The Role of Anaerobes in Patients With

Ventilator-associated Pneumonia and Aspiration

Pneumonia*
A Prospective Study
Paul E. Marik, MD, FCCP; and Pamela Careau, MT (ASCP)

Context: Aspiration of oropharyngeal material, with its high concentration of anaerobic bacteria, has been
implicated in the pathogenesis of both ventilator-associated pneumonia (VAP) and aspiration pneumonitis
(AP). Consequently, patients with these disorders are usually treated with antimicrobial agents with
anaerobic activity.
Objective: To determine the incidence of anaerobic bacteria in patients with VAP and AP.
Design: Prospective, nonrandomized, interventional study.
Setting: University-affiliated community teaching hospital.
Patients and interventions: We performed sequential blind protected specimen brush (PSB) sampling and
mini-BAL in 143 patients with 185 episodes of suspected VAP and 25 patients with AP who required
mechanical ventilation. Quantitative aerobic and anaerobic cultures were performed on all specimens.
Pneumonia was considered to be present when either > 500 cfu/mL cultured from blind PSB sampling or
> 5,000 cfu/mL cultured from mini-BAL were present.
Results: Using the predefined criteria, bacterial pneumonia was diagnosed in 63 of 185 suspected VAP
episodes (34%) and 12 of 25 patients with AP (48%). At least one dose of an antibiotic was given in the 24 h
prior to bacteriologic sampling in 106 suspected VAP episodes (57%) and in 12 patients with AP (48%).
More than one pathogen was isolated from 11 VAP and four AP patients. Pseudomonas aeruginosa,
Staphylococcus aureus, and enteric Gram-negative organisms were isolated most frequently from patients
with VAP. In the patients with AP, enteric Gram-negative organisms were isolated in patients with GI
disorders and Streptococcus pneumoniae and Haemophilus influenzae predominated in patients with
“community-acquired” aspiration. Only one anaerobic organism was isolated from the entire group of
patients; Veillonella paravula was isolated from a blind PSB specimen in a patient with suspected
aspiration pneumonia.
Conclusion: Despite painstaking effort, we were able to isolate only one anaerobic organism (nonpatho-
genic) from this group of patients. The spectrum of aerobes in patients with VAP was similar to that
reported in the literature. The organisms found in patients with AP was a reflection of the organisms likely
to colonize the oropharynx. The use of antibiotics with anaerobic coverage may not be necessary in
patients with suspected VAP and AP. Furthermore, penicillin G and clindamycin may not be the antibiotics
of choice in patients with AP. (CHEST 1999; 115:178–183)

Key words: anaerobes; aspiration pneumonitis; diagnosis; mechanical ventilation; protected specimen brush sampling; ventilator-
associated pneumonia

Abbreviations: AP 5 aspiration pneumonitis; PSB 5 protected specimen brush; VAP 5 ventilator-associated pneumonia

V entilator-associated pneumonia (VAP) and aspi-

ration pneumonitis (AP) represent a spectrum of
encountered in the ICU, with VAP occurring in
approximately 25% of patients undergoing mechan-
aspiration “syndromes.” Both are common problems ical ventilation,1–5 and AP commonly occurring in

*From the Medical and Surgical Intensive Care Unit (Dr. Marik)
and CliniTech Services (Ms. Careau), St. Vincent Hospital, Worces-
ter, MA.
Supported by the St. Vincent Hospital Critical Care Research
Fund. The authors have no financial involvement with any of the
products mentioned.
Manuscript received April 16, 1998; revision accepted July 21,
1998.
Correspondence to: Paul E. Marik, MD, FCCP, Department of
Critical Care, St. Vincent Hospital, 25 Winthrop St, Worcester,
MA 01604; e-mail: pmarik@ultranet.com
For editorial comment see page 8
patients admitted to the ICU with an altered level of
consciousness.6 AP follows macroaspiration of oro-
pharyngeal and/or gastric contents in patients with
an altered level of consciousness, dysphagia, or
bowel obstruction.6 – 8 VAP is widely believed to
result from the microaspiration of oropharyngeal

