequent
recovery in PPA pus compared with tonsillar surface and tissue. Comprehensive bacterial cultures were performed on specimens
obtained from adult patients (n = 60) with surgically verified PPA, who were prospectively enrolled at five Danish ear-nose-
throat departments. The prevalent isolates (in PPA pus) were unspecified anaerobes (73%), non-hemolytic streptococci (67%),
Streptococcus anginosus group (SAG) (40%), Corynebacterium spp. (25%), Neisseria spp. (23%), Fusobacterium spp. (22%),
Fusobacterium necrophorum (17%), Prevotella spp. (12%), and Streptococcus pyogenes (10%). The bacteria most frequently
isolated in heavy (maximum) growth were unspecified anaerobes (60%), SAG (40%), F. necrophorum (23%), and Prevotella
spp. (17%). The predominant microorganisms (those found in highest relative abundance) were unspecified anaerobes (53%),
SAG (28%), non-hemolytic streptococci (25%), F. necrophorum (15%), S. pyogenes (10%), and Prevotella spp. (10%). Four
potential pathogens were found in both heavy growth and highest relative abundance in at least 50% of cases: F. necrophorum,
Prevotella spp., SAG, and S. pyogenes. SAG, Prevotella spp., F. necrophorum, S. pyogenes, and Bacteroides spp. were
recovered with the same or higher frequency from PPA pus compared with tonsillar tissue and surface. Our findings suggest
that SAG, F. necrophorum, Prevotella, and S. pyogenes are significant pathogens in PPA development.

Keywords Parapharyngeal abscess . Microbiology . Fusobacterium . Streptococcus . Pathogens

Introduction
* Tejs Ehlers Klug Parapharyngeal abscess (PPA) refers to a collection of pus
tejsehlersklug@hotmail.com
located laterally or posteriorly to the pharyngeal constrictor
muscle. The pathogenesis of PPA is scarcely described but
1
Department of Otorhinolaryngology, Head & Neck Surgery, Aarhus likely includes lymphogenous or direct spread of bacteria
University Hospital, Palle Juul-Jensens Boulevard 99,
DK-8200 Aarhus N, Denmark
from upper airway mucosa or teeth [1]. Less frequently,
2
PPAs are reported as extensions of peritonsillar abscesses
Department of Clinical Microbiology, Aarhus University Hospital,
Aarhus, Denmark
(PTA) [2–5].
3
As with other neck abscesses derived from upper airway
Department of Otorhinolaryngology, Head & Neck Surgery,
Hospital Lillebaelt, Kolding, Denmark
mucosa (i.e., PTA), a polymicrobial mixture of aerobes and
4
anaerobes can be grown from PPA pus, when appropriate
Department of Otorhinolaryngology, Head & Neck Surgery, Aalborg
University Hospital, Aalborg, Denmark
culture methods are applied [6, 7]. These polymicrobial infec-
5
tions derived from areas that are heavily colonized raise the
Department of Otorhinolaryngology, Head & Neck Surgery, Odense
University Hospital, Odense, Denmark
following questions: which bacteria are significant pathogens,
6
which are merely non-pathogenic bystanders, and which
Department of Otorhinolaryngology, Head & Neck Surgery,
Regional Hospital West Jutland, Herning, Denmark
1462 Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470

bacteria represent contamination as the needle pass through Microbiological analyses

