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Journal of Clinical Microbiology, November 2000, p. 3932-3936, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Discrimination between Clinically Relevant and Nonrelevant
Acanthamoeba Strains Isolated from Contact Lens- Wearing
Keratitis Patients in Austria
J.
Walochnik,1
E.-M.
Haller-Schober,2
H.
Kölli,2
O.
Picher,1
A.
Obwaller,1 and
H.
Aspöck1,*
Department for Medical Parasitology, Clinical
Institute of Hygiene, University of Vienna,1
and Department of Ophthalmology, Karl-Franzens-University,
Graz,2 Austria
Received 24 April 2000/Accepted 18 August 2000
 |
ABSTRACT |
Eighteen cases of Acanthamoeba-associated
keratitis among contact lens wearers seen at the Department of
Ophthalmology, Karl-Franzens-University, Graz, Austria, between 1996 and 1999 are reviewed. The amoebae were proven to be the causative
agents in three patients. The aim of our study was to discriminate
between clinically relevant and nonrelevant isolates and to assess the
relatedness of the isolates to published strains. Altogether, 20 strains of free-living amoebae, including 15 Acanthamoeba strains, 3 Vahlkampfia
strains, and 2 Hartmannella strains, were isolated from
clinical specimens. The virulent Acanthamoeba
strains were identified as A. polyphaga and two strains of
A. hatchetti. To our knowledge this is the first
determination of keratitis-causing Acanthamoeba
strains in Austria. Clinically relevant isolates differed markedly from nonrelevant isolates with respect to their physiological
properties. 18S ribosomal DNA sequence types were determined for the
three physiologically most-divergent strains including one of the
keratitis-causing strains. This highly virulent strain exhibited
sequence type T6, a sequence type not previously associated with
keratitis. Sequence data indicate that
Acanthamoeba strains causing keratitis as well as nonpathogenic strains of Acanthamoeba in
Austria are most closely related to published strains from other parts
of the world. Moreover, the results of our study support the assumption
that pathogenicity in Acanthamoeba is a
distinct capability of certain strains and not dependent on appropriate
conditions for the establishment of an infection.
| |
INTRODUCTION |
Within the past few years
free-living amoebae of the genus Acanthamoeba
have gained increasing clinical relevance mainly as causative
agents of a very often seriously progressing keratitis. The first
ocular infections with acanthamoebae were diagnosed in 1974 (21). Since then the occurrence of keratitis due to Acanthamoeba
(Acanthamoeba keratitis) has been escalating in
correlation with the increasing number of contact lens wearers.
Contaminated contact lens care systems usually are the first step in
Acanthamoeba keratitis pathogenesis. The most
prevalent risk factors are contact lens wear, poor hygiene, and a
compromised corneal barrier. Users of extended-wear lenses are at
special risk. Nevertheless, about 10 to 15% of cases of
Acanthamoeba keratitis occur in persons who do
not wear contact lenses (15).
The prognosis for Acanthamoeba keratitis, when
the disease is diagnosed at an early stage and treated adequately, is
rather good, but fast and reliable diagnosis is of crucial importance. Clinical signs and symptoms of Acanthamoeba
keratitis are easily confused with fungal or viral keratitis. Initial
improvement or stabilization in response to topical antibacterial,
antiviral, antifungal, or corticosteroid therapy can occur, altering
the clinical picture and thus complicating diagnosis. Clinical
diagnosis should be based on the presence of keratitis with severe pain and photophobia, stromal infiltrates, radial keratoneuritis, and sometimes pseudodendriform epithelial lesions. Cysts or trophozoites, found in corneal scrapings, on contact lenses, and inside of lens storage cases, are confirmatory. Agar culture is the mainstay for
laboratory detection of Acanthamoeba
(12).
Various Acanthamoeba species have been reported
to be able to cause keratitis: A. castellanii, A. polyphaga, A. hatchetti, A. culbertsoni,
A. rhysodes, A. lugdunensis, A. quina,
and A. griffini (26). Although isolates
can easily be recognized as belonging to the genus
Acanthamoeba by their polygonal cysts, accurate
species determination is still problematic. An important step forward
in the differentiation of acanthamoebae was the division of the genus
into three morphological groups by Pussard and Pons (24).