178 Clinical Investigations in Critical Care

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 material colonized by pathogenic microorgan-
isms.9 –13 Although anaerobic bacteria are frequently
isolated from the oropharyngeal flora,14 their patho-
genetic role in VAP and AP is unclear. Many studies
have failed to isolate anaerobic bacteria in patients
with VAP.15–18 The microbiology of aspiration pneu-
monitis is based on studies performed in the 1970s
using transtracheal aspiration.19 –22 These studies
suggested that anaerobic bacteria were the major
pulmonary pathogens. Based on these studies, most
patients with AP are currently treated with either
penicillin G or clindamycin.7,8,23–25 However, a re-
cently published study questions the validity of this
treatment approach.6
Isolation of anaerobic bacteria requires adequate
transport conditions and specific growth media.
Many of the recent studies investigating the micro-
biology of VAP have not specifically taken these
measures.15–18 Transtracheal aspiration may result in
a large number of false-positive results.26 Further-
more, transtracheal aspiration cannot be performed
in intubated patients. Protected specimen brush
(PSB) sampling has been recommended as the
method of choice in isolating anaerobes in ventilated
patients.27 The aim of this study was to determine,
using strictly controlled culture conditions, the
pathogenetic role of anaerobic bacteria in patients
with suspected VAP and in patients with AP who
required endotracheal intubation.
Materials and Methods
This study was conducted between June 1996 and August 1997
in the Medical and Surgical ICUs at St. Vincent Hospital,
Worcester, MA. This study was approved by our Institutional
Review Board. As both blind PSB sampling and mini-BAL are
considered to be within the standard of care in our ICUs, the
Institutional Review Board waived the need for informed con-
sent. Patients were eligible for inclusion in this study if they had
been mechanically ventilated for more than 48 h and were
suspected to have VAP based on clinical grounds. The clinical
suspicion of VAP was based on recent and persistent pulmonary
infiltrates on the chest radiograph and at least two of the
following three clinical criteria: fever . 38.5°C, leukocytosis
(. 10 3 109/L), and purulent tracheal secretions.5,28,29 In addi-
tion, patients who required endotracheal intubation and mechan-
ical ventilation as a consequence of AP were included in the
study. The criteria for aspiration were those defined by Lorber
and Swenson20 and Bartlett et al22: namely, the presence of
alveolar infiltrates on the chest radiograph and either witnessed
aspiration or risk factors for aspiration (ie, altered level of
consciousness, dysphagia, or intestinal obstruction).
All patients underwent sequential blind PSB sampling and
mini-BAL as previously reported.30,31 Briefly, patients were
preoxygenated at 80 to 100% oxygen throughout the procedure
and monitored by continuous pulse oximetry. Midazolam was
used for sedation as needed. No local anesthesia was used. Blind
PSB sampling was performed first. Utilizing a standard microbi-
ology specimen brush (Microvasive No 1650; Boston Scientific
Corp; Watertown, MA), the catheter was inserted through the
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endotracheal tube and advanced “blindly” to approximately 35
cm or until resistance was met. A specimen was obtained by
expressing and retracting the inner catheter and brush in the
standard fashion. The brush was then placed in 1 mL of
thioglycolate broth (Remel; Lenexa, KS).
Subsequently, a mini-BAL specimen was obtained using a BAL
catheter (No. 140; Ballard Medical Products; Draper, UT). The
BAL catheter was inserted through the adapter into the endo-
tracheal tube. With the catheter just protruding into the endo-
tracheal tube, wide-bore suction tubing was connected to the end
of the catheter. The catheter stopcock was then opened to
suction, the pressure adjusted to 40 to 60 mm Hg, and the