the mucosa and tissues to the abscess?
The knowledge regarding significant pathogens associated Samples were processed in a class 2 laminar air flow safety
with PPA is very limited. The few previous studies focusing cabinet using an aseptic technique. Initially, a pilot study was
on the microbiology of PPA in adults were all retrospective, performed to determine which culture media were optimal for
and thus reported findings in routine cultures, and no attempts culturing in relation to bacterial distinction. In ten patients, pus
were made to analyze the significance of the recovered bacte- aspirates, tissue samples, and swabs were plated on 5% horse
ria [2, 5, 8, 9]. blood agar, chocolate ager, 10% horse blood agar, anaerobic
The current study was undertaken to further describe the agar (chocolate plate containing K-vitamin and cysteine), selec-
microbiology associated with PPA in adults, using compre- tive Fusobacterium agar (containing 5 mg/L nalidixic acid and
hensive aerobic and anaerobic culture methods, and to identify 2.5 mg/L vancomycin) [10], and semi-solid thioglycolate
which bacteria were the likely pathogens. (Statens Serum Institute Diagnostica, Hillerød, Denmark).
Plates were incubated at 35 °C, the first three plates in 5%
CO2, the next two plates in anaerobic atmosphere including a
metronidazole-disc (10 UG) (Oxoid, Roskilde, Denmark), and
Materials and methods the thioglycolate vial at ambient atmosphere. A Mueller-Hinton
agar with horse blood and 20 mg/L NAD (Oxiod, Roskilde,
Patients Denmark), plus selected antimicrobial discs, was also inoculat-
ed and incubated at 35 °C at 5% CO2; this plate was not used
Patients were prospectively enrolled in the study between for antimicrobial susceptibility testing but as a selective medi-
April 2016 and August 2019 at five Danish ear-nose-throat um aiding the initial differentiation of bacterial species. All
departments (Aarhus University Hospital, Odense University plates were incubated for up to 7 days and checked for growth
Hospital, Aalborg University Hospital, Hospital Lillebaelt, after 2 and 4 days. Based on the pilot study, the rest of the
and Regional Hospital West Jutland). The inclusion criteria samples were plated on 5% horse blood agar, anaerobic agar,
were (1) patients with PPA, defined as surgical finding of pus Fusobacterium agar, Mueller-Hinton agar and thioglycolate,
located laterally or posteriorly to the pharyngeal constrictor and checked for growth after 2 and 4 days. The thioglycolate
muscle, including abscesses in the parapharyngeal, medium was only cultured when the solid media showed no
retropharyngeal, visceral, and pharyngeal mucosa (the part growth after 4 days of incubation. Cultured microorganisms
laterally to the constrictor muscle) spaces, but excluding base were identified by phenotypic appearance and biochemical pro-
of tongue, floor of mouth, and submandibular space, (2) age > files according to accepted guidelines [11] and in selected cases
17 years, and (3) written and oral consent. supplemented with matrix-assisted laser desorption/ionization
Patients were categorized in subgroups based on the site of time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker
primary infection and the presence of concurrent PTA. Daltonics, Bremen, Germany). The microorganism colony
count was reported semi-quantitatively.
Species level identification was performed depending on
Specimen collection the type of material (pus, tissue, or swab), abundance of the
bacteria, and methods available for identification. In general,
After the induction of general anaestesia, swabs were bacteria belonging to the normal pharyngeal flora were de-
rubbed thoroughly on the surfaces of each of the tonsils scribed at genus level in tissue and swab cultures (e.g., non-
and placed in transport media (E-swab (Copan, Brescia, hemolytic streptococci, Neisseria spp., Corynebacterium
Italy)). The PPA was punctured through the pharyngeal spp.), whereas bacteria were identified to species level in
mucosa or externally through the skin and aspirated into pus if they were in a dominant or co-dominant quantity.
a sterile syringe. In cases of concurrent PTA, needle aspi- Species differentiation between the closely related
ration was performed through the peritonsillar mucosa to Streptococcus anginosus, Streptococcus constellatus, and
the PTA. If pus was not obtained through intact mucosa or Streptococcus intermedius is inadequate by phenotypic ap-
skin, pharyngeal (+/− ipsilateral tonsillectomy) or skin in- pearance, MALDI-TOF, and 16S rDNA gene fragment anal-
cision was performed, and pus was collected into a sterile ysis and is thus referred to as Streptococcus anginosus group.
syringe at the time of abscess perforation. In cases of ton- Most anaerobic bacteria were merged into unspecified anaer-
sillectomy, large bilateral tonsil biopsies (approximately obes, where the initial criteria were growth in anaerobic atmo-
half the volume of the tonsils) were obtained and placed sphere and sensitivity towards metronidazole. Long wave UV
in sterile containers separately. Pus aspirates, tonsillar tis- light was used to detect characteristic fluorescence of the col-
sue, and surface swabs were placed at −80 o C within onies (e.g., Fusobacterium spp. = green, Prevotella spp.,
30 min of collection. Porphyromonas spp., and Veillonella spp. = red), and
Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470 1463

pigmentation was also used for identification (e.g., Prevotella Results

spp. = black) [12]. Fusobacterium was divided into
Fusobacterium nechrophorum and Fusobacterium spp. (the Patient characteristics
latter including as per MALDI-TOF identification:
Fusobacterium nucleatum, Fusobacterium naviforme, Sixty adult patients (mean age 51 years, range 18–89 years)
Fusobacterium gondiaformans, and Fusobacterium sp.). All with surgically verified PPA were enrolled in the study.
species of Prevotella are referred to as Prevotella spp. (con- Twenty-six (43%) patients had concurrent PTA. Significantly
taining as per MALDI-TOF identification: Prevotella oris, more patients were male (n = 43, 72%) (p = 0.001, binomial
Prevotella denticola, Prevotella disiens, Prevotella probability test). The oropharynx was deemed (by the investi-
nigrescens, Prevotella baroniae, and Prevotella histocola), gator) as the primary site of infection in the majority of cases (n
Veillonella as Veillonella spp., and Bacteroides is divided into = 48, 80%), followed by the hypopharynx (n = 8, 13%), the
Bacteroides fragilis and Bacteroides spp. larynx (n = 2, 3%), and the teeth (n = 1, 2%). One patient had
unknown site of primary infection. Six (10%) patients were
Pathogenic significance previously tonsillectomized (all bilaterally). The majority of
patients had sore throat (98%), pain on swallowing (97%),
To identify likely pathogens, we considered several factors, and anamnestic fever (67%). Clinical findings included
including absolute and relative abundance of growth in cultures parapharyngeal swelling (97%, on flexible endoscopy),
from PPA pus, as well as recovery from PPA pus vs. the ton- peritonsillar (71%) and tonsillar swelling (63%), tender neck
sillar mucosa/tissue. In this regard, we assumed that frequent on palpation (67%), trismus (58%), neck swelling (43%), dys-
recovery of a microorganism in absolute and relative (compared pnea (25%), and torticollis (10%). Antibiotics were prescribed
with the other bacteria) abundance at the site of infection (PPA to 51% (30/59) of patients prior to admission, and 86% (48/56)
pus) suggests pathogenic significance [13]. To address the received antibiotics before specimens were obtained (Table 1).
problem with insignificant bystanders and contamination, we No statistically significant differences in biochemical parame-
hypothesized that more frequent recovery from PPA pus than ters were found between subgroups (based on the site of prima-
from the tonsillar mucosa points to pathogenic significance. ry infection and the presence of concurrent PTA; Table 1).
Hence, the following principles to suggest pathogenic sig-
nificance of the recovered bacteria were used in the current Number of isolates
study: (1) frequent (10% or more) recovery in PPA pus, (2)
abundant (maximal level) growth in PPA pus, (3) greater rel- The mean number of isolates was significantly higher in PTA
ative abundance in PPA pus culture compared with other mi- pus (4.3, SD 1.8) than PPA pus (3.4, SD 1.5) (p = 0.038,
croorganisms, (4) majority (50% or more) of isolates recov- Student’s t-test) and higher in ipsilateral tonsillar tissue (5.7,
ered in both absolute and relative abundance, (5) more fre- SD 1.6) and surface swabs (5.8, SD 1.6) (both p < 0.001,
quent recovery in PPA pus compared with tonsillar surface Student’s t-test). No significant differences were found between
swabs and tonsillar tissue. the mean numbers of isolates in cultures from ipsi- and contra-
lateral tonsillar tissues (p = 0.65, Student’s t-test) or from ipsi-
Statistical analyses and contralateral surface swabs (p = 0.95, Student’s t-test).
The mean number of isolates from PPA and PTA pus cul-
Statistical analyses were performed using the Fisher’s exact tures was similar between the pilot study (samples from ten
test for categorical variables (absolute number of isolates) and patients cultured on a larger variety of plates) compared with
the Student t-test and analysis of variance (ANOVA) for con- the rest of the samples (PPA 3.8 vs 3.3, p = 0.37, Student’s t-
tinuous variables (mean number of isolates and biochemical test; PTA 4.7 vs 4.3, p = 0.73).
parameters) in between-groups comparisons. The binomial
probability test was used for gender comparison. The normal- PPA pus culture findings
ity of data was assessed using quantile-quantile (QQ) plots.
Statistical significance was defined as p < 0.05. PPA pus cultures were polymicrobial in 93% (56/60) of cases.
The most prevalent isolates were unspecified anaerobes (n = 44,
Permissions 73%), non-hemolytic streptococci (n = 40, 67%), Streptococcus
anginosus group (n = 24, 40%), Corynebacterium spp. (n = 15,
The study was approved by the Ethical Committee of Aarhus 25%), Neisseria spp. (n = 14, 23%), Fusobacterium spp. (n =
County (# 1-10-72-4-16) and by the Danish Data Protection 13, 22%), F. necrophorum (n = 10, 17%), Prevotella spp. (n =
Agency (1-16-02-65-16). Informed consent was obtained 7, 12%), and Streptococcus pyogenes (n = 6, 10%) (Table 2).
from all patients, in accordance with the guidelines set by No statistically significant differences in recovery rates were
the Danish National Board of Health. found between subgroups (Table 2). Furthermore, no
1464 Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470