Since then a number of attempts to enable a more precise identification
have been made. A very promising method for identification and
phylogenetic studies is the analysis of the 18S rRNA gene sequence.
Recently Stothard et al. (27) identified 12 Acanthamoeba sequence types, the vast majority
of keratitis-causing strains belonging to sequence type T4. However, a
final system does not yet exist, and, moreover, representatives from
same species differ with respect to their pathogenicities.
We report on 18 cases of Acanthamoeba-associated
keratitis seen at the Department of Ophthalmology,
Karl-Franzens-University, Graz, Austria, between 1996 and 1999; in
three cases the amoebae were the disease-causing agents. The aim of our
study was to discriminate between clinically relevant and nonrelevant
isolates and to assess the relatedness of our isolates to published
strains by 18S ribosomal DNA (rDNA) sequence similarities.
| |
MATERIALS AND METHODS |
Patients.
Clinical specimens of keratitis patients
presenting at the Department of Ophthalmology were on a routine basis
investigated for Acanthamoeba spp. During 1996 to 1999 specimens from 18 keratitis patients, 10 women (55.6%) and 8 men (44.4%), yielded Acanthamoeba. The
definitive diagnosis of Acanthamoeba keratitis
on the basis of typical clinical signs, no response to antibacterial or
antiviral treatment, and detection of acanthamoebae in the corneal
epithelium was verified for three patients (16.7%).
All 18 patients were contact lens wearers, and for all patients cysts
were detectable in contact lens cases by lactophenol cotton blue
staining (Table 1). Thirteen patients
(72.2%) wore soft contact lenses, 3 (16.7%) wore rigid gas-permeable
lenses, and 2 (11.1%) wore both types. Patients' ages ranged from 15 to 54 years (mean, 29 years). The most prevalent clinical signs we observed were chronic keratitis and keratoconjunctivitis; clinical signs for Acanthamoeba keratitis, namely,
presence of keratitis with severe pain and photophobia, stromal
infiltrates, radial keratoneuritis, and sometimes pseudodendriform
epithelial lesions, were seen in seven patients (38.9%). In three of
these patients the definitive diagnosis of
Acanthamoeba keratitis was verified. Of the 18 patients, only these 3 showed no response to antibacterial or antiviral
treatment. Moreover, these were the only three cases with acanthamoebae
detectable in corneal scrapings.
The
Acanthamoeba keratitis patients were a
15-year-old female wearer of soft daily-wear lenses (2HAP), a
41-year-old male
with rigid gas-permeable lenses in the afflicted left
eye (11DSP),
and a 39-year-old male wearer of rigid gas-permeable
lenses in
both eyes (15SOP). In two of these cases the initial
diagnosis
had been herpes simplex virus keratitis, which did not
improve
despite antiviral therapy. The time between clinical onset and
correct diagnosis ranged from 1 to 5 weeks. Antiamoebic treatment
consisted of local application of propamidine isethionate (Brolene
eyedrops), hexamidine isethionate (Desomedine eyedrops), and bacitracin
plus neomycin (Nebacetin ointment). No perforating keratoplasty
was
necessary. The first patient recovered, with a best corrected
visual
acuity of 20/20; the visual acuity of the second patient
was 20/60 in
the affected eye; the third patient still had corneal
erosions, maybe
caused by the toxicity of the
medication.
Isolation and culture.
Contact lenses, corneal scrapings,
and swabs from contact lens cases were transferred to nonnutrient agar
plates covered with 100 µl of a 24-h-old culture of Escherichia
coli in brain heart infusion medium. The plates were sealed and
incubated at 30°C for 14 days and examined every 48 h for
amoebal growth. Positive cultures were diluted in order to eliminate
coexisting ciliates, flagellates, bacteria, and fungi by harvesting
amoebae at a noncontaminated site of the plate with a sterile
cotton-tipped applicator and transferring the amoebae to a fresh plate.
All isolates were cloned with the use of a micromanipulator and
incubated at various temperatures (30, 34, 37, and 42°C). They were
examined daily by phase-contrast microscopy, and amoebal growth and
temperature tolerances were recorded.
Identification and characterization.