stopcock then closed. The catheter was then advanced into the
mainstem bronchus of the lung suspected to have pneumonia by
orienting the directional tip. The tip of the catheter was flushed
with 2 mL of sterile physiologic saline solution. The inner
catheter was then advanced until resistance confirmed a wedge
position. A Luki sputum trap (Davis & Geck; Wayne, NJ) was
placed in line with the catheter and wide-bore suction tubing.
With the inner catheter in a wedged position, 20 mL of sterile
physiologic saline solution was instilled into the lung and allowed
to dwell for approximately 20 s. The stopcock was then turned
into the open position, allowing aspiration of lavage fluid. If no
return was obtained, another 20-mL aliquot was injected and
suctioned. This procedure was repeated until at least 2 to 3 mL
of return was obtained. No attempt was made to radiographically
determine in which lobe of the lung the blind PSB/mini-BAL
sampling was performed.
Microbiologic Processing
The respiratory specimens were transported immediately by
hand, and processed by our microbiology laboratory within 20
min of sampling. Quantitative culture was performed on the blind
PSB and mini-BAL specimens using the calibrated loop meth-
od.27 After gently vortexing the samples, each specimen was
inoculated onto two sets of media using first a 0.01-mL and then
a 0.1-mL calibrated loop. Final organism dilutions were 1:100
and 1:10, respectively. Routine sets of culture media inoculated
included 5% sheep blood agar plates, chocolate agar plates,
MacConkey agar plates and CDC anaerobic blood agar plates
(Remel). Blood and MacConkey agar plates were incubated at
35°C in a 5% CO2 incubator for a total of 48 h.
Specimens submitted for anaerobic culture were inoculated on
prereduced commercial media, which are maintained prior to
inoculation in jars flushed with nitrogen gas (anaerobic holding
jars). After inoculation, agar media was immediately placed into
a self-contained jar with a commercial gas-generating system and
an anaerobic indicator strip (BBL anaerobic GasPak Plus; Becton
Dickinson Microbiology Systems; Sparks, MD). The indicator is
monitored throughout the culture process. Anaerobic cultures
are incubated in monitored 35 to 37°C incubators for 48 h prior
to first examination. Negative cultures are incubated for an
additional 3 days. For quality control, various anaerobic organ-
isms are periodically subcultured in the same manner. Recovery
of these organisms validates the ability of the system to recover a
variety of anaerobic species.
All organisms recovered from cultures were quantitated. Those
bacterial isolates recognized as potential respiratory pathogens
and recovered at the 10 cfu/mL threshold were identified as
completely as possible using either the Vitek System (BioMerieux
Vitek; Hazelwood, MO) or standard manual laboratory proce-
dures. Anaerobic organisms were identified using the Innovative
Diagnostic Systems (Remel). Antibiotic susceptibility testing was
performed using either the Vitek System or by the Kirby-Bauer
disk diffusion method for aerobic and microaerophilic organisms.
No antibiotic testing was performed on anaerobic organisms.
CHEST / 115 / 1 / JANUARY, 1999 179
 Data Collection and Analysis Table 1—Clinical Features of Patients with Suspected
VAP and AP
In patients who were investigated for repeat episodes of
suspected VAP, each episode was recorded separately. The VAP AP
patients’ basic demographic data, major admitting diagnoses, (n 5 143) (n 5 25)
acute physiology and chronic health evaluation II score, WBC
count, radiographic changes, and concurrent antibiotics were Age, yr 67 6 15 58 6 21
recorded in a computerized database (Access 97; Microsoft; Male:female ratio 1.8:1 1:1
Redmond, WA). For the purposes of this study, patients with APACHE II score* 16 6 5 12 6 3
either . 500 cfu/mL cultured from blind PSB sampling or WBC count, 3 109/L† 14.8 6 3.4 13.2 6 2.7
. 5,000 cfu/mL cultured from mini-BAL were considered to Lobes involved†