Table 1 Clinical and biochemical

characteristics of 60 patients with Primary site of infection All Oropharynx Hypopharynx Other1
parapharyngeal abscess stratified n = 60 n = 48 n=8 n=4
by primary site of infection and
presence of concurrent + PTA - PTA
peritonsillar abscess (PTA) n=26 n=22

Males 43 (72%) 18 (69%) 17 (77%) 5 (63%) 3 (75%)

Age, mean (SD)2 51.3 (19) 47.3 (21) 54.2 (16) 48.3 (17) 67.0 (10)
Duration of symptoms, median (days) 4.5 5.0 4.0 4.0 4.5
Antibiotic treatment prior to admission3 51% 60% 40% 63% 25%
Penicillin V 40% 48% 40% 38% 0%
Penicillin V + metronidazole 9% 12% 0% 25% 0%
Amoxicillin-clavulanate 2% 0% 0% 0% 25%
Antibiotic treatment prior to obtaining 86% 87% 82% 88% 75%
specimens4
Tobacco smoking (current)5 41% 46% 38% 25% 50%
Temperature, mean °C (SD)6 38.0 (0.8) 38.0 (0.7) 37.9 (0.8) 38.1 (1.0) 39.3
Biochemistry, mean (SD)
C-reactive protein (mg/L)7 186 (106) 177 (111) 176 (104) 192 (55) 299 (141)
Leukocyte count (× 109/L)8 15.4 (3.6) 14.6 (2.8) 15.1 (4.2) 17.2 (3.9) 18.3 (1.8)
Neutrophil count (× 109/L)9 12.4 (3.2) 11.9 (2.5) 11.8 (3.5) 14.3 (4.0) 15.7 (2.5)
1
The larynx (n = 2), tooth (n = 1), unknown (n = 1)
2
Subgroup comparison: p = 0.19, ANOVA
3
Three patients with missing information (n = 57)
4
Pencillin (n = 17), penicillin and metronidazole (n = 14), cefuroxime and metronidazole (n = 10), cefuroxime (n
= 5), and ampicillin (n = 2)
5
n = 59
6
n = 36
7
Subgroup comparison: p = 0.18, ANOVA), n = 59
8
Subgroup comparison: p = 0.15, ANOVA), n = 59
9
Subgroup comparison: p = 0.07, ANOVA), n=55

significant impact of smoking, age (cut off 50 years), or the anaerobes (n = 32, 53%), Streptococcus anginosus group
presence of PTA was found on culture findings. (n = 17, 28%), non-hemolytic streptococci (n = 15, 25%),
F. necrophorum (n = 9, 15%), S. pyogenes (n = 6, 10%),
and Prevotella spp. (n = 6, 10%) (Table 3).
Abundant growth

