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Journal of Clinical Microbiology, November 1998, p. 3417-3419, Vol. 36, No. 11
Center for Genomic
Sciences1 and
Department of
Otolaryngology,4 Allegheny University of the
Health Sciences, Pittsburgh, Pennsylvania 15212, and
Department
of Pathology,
Received 11 May 1998/Returned for modification 9 July 1998/Accepted 4 August 1998
The presence of endotoxin (detected by the Limulus
amebocyte lysate assay) was compared to the presence of viable
Haemophilus influenzae and Moraxella
catarrhalis (detected by PCR) in 106 middle-ear effusions from
pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested
positive for one or both of the gram-negative bacteria H. influenzae and M. catarrhalis. The data suggest
that viable gram-negative bacteria, detectable by PCR but often
undetectable by culture, may be the source of endotoxin in middle-ear
effusions.
Otitis media is the most common
reason for an ill child to visit a health-care provider and receive
antimicrobial treatments or surgery (16, 17). Chronic otitis
media with effusion (OME), the persistence of fluid in the middle ear
with minimal constitutional symptoms, can lead to significant hearing
loss and delayed speech development in pediatric patients (2,
8). Common reasons for the onset of OME include dysfunction of
the eustachian tube, immaturity of the immune system, allergic
response, and infection.
Streptococcus pneumoniae, Haemophilus
influenzae, and Moraxella catarrhalis are the
most common bacteria cultured from middle-ear-effusion aspirates;
however, the majority of effusions (40 to 60%) are negative by culture
for any bacterium (1, 5, 7). Recent application of PCR has
allowed for the simultaneous and specific detection of DNA from
S. pneumoniae, H. influenzae, and
M. catarrhalis in culture-sterile specimens from
pediatric patients with chronic OME (13). Additional studies
demonstrated that in the chinchilla middle ear, purified DNA and DNA
from pasteurized bacteria are rapidly degraded, as demonstrated by the
fact that they are not detectable by PCR (14). Conversely,
inoculated live bacteria persist for weeks, and their DNA is detectable
by PCR during that time, though after treatment with antibiotics the
effusions in which this DNA is detected are sterile as determined by
culture. These experiments showed that any bacterial DNA detected by
PCR in middle-ear effusions comes from viable bacteria. Furthermore, the bacteria within culture-sterile pediatric middle-ear effusions have
been shown to be metabolically active by means of reverse transcriptase
PCR-based assays (15). Viable, metabolically active H. influenzae, the most common bacterium isolated from
the effusions of patients with OME, was present in approximately 30%
of culture-sterile middle-ear effusions and 100% of culture-positive
middle-ear effusions (15).
Endotoxin, a lipooligosaccharide complex on the outer surfaces of most
gram-negative bacteria, is thought to be largely responsible for the
inflammatory response and the accumulation of fluid in the middle ear
in OME patients (4). However, it has been unclear whether
the effusion-inducing endotoxin can persist in the middle ear only as a
component of viable bacteria or whether it can persist as a component
of nonviable bacteria or remain free in suspension in the absence of
demonstrable intact bacteria. In a previous study, endotoxin was
present in 80% of all middle-ear effusions, including 67% of
effusions that were negative as determined by culture for any bacterium
(5). This result indicated that culturable bacteria were not
present in the majority of endotoxin-positive specimens. However, the
culture method used to detect bacteria in that earlier study is less
sensitive than PCR and would not have identified viable, nondividing
bacteria associated with a biofilm. We previously demonstrated that PCR
detection of bacteria correlates nearly 100% with the presence of
metabolically active pathogens (15). Viable, nondividing
gram-negative bacteria may harbor endotoxin, but because they are not
detectable by culture they would have been overlooked as a source of
lipooligosaccharide.
In our study, we attempted to correlate the detection of endotoxin,
using the Limulus amebocyte lysate (LAL) assay, with the presence of viable H. influenzae and M. catarrhalis, as detected by PCR. We hypothesize that the greater
sensitivity of PCR over culture will more definitely establish the
origins of bacterial endotoxin in chronic OME.