Amoebae were identified
as belonging to one of the cyst morphological groups
(Acanthamoeba sp. groups I to III) established by Pussard and Pons (24), and species determination was
performed according to the identification key of Page (23).
Differentiation was achieved mainly on the basis of cyst size, number
of opercula, and temperature tolerance.
Moreover, all isolates were examined for their cytopathic effects to a
human cell line (HEp-2). Amoebae were axenized by harvesting
cysts from
the plate cultures, incubating them in 3% HCl overnight
in order to
eliminate the bacteria, and transferring the amoebae
into liquid
culture. As a liquid medium we used proteose peptone-yeast
extract-glucose (
23). We cultured the amoebae in
150-cm
2 tissue culture flasks (Corning Costar, Bodenheim,
Germany) at
30°C. Trophozoites were harvested from the axenic
cultures by
centrifugation (500 ×
g for 7 min) and
transferred onto a monolayer
of HEp-2 cells in an amoeba/cell ratio of
1/10. The amoebae were
designated as highly cytopathic when the
monolayer was completely
lysed after 24 to 48
h.
Molecular biology analysis.
18S rDNA sequence analysis was
performed for three isolates most divergent with respect to their
physiological properties (strains 4RE, 9GU, and 11DS) in order to
determine the differences of these strains from and their relatedness
to the published strains from other parts of the world. The 4RE and the
9GU strains were derived from contact lens cases of
non-Acanthamoeba keratitis patients, while the
11DS strain was isolated from the corneal scraping of
Acanthamoeba keratitis patient 11DSP.
For molecular biology investigations amoebae (~10
6 cells)
were harvested from actively growing axenic cultures by centrifugation
at 500 ×
g for 7 min. Whole-cell DNA was isolated by a
modified
UNSET procedure (
14). Briefly, the pellet was
resuspended in
500 µl of UNSET lysis buffer, overlaid with 500 µl
of phenol-chloroform-isoamylalcohol
(PCI), and shaken gently for 5 h. DNA was extracted by multiple
PCI extraction, precipitated in
alcohol, air dried and resuspended
in 30 µl of sterile
double-distilled water. The 18S rRNA gene
was amplified using the SSU1
and SSU2 primers (
9), complementary
to the 5' and 3' ends of
the gene, respectively, and a standard
amplification program (30 cycles; 95°C for 1 min, 50°C for 2 min,
72°C for 3 min).
Amplification of the 18S rRNA gene was visualized
with ethidium bromide
in an agarose gel electrophoresis. The amplified
gene was sequenced
stepwise by direct sequencing from the PCR
product using the Thermo
Sequenase II sequencing kit (Amersham
Pharmacia Biotech GmbH, Vienna,
Austria) and subsequent construction
of complementary internal primers.
Sequences were obtained from
both strands. Sequencing was carried out
in a 310 ABI PRISM automated
sequencer (PE Applied Biosystems, Langen,
Germany).
Sequence data were processed with the GeneDoc (
22) sequence
editor, and sequences were compared to the ones of published
strains
using a BLAST search (
2). ClustalX (
29) was used
for pairwise alignment and calculation of the percentage of sequence
dissimilarity.
Nucleotide sequence accession numbers.
Sequence data
reported in this paper were deposited in GenBank and are
available under the following reference numbers: strain 4RE, AF251937;
strain 9GU, AF251938; strain 11DS, AF251939.
| |
RESULTS |
Identification and characterization of isolates.
In all,
20 strains of free-living amoebae including 15 Acanthamoeba strains, 3 Vahlkampfia strains, and 2 Hartmannella strains were isolated from clinical specimens (Table
2). Four specimens revealed two
variant strains of free-living amoebae each (2HAP, 6DOP, 7TOP, and
8PRP), and in two cases culture was unsuccessful (12JOP and 13PTP).