Among the 35 PPA patients who had heavy growth (the max-
Majority of isolates recovered in both heavy growth
imal level of growth) of one or more microorganisms, the
and relative abundance
bacteria most frequently recovered in heavy growth in PPA
pus specimens were unspecified anaerobes (n = 21, 60%),
Four potential pathogens were found in both heavy growth
Streptococcus anginosus group (n = 14, 40%),
and highest relative abundance in at least 50% of cases:
F. necrophorum (n = 8, 23%), and Prevotella spp. (n = 6,
F. necrophorum (90% of isolates were found in heavy
17%) (Table 3).
growth/80% of isolates were found in highest relative
abundance), Prevotella spp. (86%/86%), Streptococcus
Growth in relative abundance to other anginosus group (71%/58%), and S. pyogenes (100%/
microorganisms 50%) (Table 3). In addition, a number of infrequent recov-
eries were also found in both relative and absolute abun-
The most frequently recovered predominant microorgan- dance (Bacteroides sp. (n = 1), Eggerthia catenaformis
isms (most abundant in relation to other isolates in the (n = 1), Capnocytophaga sp. (n = 1), and
same culture) in PPA pus specimens were unspecified Lachnoanaerobaculum orale (n = 1)) (Table 3).
Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470 1465

Table 2 Culture findings in

parapharyngeal pus aspirates Primary site of infection All Oropharynx Hypopharynx Other1
among 60 patients with n = 60 n = 48 n=8 n=4
parapharyngeal abscess stratified
by primary site of infection and Microorganisms +PTA −PTA
presence of concurrent n = 26 n = 22
peritonsillar abscess (PTA)
Aerobic bacteria
Streptococcus pyogenes 6 (10%) 1 (4%) 4 (18%) 1 (25%)
Streptococcus anginosus group 24 (40%) 12 (46%) 10 (45%) 1 (13%) 1 (25%)
Non-hemolytic streptococci 40 (67%) 17 (65%) 14 (64%) 6 (75%) 3 (75%)
Streptococcus pneumoniae 2 (3%) 2 (9%)
Haemophilus parainfluenzae 1 (2%) 1 (5%)
Staphylococcus aureus 2 (3%) 1 (4%) 1 (5%)
Coagulase-negative staphylococci 4 (7%) 2 (8%) 1 (5%) 1 (13%)
Eikenella corrodens 4 (7%) 2 (8%) 1 (5%) 1 (13%)
Neisseria spp. 14 (23%) 2 (8%) 7 (32%) 5 (63%)
Escherichia coli 2 (3%) 2 (8%)
Proteus vulgaris 1 (2%) 1 (4%)
Aggregatibacter aphrophilus 1 (2%) 1 (5%)
Corynebacterium spp. 15 (25%) 6 (23%) 6 (27%) 2 (25%) 1 (25%)
Trueperella pyogenes 1 (2%) 1 (5%)
Gemella spp. 1 (2%) 1 (13%)
Anaerobic bacteria
Fusobacterium necrophorum 10 (17%) 5 (19%) 3 (14%) 2 (25%)
Fusobacterium spp. 13 (22%) 4 (15%) 7 (32%) 2 (50%)
Prevotella spp. 7 (23%) 5 (19%) 2 (1%)
Bacteroides fragilis 1 (2%) 1 (5%)
Bacteroides spp. 1 (2%) 1 (4%)
Bifidobacterium longum 1 (2%) 1 (4%)
Eggerthia catenaformis 1 (2%) 1 (5%)
Capnocytophaga spp. 1 (2%) 1 (4%)
Actinomyces spp. 2 (3%) 1 (13%) 1 (25%)
Alloscardovia omnicolens 1 (2%) 1 (4%)
Lachnoanaerobaculum orale 1 (2%) 1 (13%)
Anaerobes (unspecified) 44 (73%) 17 (65%) 18 (82%) 7 (88%) 2 (50%)
Fungi
Candida 1 (2%) 1 (4%)
Number of isolates, mean 3.4 3.2 3.7 3.5 2.8
Polymicrobial 56 (93%) 22 (85%) 22 (100%) 8 (100%) 4 (100%)
1
The larynx (n = 2), tooth (n = 1), unknown (n = 1)

PPA pus versus tonsillar tissue and surface swab compared with tonsillar tissue and tonsillar surface swabs
findings (Table 4). In contrast, Corynebacterium spp., Neisseria spp.,
and Hemophilus spp. were recovered from PPA pus much less
In 53 patients, a complete set of ipsilateral cultures was per- frequently compared with tonsillar tissue and swab (Table 4).
formed (including PPA pus, ipsilateral tonsillar tissue, and
ipsilateral tonsillar swab). Disregarding isolates detected in The impact of antibiotic treatment prior to admission
only one patient, Streptococcus anginosus group, Prevotella
spp., F. necrophorum, S. pyogenes, and Bacteroides spp. were The mean number of isolates in PPA cultures among pa-
recovered with the same or higher frequency from PPA pus tients without antibiotic treatment prior to admission was
1466 Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470

Table 3 Predominant
microbiologic findings in Microorganisms Relative Absolute Percentage of isolates found in relative/
parapharyngeal abscess (PPA) abundance1 n = 60 abundance2 absolute abundance3
pus based on semi-quantitative n = 35
cultures
Aerobic bacteria
Streptococcus pyogenes 6 3 100%/50%
Streptococcus 17 14 71%/58%
anginosus group
Non-hemolytic 15 6 38%/15%
streptococci
Streptococcus 1 50%/0%
pneumoniae
Staphylococcus 1 25%/0%
epidermidis
Eikenella corrodens 2 50%/0%
Aggregatibacter 0%/0%
aphrophilus
Neisseria spp. 2 14%/14%
Escherichia coli 0%/0%
Corynebacterium spp. 2 13%/0%
Gemella spp. 0%/0%
Anaerobic bacteria
Fusobacterium 9 8 90%/80%
necrophorum
Fusobacterium spp. 5 4 38%/31%
Prevotella spp. 6 6 86%/86%
Bacteroides spp. 1 1 50%/50%
Bifidobacterium 1 100%/0%
longum
Eggerthia catenaformis 1 1 100%/100%
Capnocytophaga spp. 1 1 100%/100%
Actinomyces spp. 1 1 100%/100%
Lachnoanaerobaculum 1 1 100%/100%
orale
Anaerobes 32 21 73%/48%
(unspecified)
Fungi
Candida 1 50%/0%
1
Number (%) of microorganisms isolated in maximum (either heavy (n = 35), moderate (n = 19), or sparse (n =
6)) semi-quantitative growth among patients with PPA
2
Number (%) of microorganisms isolated in heavy semi-quantitative growth among patients with PPA
3
See Table 2 for total number of microorganisms