Patient population.
A total of 106 middle-ear-effusion
specimens were collected from pediatric outpatients at Children's
Hospital of Pittsburgh during myringotomy and tube placement for
chronic OME. Patients ranged in age from 3 months to 13 years.
Acquisition of clinical specimens.
The external ear canal was
disinfected and desquamated by placing 70% isopropyl alcohol in the
ear canal for 1 min. Immediately following myringotomy, the effusion
was suctioned from the middle-ear cleft with a sterile 14-gauge Baxter
Quick-Cath cover attached to a Senturia trap. In order to clear the
catheter of viscous material, prior to the procedure 50 µl of sterile
saline was added to each autoclaved collection tube and used to dilute
the effusion aspirate. Each sample was snap-frozen on dry ice and
transported to the Center for Genomic Sciences (Pittsburgh, Pa.), where
it was stored at Preparation of clinical specimens for PCR.
The sensitivity and
specificity of the PCR assay for H. influenzae and
M. catarrhalis were established as previously described (13). After the effusions were thawed, 50 µl was aliquoted
for the endotoxin assay, and the remainder was used for the DNA
extraction. The Trizol LS reagent (Life Technologies, Gaithersburg,
Md.) protocol for RNA and DNA extraction was used according to the
manufacturer's recommendations, with one modification. The nucleic
acid specimens used for multiplex PCR were not treated with DNase.
PCR.
Multiplex PCR and amplification analyses were
performed for H. influenzae with primer set
HI-IV/V/VIB and for M. catarrhalis with primer
set MCAT51/52/53 as described previously (13).
LAL endotoxin assay.
The presence of endotoxin was determined
with the BioWhittaker (Walkersville, Md.) QCL-1000 third-generation
chromogenic LAL assay. The assay was performed as previously described,
with several modifications (11). Endotoxin concentrations
were determined with a Perkin-Elmer spectrophotometer; the absorbance
of each specimen was measured at 405 nm and was used to calculate the endotoxin concentration. Escherichia coli standardization
dilutions ranged from 0.1 to 1.0 endotoxin units (EU) per ml (9 EU = 1 ng). A calculated value of 0.1 EU/ml was considered the threshold
for endotoxin positivity in the specimens. Arithmetically positive values below this threshold were recorded, but their significance is
unknown. Arithmetically negative values calculated for endotoxin were
assumed to be 0.0 EU/ml. All effusion samples were tested in groups of
20 (except for the last group of 6). To insure against endotoxin
contamination, each group of 20 samples included a sterile-water sample
as a negative control. The assays were performed independently: PCR was
done without knowledge of the LAL assay results and vice versa.
Endotoxin concentrations.
Endotoxin concentrations were
determined quantitatively by using the LAL assay E. coli
standard dilutions, and a correlative analysis was performed on 106 middle-ear effusions from patients with chronic OME to compare
endotoxin concentrations obtained in the presence of H. influenzae and M. catarrhalis (as detected by
PCR).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Correlation between Presence of Viable Bacteria and
Presence of Endotoxin in Middle-Ear Effusions
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-lactamase-producing organisms. Treatment
was not influenced by the results of this study.
80°C before molecular analyses.
TABLE 1.
Endotoxin concentrations in specimens of pediatric
middle-ear effusions
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Discussion. The percentage of endotoxin-positive middle-ear-effusion specimens (76.4%) is comparable to the result (80%) reported by DeMaria et al. and Willet et al. (5, 18). The PCR technique for the detection of H. influenzae and M. catarrhalis, however, identified bacteria in 81.5% of endotoxin-positive effusions, while in the previous study (5) culture had detected bacteria in only 33% of the effusions. The greater sensitivity of PCR (247%) over culture in these studies closely reflects the results seen by Post et al. (268%) (12) and Rayner et al. (264%) (15), who directly compared PCR and culture data for all middle-ear effusions.