Fourteen of the 15 Acanthamoeba isolates were identified as belonging to Acanthamoeba sp.
group II. However, several strains exhibited rather varied cyst
morphologies with respect to size and number of opercula although
they were derived from a clone. These strains were classified according
to the average cyst morphology. One strain (18MA) was designated
A. lenticulata (morphological group III), the cysts being
rather small and round. No isolate exhibited a group I morphology. In
all, A. hatchetti was identified six times, A. rhysodes and A. polyphaga were each identified
three times, A. triangularis was identified twice, and
A. lenticulata was identified once.
Acanthamoeba spp. had been proven to be of
clinical relevance in three cases (2HAP, 11DSP, and 15SOP). All of
these isolates
showed an
Acanthamoeba group II
cyst morphology. In one case (2HAP)
two different strains of
Acanthamoeba,
A. rhysodes
and
A. hatchetti,
were detected. The other keratitis-causing
isolates were identified
as
A. hatchetti (11DS) and
A. polyphaga (15SO).
Temperature tolerance tests revealed that all 20 isolates of
free-living amoebae grew at 30°C; 13 of them also grew at
34°C,
11 strains grew at 37°C, and 2 strains grew at 42°C. All of
the
thermophilic strains were among the
Acanthamoeba isolates. The
two
Vahlkampfia strains and the
Hartmannella strain
showed no
growth above 30°C. Five isolates (2HAB, 9GU, 11DS,
15SO, and 16KV)
showed cytopathic effects against monolayers of HEp-2
cells. Strains
2HAB, 11DS, and 15SO had been isolated from
Acanthamoeba keratitis
patients, yet two strains
(9GU and 16KV) isolated from patients
without
Acanthamoeba keratitis also exhibited moderate
cytopathic
effects (monolayer lysis after 3 to 4 days). From the two
different
Acanthamoeba strains isolated from
patient 2HAP only one (2HAB)
showed pathogenicity-related physiological
characteristics. Eventually
only this strain was responsible for the
infection, while the
other strain (2HAA), with no
pathogenicity-associated characteristics,
was only a
concontaminant.
The clinically relevant strains 2HAB, 11DS, and 15SO not only showed
high-temperature tolerance and high cytopathic effects
but also
generally exhibited far higher growth rates than the
other
isolates.
Molecular biology analysis.
All strains investigated revealed
more than 97% sequence identity to classified published
strains (Table 3) and could thus be
assigned to one of the 12 published sequence types, as sequence types
differ from one another by at least 5% (27). Nonpathogenic strain 4RE (GenBank accession no. AF251937), having been identified as
A. hatchetti, displayed sequence type T11, with 99.3%
identity to the BH-2 A. hatchetti strain isolated in
brackish water in the United States (GenBank accession no. AF019068).
The 9GU strain (GenBank accession no. AF251938), which did not cause disease but which is pathogenic to tissue culture, showed 98.6% identity to the Castellani strain of A. castellanii (GenBank
accession no. U07413), which has sequence type T4. This strain of
A. castellanii has been isolated from a yeast culture in
the United Kingdom and is the type strain for the species
A. castellanii. We thus reclassified our isolate,
initially classified as A. polyphaga, as A. castellanii. Most of the published A. polyphaga isolates show sequence type T4, as do most of the
A. castellanii isolates. Stothard et al.
(27) proposed to reclassify all sequence type T4 isolates as
A. castellanii. Strain 11DS (GenBank accession no.
AF251939), isolated from the contact lens case of a patient with
serious Acanthamoeba keratitis, showed an
18S rDNA sequence with highest identity (97.7%) to the 2802 strain of A. palestiniensis with sequence type T6,
isolated in a swimming pool in France (GenBank accession no. AF019063).
However, the 11DS strain exhibits a typical A. hatchetti
morphology, with most of the cysts being rectangular (Fig.
1), and the isolate showed the ability to
grow at 42°C, while A. palestiniensis is described as
showing no growth even at 37°C (23). Moreover,
because A. hatchetti and A. palestiniensis are
described as polyphyletic (27), we prefer not to
reclassify this isolate.
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FIG. 1.
Cysts of the 11DS isolate with typical A. hatchetti morphology, as shown by phase-contrast microscopy.
Magnification, ×1,000.
|
|
The sequences of our strains clearly diverged from one another, all
exhibiting different sequence types, namely, T4, T6, and
T11. The 4RE
and the 9GU strains were more closely related to
one another (5.57%
dissimilarity) than either was to the 11DS
strain. The 4RE and 9GU
strains showed 9.68 and 9.40% sequence
dissimilarities,
respectively, to the 11DS
strain.
| |
DISCUSSION |
Identification and characterization of isolates.