higher (3.7) compared with patient with antibiotic treat- Discussion

ment (3.1), although this was not statistically significant
(p = 0.16, Student’s t-test). Similarly, polymicrobial Suggested significant pathogens
growth was found in all PPA pus cultures from patients
without antibiotic treatment before admission compared Based on extensive cultures from PPA aspirates obtained with
with 86% in patients, who had received antibiotics (p = a focus to minimize the contamination from surrounding tis-
0.11, Fisher’s exact test). S. pyogenes was recovered sig- sues, a polymicrobial aerobic and anaerobic flora was found in
nificantly more frequently among patients without antibi- the vast majority (93%) of PPAs, and the most frequently
otic treatment prior to admission (21%) compared with recovered bacteria were non-hemolytic streptococci (includ-
patients treated with antibiotics (0%) (p = 0.01, Fisher’s ing the Streptococcus anginosus group) and unspecified
exact test). anaerobes.
Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470 1467

Table 4 Bacterial findings in

cultures from parapharyngeal PPA pus Tonsillar tissue Tonsillar surface swab
abscess (PPA) pus, tonsillar tissue
(ipsilateral to PPA), and tonsillar Aerobic bacteria
surface swab (ipsilateral to PPA) Streptococcus pyogenes 5 4 5
among 53 PPA patients with all Streptococcus anginosus group 21 20 8
three specimens collected
Non-hemolytic streptococci 36 50 51
Streptococcus pneumoniae 2 1 1
Streptococcus agalactiae 2 2
Haemophilus influenzae 3 1
Haemophilus parainfluenzae 1 4 10
Haemophilus haemolyticus 1
Haemophilus parahaemolyticus 1
Staphylococcus aureus 2 7 9
Staphylococcus lugdunensis 1
Coagulase-negative staphylococci 3 25 31
Eikenella corrodens 3 8 10
Aggregatibacter aphrophilus 1
Neisseria spp. 12 38 34
Escherichia coli 2 3 3
Proteus vulgaris 1
Corynebacterium spp. 13 39 38
Mycoplasma sp.1 1 1
Gemella sp. 1 1
Anaerobic bacteria
Fusobacterium necrophorum 8 8 7
Fusobacterium spp. 13 18 8
Prevotella spp. 8 5 5
Bacteroides spp. 22 23 34
Bifidobacterium longum 1
Eggerthia catenaformis 1
Capnocytophaga spp. 1 1 2
Actinomyces spp. 1 1 1
Veillonella spp. 8 7
Lactobacillus spp. 2 3
Parvimonas micra 1
Lachnoanaerobaculum orale 1
Alloscardovia omnicolens 1
Anaerobes (unspecified) 37 49 48
Fungi
Candida 2 4 14
No. isolates, mean (SD) 3.4 (1.5) 5.8 (1.6) 5.9 (1.6)
Polymicrobial 50 (94%) 52 (98%) 53 (100%)
1
M. hominis (n = 1)
2
B. fragilis (n = 1), B. pyogenes (n = 1)
3
B. fragilis (n = 2)
4
B. fragilis (n = 3)

Focusing on the absolute and relative abundance of the F. necrophorum stand out as the prevalent recoveries. To fur-
obtained microorganisms, unspecified anaerobes, ther pinpoint the likely pathogens and deduct the massive
Streptococcus anginosus group, Prevotella spp., and commensal flora of the pharynx, we (1) calculated the
1468 Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470