have pneumonia.5 The patients’ antibiotic regimens were tailored Right lower lobe 119 21
according to the sensitivities of the pathogen(s) isolated. In Right middle lobe 3 —
patients with negative cultures, the decision to continue or stop Left lower lobe 22 1
the antibiotics was made by the patient’s attending physician. Bilateral 41 3
At the end of the data collection, summary statistics were Primary diagnoses
compiled to allow a description of the study population. x2 COPD/pneumonia 38 1
analysis was used to compare categorical data. Unless otherwise Post-bowel surgery 21 —
stated, all data are expressed as mean 6 SD, with statistical Congestive heart failure 23 1
significance declared for p values of 0.05 or less. Sepsis syndrome/septic shock 15 —
Cerebrovascular accident 12 2
Post-peripheral vascular surgery 11 —
Post-thoracic surgery 6 —
Results Head injury 6 1
Drug overdose 2 6
During the study period, 185 paired specimens Delirium tremens 1 5
from 143 patients with suspected VAP and 25 paired Seizures 1 4
specimens from 25 patients with AP were obtained. Small bowel obstruction 3 3
One patient with bacteriologically proven AP subse- Other 4 2
quently underwent blind PSB sampling /mini-BAL *APACHE 5 acute physiology and chronic health evaluation.
for suspected VAP (the cultures were negative). The †n 5 185 for VAP, ie, 185 episodes of suspected VAP in 143 patients.
clinical characteristics of both groups of patients are
listed in Table 1. Blind PSB sampling and mini-BAL
were performed within 24 h of the suspected episode of an antibiotic in the previous 24 h and seven
of aspiration in the patients with AP. Blind PSB patients who had not (42 vs 54%, respectively; not
sampling and mini-BAL were performed 5 6 4 days significant).
after endotracheal intubation in the patients with More than one pathogen was isolated from 11 VAP
suspected VAP. and four AP patients. The bacterial isolates from
Using the predefined quantitative culture thresh- both groups of patients are listed in Table 2. Pseudo-
olds, bacterial pneumonia was confirmed in 63 of the monas aeruginosa, Staphylococcus aureus, and en-
185 suspected VAP episodes (34%) and in 12 of the teric Gram-negative organisms were isolated most
25 patients with AP (48%). One patient had two frequently from the patients with VAP. In the pa-
distinct episodes of VAP, each with different organ- tients with AP, enteric Gram-negative organisms
isms. At least one dose of an antibiotic had been were isolated in patients with GI disorders, and
given in the 24 h prior to bacteriologic sampling in Streptococcus pneumoniae and Haemophilus influ-
106 suspected VAP episodes (57%) and in 12 AP enzae predominated in patients with “community-
patients (48%). The 106 patients with suspected VAP acquired” aspiration. The isolates in patients with AP
received the following antibiotics (with number in grouped according to their primary disease are listed
parentheses) prior to bacteriologic sampling: ceftazi- in Table 3. Only one anaerobic organism was isolated
dime (n 5 67), aztreonam (n 5 16), ceftriaxone from the entire group of patients studied; Veillonella
(n 5 15), gentamicin (n 5 8), vancomycin (n 5 16), paravula was isolated from a blind PSB specimen in
and clindamycin (n 5 63). The 12 patients with a patient with suspected AP.
suspected AP all received clindamycin in the 24 h
prior to blind PSB sampling /mini-BAL. In the group
of patients with suspected VAP, pneumonia was Discussion
diagnosed in 32 instances in which an antibiotic had
been given in the previous 24 h, compared with 31 Anaerobic bacteria are considered to be common
instances in which no antibiotic had been given (30 pulmonary pathogens, and they are believed to play
vs 39%, respectively; not significant). Similarly, in a major role in aspiration and nosocomial pneumo-
the group of patients with AP, a bacterial pathogen nia.8,23,25,32 These beliefs are based on a handful of
was isolated in five patients who had received a dose studies conducted in the 1970s, when transtracheal