Altogether 15 strains of Acanthamoeba, 3 strains of
Vahlkampfia, and 2 strains of Hartmannella were
isolated. During the last few years it has become apparent that
Vahlkampfia and Naegleria (11) as well
as Hartmannella (1, 16) can cause keratitis or at
least be associated with keratitis. However, neither the Hartmannella strain nor the Vahlkampfia strains
were of clinical relevancy in our study.
In two cases the amoebae could not be grown in vitro. This may
partially be due to the fact that patients had already been
treated for
bacteria, and antibacterial and antifungal treatment
is at least partly
effective against free-living amoebae. On the
other hand, contact lens
disinfectants, even if not used properly,
compromise amoebal viability.
Moreover, amoebae penetrate the
cornea during the course of infection,
protruding up to Descemet's
membrane. It might therefore in some cases
be impossible to isolate
viable amoebae by scraping. Several studies
report on unsuccessful
attempts to culture amoebae from clinical
specimens (
15,
26).
In general most of the acanthamoebae isolated were identified as
belonging to
Acanthamoeba sp. group II, which
also is reported
to be the most prevalent group of these microorganisms
(
23).
In a former study we could demonstrate that in the
area of Vienna
Naegleria seems to be the predominant genus
in environmental habitats,
whereas probing of shower heads and tap
water mainly revealed
amoebae of the genera
Acanthamoeba and
Hartmannella (our
unpublished
data). This supports the assumption that, in
Acanthamoeba keratitis,
infection is primarily
acquired via contaminated contact lenses
and lens cases, the amoebae
deriving mainly from tap water, rather
than by outdoor swimming.
Domestic tap water as source of
Acanthamoeba sp.
in
Acanthamoeba keratitis was described for the
first time
in 1990 (
17). Interestingly two of the
Acanthamoeba keratitis
patients (
n = 3) discussed here wore rigid gas-permeable contact
lenses in the
afflicted eyes. Usually wearers of soft lenses are
more likely to
acquire
Acanthamoeba keratitis, as the
hydrophilic
material seems to support attachment and survival of cysts.
However,
several cases of
Acanthamoeba keratitis
in wearers of rigid gas-permeable
contact lenses have been reported
(
4,
25). A case of
Acanthamoeba keratitis occurring in a wearer of daily-disposable contact lenses
has
also been documented recently (
31). We did not observe any
correlation to age or sex, and in none of the three patients did
surgical treatment become necessary. In a study from the United
Kingdom
severe visual loss was seen in about 15% of the patients
(
25).
Morphological determination was rather difficult in some cases as
cysts, although all deriving from one clone, had varied
morphologies,
and generally intraspecific polymorphism is rather
common
among acanthamoebae (
3). The four
Acanthamoeba strains
isolated from the contact
lens cases of the three
Acanthamoeba keratitis
patients were identified by cyst morphology as
A. rhysodes,
A. polyphaga, and two strains of
A. hatchetti. All of these species
are known to cause keratitis
(
26). To our knowledge this is
the first determination of
keratitis-causing strains in Austria.
In 1989, Huber-Spitzy et al.
described a case of
Acanthamoeba keratitis in a
37-year-old woman in Austria (
13). They detected
cysts
of
Acanthamoeba in the corneal epithelium, but
the amoeba
was neither isolated nor identified to the species
level.
Clinical relevance of strains.
Remarkably, in the majority of
keratitis cases we investigated, the acanthamoebae were of no clinical
relevance. Although Acanthamoeba keratitis has
become of increasing importance within the last 10 years, correlating
to the greater number of contact lens wearers, it is still a rare
disease. The annualized incidence of
Acanthamoeba keratitis is estimated as
0.14 per 100,000 individuals (25).
Also other studies revealed that the majority of people do not develop
a keratitis in spite of coming into contact with acanthamoebae;
the
prevalence of acanthamoebae and other free-living amoebae
in
asymptomatic contact lens wearers has been reported frequently
(
8,
18). Even from the nasal mucosae of healthy individuals
different
strains of
Acanthamoeba spp. could be isolated
(
20).