percentage of each bacterial strain, which was most common- anaerobes (Peptostreptococcus spp., Bacteroides (currently
ly recovered in both absolute and relative abundance and (2) named Prevotella) melaninogenicus, and unspecified) [8].
compared the findings in PPA pus with tonsillar tissues and Among three patients with PTA and PPA, Ohori et al. reported
surface swabs. These analyses suggested that the two ahd positive cultures for S. constellatus, alone and in
Streptococcus anginosus group, F. necrophorum, Prevotella combination with P. melaninogenica in each case. Thapar
spp., and S. pyogenes were the major pathogens. Fifty-three et al. found S. pyogenes and mixed anaerobes in blood cul-
percent (32/60) of patients had one or more of these four tures from a 24-year-old woman with PPA [17]. Lastly, in an
bacterial strains recovered in absolute or relative abundance earlier retrospective study from our own group of 61 PPA
from their PPA, and one or more of the bacteria were obtained patients, 28 patients had positive culture findings from pus
in 67% (40/60) of patients (regardless of abundance). The less aspirate or pus swabs, and the predominant bacterial species
frequent detection of Streptococcus anginosus group from were S. pyogenes, viridans group streptococci,
tonsillar swabs compared with PPA pus and tonsillar tissue F. necrophorum, and unspecified anaerobes [5]. Hence, a
might suggest that Streptococcus anginosus group is part of wide variety of bacterial species has been grown from PPA
the commensal flora of the tonsillar crypts and therefore not pus specimens, but the very limited previous evidence points
sampled adequately by tonsillar swaps. Alternatively, these in the same direction as the findings in the current study. It is
findings could support the pathogenic role of Streptococcus noteworthy that 35 of 79 (44%) PPA patients in these previous
anginosus group in the anaerobe micro-conditions of PPA. studies had concomitant PTA, which is in accordance with the
Unspecified anaerobes and non-hemolytic streptococci (other current study (43%), and that the recovered bacterial flora is
than the Streptococcus anginosus group) were also frequently comparable for those with or without concomitant PTA.
recovered, and further investigations are warranted to explore
the potential roles for subgroups of bacteria within these cat- Previous evidence to suggest pathogenic importance
egories. S. pyogenes was recovered significantly less frequent- of individual bacteria in throat infections
ly in patients who were treated with antibiotics prior to admis-
sion (0%) compared with untreated patients (21%), which The significance of S. pyogenes, F. necrophorum, Prevotella
may suggest that this pathogen is underestimated in our spp., and the Streptococcus anginosus group in PPA has only
findings. had limited attention in previous studies. However, it seems
reasonable to extrapolate from other throat infections to PPA.
Previous studies of PPA microbiology S. pyogenes is widely recognized as the major pathogen in
throat infections. In acute tonsillitis, it has been recovered
Itzhak Brook performed extensive cultures on PPA pus spec- more frequently among patients than healthy controls, the de-
imens from 14 children aged 1–6 years in the only previous velopment of specific antibodies has been documented, and
prospective microbiologic study of PPA patients [6]. A antibiotics reduce the duration of symptoms and the risk of
polymicrobial mixture of aerobes and anaerobes was found PTA and acute rheumatic fever [18–22]. In PTA, S. pyogenes
in 12 cases, and only anaerobes were detected in the latter has been consistently detected in pus aspirate studies, the de-
two cases. The predominant bacteria were Bacteroides spp. velopment of specific antibodies has been documented, and it
(n = 32), Peptostreptococcus spp. (n = 18), alpha- and has been recovered more frequently in tonsillar core tissue
gamma-hemolytic streptococci (n = 14), F. nucleatum (n = from PTA patients than controls [7, 23–25].
6), Staphylococcus aureus (n = 5), and Fusobacterium spp. F. necrophorum is a well-characterized pathogen known to
(n = 5). Retrospective, pediatric studies from the same period cause Lemierre’s syndrome [26]. The role for F. necrophorum
(late 80s) reported similar findings [14, 15]. It is well de- in acute tonsillitis is unclarified, but a number of studies report
scribed in other pharyngeal infections (i.e., acute tonsillitis more frequent detection among patients than healthy controls
and PTA) that the prevalent pathogens are closely associated [27]. Numerous findings suggest that it is a significant patho-
with patient age, and it is doubtful that these pediatric findings gen in PTA, given high recovery rates in PTA pus aspirates,
can be extrapolated to adults [7, 16]. more frequent isolation from PTA patients compared with
Only a few studies of adult PPA patients include informa- controls, significantly higher inflammatory markers in
tion on the microbiology, and no previous attempts to exhaus- F. necrophorum–positive patients compared with patients in-
tively define the bacteriology have been done [2, 5, 8, 9, 17]. fected with other bacteria, and the development of specific
Hence, previous studies were retrospective and described rou- antibodies [7, 25, 28]. In the current study, F. necrophorum
tine culture findings. Sethi et al. reported the recovery of four was found in 17% of PPA pus specimens, which is consider-
Klebsiella pneumoniae, one Pseudomonas aeruginosa, and ably less than the previous studies of PTA, where anaerobic
one S. aureus among nine patients [9]. Alaani et al. described cultures were performed (38–58%) [7, 29]. This less prevalent
five PPA cases with different combinations of streptococci role of F. necrophorum may be related to the fact that this
(beta-hemolytic, milleri group, and unspecified) and anaerobe has a high preponderance among patients aged 15–
Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470 1469

30 years, which coincides with the highest incidences of unspecified, anaerobes are also likely to play a role as are other
Lemierre’s syndrome, PTA, and acute tonsillitis but is consid- less prevalent bacteria. Based on our findings, we recommend
erably younger than the majority of PPA patients [26, 27, 30]. that the antibiotic regime for PPA patients include the cover-
In that regard, it is noteworthy that F. necrophorum also age of the described likely pathogens, e.g., benzylpenicillin (to
seems to play a role in PPA and that the mean age of cover streptococci and F. necrophorum) and metronidazole
F. necrophorum–positive patients was 48 years in the current (to cover Prevotella spp., F. necrophorum, and other anaer-
study and not significantly different from patients with other obes). We suggest utilization of a combination of highly fo-
bacterial findings. cused culture-based and non-culture-based methods to com-
The significance of Prevotella spp. in throat infections is prehensively unveil the microbial pathogenesis of PPA.
uncertain. In the 1990s, Brook and colleagues performed a
number of serological studies and reported an increase (at least
a doubling) in antibody levels to Prevotella intermedia in Authors’ contribution TEK: initiation and design of the study, inclusion
of patients; analysis and interpretation of the results; drafting and approv-
patients with PTA, peritonsillar cellulitis, infectious mononu- al of the manuscript; accountable for all aspects of the work. TG and CA:
cleosis, acute tonsillitis, and recurrent non-streptococcal ton- design and conduction of microbiological analyses; analysis and interpre-
sillitis [31–34]. tation of the results; critical revision; approval of the manuscript; account-
The Streptococcus anginosus group is known to cause a able for all aspects of the work. PH, MNP, ALC, and AK: design of the
study, inclusion of patients; critical revision; approval of the manuscript;
variety of different human infections, but the pathogenic im-
accountable for all aspects of the work. CD, NKP, SM, SP, SF, and HD:
portance in throat infections is previously undocumented [35]. inclusion of patients; critical revision; approval of the manuscript; ac-
countable for all aspects of the work. MR: analysis and interpretation of
Limitations the results; critical revision; approval of the manuscript; accountable for
all aspects of the work.