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 Table 2—Isolates Recovered From Patients With VAP gested that this low yield may be related to the fact
and AP that anaerobes are unlikely to survive in these solu-
VAP Isolates AP Isolates tions.27 Doré et al33 recently reported the results of
(n 5 74) (n 5 17) a study in which they performed bronchoscopically
P aeruginosa 16 — directed PSB sampling in patients with suspected
MSSS 15 2 VAP. Anaerobic transport broth and anaerobic cul-
Enterobacter spp 8 3 ture media were used to promote the recovery of
S pneumoniae 5 2
anaerobic bacteria. These authors recovered anaer-
MRSA 4 —
Streptococcus agalactiae 4 — obic bacteria in 23% of patients with VAP. The major
H influenzae 3 2 anaerobic strains that they isolated were Prevotella
Stenotrophomonas maltophilia 3 — melaninogenica, Fusobacterium nucleatum, and V
Acinetobacter baumannii 3 —
parvula. While Prevotella and Fusobacterium spp
Klebsiella pneumoniae 3 2
Escherichia coli 1 2 are regarded as pulmonary pathogens, Veillonella
Flavobacterium spp — 2 spp are considered to be nonpathogenic.34,35 The
Serratia sp 1 1 disparate finding between this study and our present
V paravula† — 1 study is unclear. On the day of the PSB sampling,
Other 8 —
47% of the patients in the study by Doré et al were
*MSSS 5 methicillin-sensitive S aureus; MRSA 5 methicillin-resis- receiving antimicrobial therapy, but none were re-
tant S aureus. ported to be receiving clindamycin. It is noteworthy
†Nonpathogenic anaerobe.
that 35% of the patients in whom these authors
isolated an anaerobe were receiving antibiotics at the
time of microbiologic sampling. The only apparent
aspiration was used for the collection of “uncontam- difference in study design is that the PSB sampling
inated” respiratory secretions 19 –22,32 Consequently, in our study was performed “blind” (nondirected),
antimicrobial agents with anaerobic coverage have while in the study by Doré et al, sampling was
been recommended in patients with aspiration pneu- performed through a bronchoscope. The dependent
monia and nosocomial pneumonia.7,8,23–25 The re- segments of the right lower lobe are predominantly
sults of our study question this common clinical involved in aspiration syndromes, and the blind
practice. Despite painstaking effort, we recovered technique will likely sample these segments.6,24,36
only a single nonpathogenic anaerobe from a diverse We and others have previously demonstrated an
population of hospitalized ICU patients with sus- excellent concordance between the two techniques
pected pneumonia. and this factor seems unlikely to account for the
A number of studies using bronchoscopically di- different findings.15,30,37
rected PSB sampling and anaerobic culture media Mier et al6 performed bronchoscopy and PSB
have failed to isolate anaerobic bacteria in patients sampling in a group of intubated ICU patients with
suspected to have VAP or have reported an inci- aspiration pneumonia. As in our study, anaerobic
dence of less than 2%.15–18 However, in all these transport media and strict anaerobic culture tech-
studies, physiologic saline solution or Ringer’s lactate niques were employed. These investigators failed to
was used as the transport media. It has been sug- isolate a single anaerobe; the spectrum of aerobic