Apart from such widely accepted risk factors as
extended contact
lens wear and microlesions in the cornea, certainly
the immune
status of the patient may play a fundamental role in the
course
of infection and may result in enhanced susceptibility to
developing
an
Acanthamoeba keratitis. It was
shown that nearly 50% of healthy
individuals carry antibodies against
Acanthamoeba, probably due
to the ubiquity of
this microorganism (
28).
Moreover, apart from differences in predisposition of patients,
one can assume that amoeba strains vary in pathogenicity.
Mazur
et al. demonstrated in an animal model a clear correlation
between the
occurrence of eye infections and the degree of virulence
of the
strains after installation into the brain (
19), which
suggests that pathogenicity is not so much dependent on
environmental
conditions but rather represents a distinct
characteristic of
certain strains. Nevertheless the initial
infective dose is certainly
of considerable importance. The factors
which prime amoebae for
pathogenicity are poorly
understood. Temperature tolerance (
10)
and cell culture
pathogenicity (
3,
5,
6) seem to be related
to pathogenicity
potential. Temperature tolerance is widely accepted
to be a
prerequisite for pathogenicity in granulomatous amoebic
encephalitis
caused by
Acanthamoeba, as the human body
temperature
is 37°C. However, the human eye has a mean temperature of
only
around 34°C. Yet data presented here indicate that clinically
relevant strains not only show higher temperature tolerances but
also
generally yield far higher growth rates, which certainly
is of major
importance for the establishment of infection. Temperature
tolerance
among keratitis-causing
Acanthamoeba strains is
rather
conspicuous (
3,
7). Moreover, the keratitis-causing
acanthamoebae
described here were highly cytopathic to HEp-2 cells,
producing
complete destruction of the monolayer in 1 to 2 days.
Altogether,
there seems to be a correlation between clinical relevance
and
pathogenicity-related physiological
characteristics.
The occurrence of free-living amoebae of nonpathogenic strains in
contact lens cases, however, is still of medical interest,
as they can
harbor bacteria inside their cysts, protecting them
from disinfectants,
and thus function as vectors. In a recent
study we showed that viable
Pseudomonas aeruginosa, one of the
major ocular pathogens,
can survive in and be reisolated from
cysts of
Acanthamoeba (
30).
Molecular biology analysis.
Interestingly, the three
investigated strains showing the most-diverse physiological capacities
also differed with respect to their 18S rDNA sequences, exhibiting
three different sequence types. The two strains with no clinical
relevancy, both classified as group II acanthamoebae, showed sequence
type T4 (strain 9GU) and sequence type T11 (strain 4RE). Sequence types
T3, T4, and T11 are most closely related, and all represent
morphological group II (27). T4 is reported to be the
sequence type isolated most frequently. Moreover, the majority of
keratitis-causing Acanthamoeba strains are
reported to belong to sequence type T4 (27).
The keratitis-causing and highly cytopathic strain 11DS, identified as
A. hatchetti, morphological group II, exhibited sequence
type T6. Sequence type T6 is represented by a single strain, which
had
been identified morphologically as
A. palestiniensis
belonging
to morphological group III. Stothard et al. also report on a
strain
presumed to be group II but with a group III sequence. The
authors
assume that
A. palestiniensis and
A. hatchetti are polyphyletic
(
27). However, a final
system does not yet exist, and most probably
diversification will still
be necessary. T6 isolates have not
previously been reported
to cause keratitis. Also no keratitis-causing
strain is known to
exhibit sequence type T2, which is the sequence
type most closely
related to sequence type
T6.
Altogether the results of our study support the assumption that
pathogenicity in
Acanthamoeba seems to be a
distinct capability
of certain strains and not so much dependent on
appropriate conditions
for the establishment of an infection. Moreover,
data presented
here indicate that
Acanthamoeba
keratitis-causing strains as well
as nonpathogenic strains of
Acanthamoeba in Austria are most closely
related
to published strains from other parts of the
world.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department for
Medical Parasitology, Clinical Institute of Hygiene, University of
Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria. Phone:
0043-1-4277-79430. Fax: 0043-1-4277-9794. E-mail:
Horst.Aspoeck{at}univie.ac.at.
| |
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Journal of Clinical Microbiology, November 2000, p. 3932-3936, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