The current study has several limitations. The relatively lim-

Funding This work was supported by the Lundbeck Foundation (Grant
ited number of patients reflects the relative low incidence of number: R185-2014-2482), Fonden for lægevidenskabens fremme, and
PPA. Our findings are limited to adults, and the pathogens Ørelæge Hans Skovby’s og Hustru Emma Skouby’s Fond.
associated with PPA in children are likely different. Due to
the large number of specimens and to homogenize processing, Data Availability Anonymized data can be obtained from the correspond-
specimens were kept at −80 °C until cultures were made. ing author upon request.
Although studies on the effect of freezing specimens do not
Code availability Not applicable.
seem to alter the ability to isolate microorganisms, some bac-
teria may not have been detected, i.e., fastidious bacteria or
Declarations
bacteria, which had been killed before the collection of spec-
imens (most patients were treated with antibiotics). This study Ethics approval The study was approved by the Ethical Committee of
relies on classical bacteriology where a correct and clinically Aarhus County (# 1-10-72-4-16).
relevant species distinction depends on the skills of the clinical
laboratory technician. Initial identification of organisms is Consent to participate Informed consent was obtained from all patients.
grounded in subjective criteria supplied with biochemical re-
actions, and experience is established through the years [36]. Consent for publication Informed consent was obtained from all
patients.
To minimize this processing bias, the same very experienced
technician processed all samples in the current study. The use Conflict of interest The authors declare that they have no conflict of
of selective and differential media for initial processing has interest.
been useful for isolation, and MALDI-TOF MS can provide
Open Access This article is licensed under a Creative Commons
an accurate identification [11, 37]. An exhaustive species dis-
Attribution 4.0 International License, which permits use, sharing, adap-
tinction using culture-based methods is difficult [11, 37, 38], tation, distribution and reproduction in any medium or format, as long as
and non culture-based methods, such as whole genome se- you give appropriate credit to the original author(s) and the source, pro-
quencing and microbiome analysis, will provide more detailed vide a link to the Creative Commons licence, and indicate if changes were
information on bacteria in polymicrobial cultures. made. The images or other third party material in this article are included
in the article's Creative Commons licence, unless indicated otherwise in a
credit line to the material. If material is not included in the article's
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Our findings suggest that Streptococcus anginosus group,
F. necrophorum, Prevotella spp., and S. pyogenes are signif-
icant pathogens in the development of PPA. Additional, yet
1470 Eur J Clin Microbiol Infect Dis (2021) 40:1461–1470