Bacterial endotoxin has been attributed to the release of inflammatory mediators by macrophages (10). Earlier work showed that residual endotoxin from pasteurized bacteria could induce an effusion in the bullae of the chinchilla (4). Additionally, it has been shown that the majority of endotoxin-positive effusions from patients with OME (67%) are sterile as determined by culture for any bacteria (5). These studies led to the postulate that residual endotoxin might cause OME to persist in the absence of viable bacteria (5). The PCR-based assays for the detection of bacteria used in this study show that viable bacteria exist in a larger percentage of endotoxin-positive effusions (81.5%) than had been seen with the culture method of detection (33%). Thus, while the presence of endotoxin is most likely important for the persistence of OME (5), our results suggest that viable, nonculturable gram-negative bacteria produce the endotoxin. The correlation of average endotoxin concentration and PCR detection of bacteria indicates that there is an increase in endotoxin concentration when more than one species of bacteria exist in an effusion. In the samples where only one of the two bacteria tested for by PCR was detected, the average concentration of endotoxin was approximately 0.9 EU/ml. When both bacteria were detected, endotoxin increased approximately 75% to 1.6 EU/ml. However, the endotoxin concentrations of many of the samples that tested positive for both bacteria had absorbance readings that exceeded the linear range of the spectrophotometer. Therefore, the measured increase in average concentration is probably an underrepresentation of the actual increase. Further studies will be needed to determine the exact endotoxin concentrations of PCR-positive specimens. The correlation between bacterial detection by PCR and endotoxin detection by LAL assay was 70.8% (75 of 106 specimens). There were 31 discordant specimens, 16 of which were negative for endotoxin (below the LAL assay detection threshold) and positive for bacteria (as determined by PCR). These discordant specimens might indicate a limitation of the LAL assay. Levin et al. demonstrated that some proteins can act as inhibitors of the LAL assay, thereby reducing the endotoxin concentration measurement (9). The remaining 15 discordant specimens were positive for endotoxin and negative for bacteria by PCR. These samples might be indicative of endotoxin embedded within nonviable bacteria, residual endotoxin, nonspecificity of the LAL assay (6), or, more likely, the existence of some additional gram-negative bacteria not tested for by PCR in this study. The PCR and endotoxin data in this study support the hypothesis that chronic OME is a biofilm disease. A biofilm is a community of bacteria that have adhered to a surface or to each other (3). The attachment of bacteria to a surface triggers the expression of a cassette of genes, which results in the formation of a biofilm. The "biofilm phenotype" confers reduced metabolic activity and enhanced antibiotic resistance in comparison with the planktonic phenotype. The existence of a biofilm in the middle ear in patients with chronic OME would explain why bacteria are detectable by PCR and not by culture (15), since biofilm bacteria have been demonstrated to be highly resistant to growth in standard planktonic culture, probably because of differences in gene expression (3). It can also be postulated that, following the death of bacteria within a biofilm, detection by PCR would cease and endotoxin would persist embedded within the biofilm matrix until that matrix degraded.| |
ACKNOWLEDGMENTS |
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We thank T. F. DeMaria of the Department of Otolaryngology, The Ohio State University, for critical review of the manuscript. We acknowledge S. Dixit of the Center for Genomic Sciences for the collection of effusion specimens.
This work was supported by NIDCD grant DC02148 (G.D.E.), NIDCD grant DC02398 (J.C.P.), and the Center for Genomic Sciences at Allegheny University of the Health Sciences. A portion of this study was performed at the University of Pittsburgh School of Medicine Center for Genomic Sciences.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Genomic Sciences, Allegheny University of the Health Sciences, Allegheny Campus, Rm. 735, 4 Allegheny Center, Pittsburgh, PA 15212. Phone: (412) 330-4678. Fax: (412) 330-4630. E-mail: gehrlich{at}pgh.auhs.edu.
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Journal of Clinical Microbiology, November 1998, p. 3417-3419, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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