Table 3—Isolates in Patients With AP According to Primary Disease

No. of Patients* CAP‡ Bacterial Isolates (n 5 17)

Drug overdose 6 (1) 6 S aureus, V paravula†

Seizures 4 (2) 4 Enterobacter spp, H influenzae
Delirium tremens 5 (3) 4 S pneumoniae (2), H influenzae, S aureus, K pneumoniae
Small bowel obstruction 3 (3) 1 E coli (2), Enterobacter spp, Flavobacterium spp
Cerebrovascular accident 2 —
COPD/pneumonia 1 1
Congestive heart failure 1 1
Head injury 1 (1) — Serratia spp
Liver failure 1 (1) — K pneumoniae
Ileus post-bowel surgery 1 (1) — Flavobacterium spp, Enterobacter spp
*Number with bacterial pneumonia in parentheses.
†Nonpathogenic anaerobe.
‡CAP 5 community-acquired pneumonia. Aspiration pneumonitis suspected within 72 h of admission to hospital.

CHEST / 115 / 1 / JANUARY, 1999 181

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 organisms recovered was remarkably similar to that
recovered in our study. The results of our study
differ markedly from the findings of Bartlett and
others, whose studies in the 1970s form the basis for
current therapy.7,19 –25 These studies used transtra-
cheal aspirates to obtain “uncontaminated” pulmo-
nary secretions. It is possible that the organisms
recovered by transtracheal aspiration represent oro-
pharyngeal flora that contaminated the trachea dur-
ing the procedure (due to aspiration) or bacteria that
have colonized the trachea, rather than representing
true pulmonary pathogens. With PSB sampling,
however, uncontaminated lower respiratory tract
secretions are obtained.28,38 This postulate is sup-
ported by a study by Moser et al,26 who demon-
strated discrepancies between bacteria recovery by
transtracheal aspiration and by transthoracic needle
aspiration in dogs with experimental pneumonia.
Less than 50% of patients with either suspected
VAP or AP in our study had bacteriologically proven
infection. This observation has been well docu-
mented in previous studies.6,18,28,30,39 Furthermore,
these studies have demonstrated that it is not possi-
ble, on clinical grounds alone, to determine which
patients have bacterial pneumonia. In our study, the
right lower lobe was the most commonly involved
site. This is not surprising, as the dependent areas of
the right lower lobe are the most frequently involved
bronchopulmonary segments in patients with aspira-
tion syndromes.6,24,36
In order to increase the diagnostic sensitivity of
blind PSB sampling and mini-BAL, we used a lower
quantitative threshold than has traditionally been
used.15–18 This is in keeping with observations of a
number of recent studies and is unlikely to have
changed the findings of our study. Timsit and col-
leagues5 have demonstrated that the classic PSB
threshold of 1,000 cfu/mL has a sensitivity of only
67%. When these investigators lowered the thresh-
old to 500 cfu/mL, the sensitivity increased to 81%
with only a moderate decrease in specificity. Simi-
larly, Dreyfuss et al40 followed 29 patients with
suspected VAP who had a PSB culture between 10
and 1,000 cfu/mL. Repeat PSB sampling in 12 of
these patients isolated the same organism that was
found in the first PSB specimen in a concentration of
. 1,000 cfu/mL, and these patients were subse-
quently treated for pneumonia.
The results of our study suggest that while anaer-
obes are quantitatively important oropharyngeal
commensals, these organisms may be unimportant
pulmonary pathogens in patients with VAP and
aspiration pneumonia. The bacteriology of these
syndromes reflects the aerobic organisms that are
likely to colonize the patients at the time of aspira-
tion. These results imply that antimicrobial agents
182
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with anaerobic coverage may no longer be required
in the treatment of these patients. Furthermore,
penicillin G and clindamycin, the traditional antimi-
crobials of choice in patients with AP, provide
inadequate antimicrobial coverage. A major limita-
tion of our study is that 35% of our patients had
received an antibiotic with anaerobic activity in the
24 h prior to microbiologic sampling. Furthermore,
it is possible that anaerobic bacteria were missed
during the sampling procedure or not identified by
culture grown out despite our rigorous efforts. We
hope the findings of our study will spur clinical trials
examining the specific utility of adding anaerobic
drugs to the standard antibacterial regimens used in
patients with suspected VAP or aspiration pneumonia.
ACKNOWLEDGMENT: The authors acknowledge with grati-
tude the excellent work done by the Respiratory Therapy De-
partment at St. Vincent Hospital and the CliniTech microbiology
technicians.
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