References rheumatic fever by treatment of the preceding streptococcal infec-

tion with various amounts of depot penicillin. Am J Med 10:673–
695
1. Sichel JY, Attal P, Hocwald E, Eliashar R (2006) Redefining
21. Stjernquist-Desatnik A, Prellner K, Christensen P (1987) Clinical
parapharyngeal space infections. Ann Otol Rhinol Laryngol 115:
and laboratory findings in patients with acute tonsillitis. Acta
117–123
Otolaryngol 104:351–359
2. Ohori J, Iuchi H, Nagano H, Umakoshi M, Matsuzaki H, Kurono Y
22. Del Mar CB, Glasziou PP, Spinks AB (2006) Antibiotics for sore
(2019) The usefulness of abscess tonsillectomy followed by
throat. Cochrane Database Syst Rev (4):CD000023
intraoral drainage for parapharyngeal abscess concomitant with
23. Klug TE (2017) Peritonsillar abscess: clinical aspects of microbiol-
peritonsillar abscess in the elderly. Auris Nasus Larynx S0385-
ogy, risk factors, and the association with parapharyngeal abscess.
8146:30283–30284. https://doi.org/10.1016/j.anl.2019.06.003
Dan Med J 64(3):B5333
3. Page C, Chassery G, Boute P, Obongo R, Strunski V (2010)
24. Flodström A, Hallander HO (1976) Microbiological aspects on
Immediate tonsillectomy: indications for use as first-line surgical
peritonsillar abscesses. Scand J Infect Dis 8:157–160
management of peritonsillar abscess (quinsy) and parapharyngeal
abscess. J Laryngol Otol 124:1085–1090 25. Klug TE, Henriksen JJ, Rusan M, Fuursted K, Krogfeldt K, Ovesen
T, Struve C (2014) Antibody development to Fusobacterium
4. Monobe H, Suzuki S, Nakashima M, Tojima H, Kaga K (2007)
Peritonsillar abscess with parapharyngeal and retropharyngeal in- necrophorum in patients with peritonsillar abscess. Eur J Clin
volvement: incidence and intraoral approach. Acta Otolaryngol Microbiol Infect Dis 33:1733–1739. https://doi.org/10.1007/
Suppl 559:91–94 s10096-014-2130-y
5. Klug TE, Fischer AS, Antonsen C, Rusan M, Eskildsen H, Ovesen 26. Riordan T (2007) Human infection with Fusobacterium
T (2014) Parapharyngeal abscess is frequently associated with con- necrophorum (Necrobacillosis), with a focus on Lemierre's syn-
comitant peritonsillar abscess. Eur Arch Otorhinolaryngol 271: drome. Clin Microbiol Rev 20:622–659
1701–1707. https://doi.org/10.1007/s00405-013-2667-x 27. Klug TE, Rusan M, Fuursted K, Ovesen T, Jorgensen AW (2016) A
6. Brook I (1987) Microbiology of retropharyngeal abscesses in chil- systematic review of Fusobacterium necrophorum-positive acute
dren. Am J Dis Child 141:202–204 tonsillitis: prevalence, methods of detection, patient characteristics,
7. Klug TE, Henriksen JJ, Fuursted K, Ovesen T (2011) Significant and the usefulness of Centor Score. Eur J Clin Microbiol Infect Dis
pathogens in peritonsillar abscesses. Eur J Clin Microbiol Infect Dis 35:1903–1912
30:619–627 28. Ehlers Klug T, Rusan M, Fuursted K, Ovesen T (2009)
8. Alaani A, Griffiths H, Minhas SS, Olliff J, Lee AB (2005) Fusobacterium necrophorum: most prevalent pathogen in
Parapharyngeal abscess: diagnosis, complications and management peritonsillar abscess in Denmark. Clin Infect Dis 49:1467–1472
in adults. Eur Arch Otorhinolaryngol 262:345–350 29. Jousimies-Somer H, Savolainen S, Makitie A, Ylikoski J (1993)
9. Sethi DS, Stanley RE (1991) Parapharyngeal abscesses. J Laryngol Bacteriologic findings in peritonsillar abscesses in young adults.
Otol 105:1025–1030 Clin Infect Dis 16(Suppl 4):S292–S298
10. Bank S, Nielsen HM, Mathiasen BH, Leth DC, Kristensen LH, 30. Klug TE (2014) Incidence and microbiology of peritonsillar ab-
Prag J (2010) Fusobacterium necrophorum – detection and identi- scess: the influence of season, age, and gender. Eur J Clin
fication on selective agar. APMIS 118:994–999. https://doi.org/10. Microbiol Infect Dis 33:1163–1167. https://doi.org/10.1007/
1111/j.1600-0463.2010.02683.x s10096-014-2052-8
11. Versalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML, 31. Brook I, Foote PA Jr, Slots J, Jackson W (1993) Immune response
Warnock DW. Manual of clinical microbiology, 10th Edition. to Prevotella intermedia in patients with recurrent nonstreptococcal
https://doi.org/10.1128/9781555816728 tonsillitis. Ann Otol Rhinol Laryngol 102:113–116
12. Nagy E, Boyanova L, Justesen US (2018) How to isolate, identify, 32. Brook I, Foote PA Jr, Slots J (1996) Immune response to anaerobic
and determine antimicrobial susceptibility of anaerobic bacteria in bacteria in patients with peritonsillar cellulitis and abscess. Acta
routine laboratories. Clin Microbiol Infect 24:1139–1148. https:// Otolaryngol 116:888–891
doi.org/10.1016/j.cmi.2018.02.008 33. Brook I, Deleyva F (1996) Immune response to Fusobacterium
13. Ehrlich GD, Hiller NL, Hu FZ (2008) What makes pathogens path- nucleatum and Prevotella intermedia in patients with infectious
ogenic. Genome Biol 9:225 mononucleosis. J Med Microbiol 44:131–134
14. Dodds B, Maniglia AJ (1988) Peritonsillar and neck abscesses in 34. Brook I, Foote PA, Slots J (1997) Immune response to
the pediatric age group. Laryngoscope 98:956–959 Fusobacterium nucleatum, Prevotella intermedia and other anaer-
15. Asmar BI (1990) Bacteriology of retropharyngeal abscess in chil- obes in children with acute tonsillitis. J Antimicrob Chemother 39:
dren. Pediatr Infect Dis J 9:595–597 763–769
16. McIsaac WJ, White D, Tannenbaum D, Low DE (1988) A clinical 35. Fazili T, Riddell S, Kiska D, Endy T, Giurgea L, Sharngoe C,
score to reduce unnecessary antibiotic use in patients with sore Javaid W (2017) Streptococcus anginosus group bacterial infec-
throat. CMAJ 158:75–83 tions. Am J Med Sci 354:257–261. https://doi.org/10.1016/j.
17. Thapar A, Tassone P, Bhat N, Pfleiderer A (2008) Parapharyngeal amjms.2017.05.011
abscess: a life-threatening complication of quinsy. Clin Anat 21: 36. Baron EJ (2002) Speculation on the microbiology laboratory of the
23–26 future. Clin Infect Dis 35(Suppl 1):S84–S87
18. Christensen AM, Thomsen MK, Ovesen T, Klug TE (2014) Are 37. Citron DM, Appelbaum PC (1993) How far should a clinical labo-
procalcitonin or other infection markers useful in the detection of ratory go in identifying anaerobic isolates, and who should pay?
group A streptococcal acute tonsillitis? Scand J Infect Dis 46:376– Clin Infect Dis 16(Suppl 4):S435–S438. https://doi.org/10.1093/
383 clinids/16.supplement_4.s435
19. Dagnelie CF, van der Graaf Y, De Melker RA (1996) Do patients 38. Rosenblatt JE (1976) Isolation and identification of anaerobic bac-
with sore throat benefit from penicillin? A randomized double-blind teria. Hum Pathol 7:177–186. https://doi.org/10.1016/s0046-
placebo-controlled clinical trial with penicillin V in general prac- 8177(76)80021-4
tice. Br J Gen Pract 46:589–593
20. Wannamaker LW, Rammelkamp CH Jr, Denny FW, Brink WR, Publisher’s note Springer Nature remains neutral with regard to jurisdic-
Houser HB, Hahn EO, Dingle JH (1951) Prophylaxis of acute tional claims in published maps and institutional affiliations.