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Applied and Environmental Microbiology, July 2001, p. 3325-3327, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3325-3327.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Incidence of Klebsiella Species in
Surface Waters and Their Expression of Virulence Factors
R.
Podschun,1,*
S.
Pietsch,1
C.
Höller,2 and
U.
Ullmann1
Department of Medical Microbiology and
Virology1 and Department of Hygiene and
Environmental Medicine,2 University of Kiel,
Kiel, Germany
Received 30 November 2000/Accepted 6 May 2001
 |
ABSTRACT |
To investigate the occurrence of different Klebsiella
spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species
being Klebsiella pneumoniae. A comparison of these isolates
to a group of 207 clinical K. pneumoniae isolates
demonstrated that water isolates of K. pneumoniae, unlike
those of K. oxytoca and K. planticola, are as
capable as clinical isolates of expressing putative virulence factors
such as serum resistance and capsular polysaccharides, pili, and siderophores.
| |
TEXT |
Bacteria of the genus
Klebsiella are a frequent cause of nosocomial infections
(8). Klebsiella spp. are ubiquitous in nature. Their nonclinical habitats encompass the gastrointestinal tract of
mammals as well as environmental sources such as soil, surface waters,
and plants (1). Environmental isolates have been described as being indistinguishable from human clinical isolates with respect to
their biochemical reactions and virulence (12). While the medical significance of Klebsiella obtained in the natural
environment is far from clear, such habitats are thought to be
potential reservoirs for the growth and spread of these bacteria which
may colonize animals and humans (11).
At present the genus Klebsiella is subdivided into five
species. Clinically, the most important species are Klebsiella
pneumoniae and K. oxytoca, while K. ornithinolytica, K. terrigena, and K. planticola are
rarely isolated from human clinical specimens (6, 18).
K. planticola and K. terrigena are considered to
be environmental species, as reflected in their species designations.
In contrast to K. pneumoniae, neither species grows at
elevated temperatures, such as at 44.5°C.
To date, however, studies on the frequency of Klebsiella in
nonclinical habitats have focused on K. pneumoniae or have
not identified isolates to the species level at all (2, 3, 4, 5,
10, 11, 12, 19). No data are currently available on the
incidence of the various Klebsiella spp. in environmental habitats.
The purpose of the present study was to determine the occurrence and
distribution of the five Klebsiella species in natural surface waters. Having done this, we investigated whether environmental isolates are capable of producing putative Klebsiella
virulence factors, such as pili, serum resistance properties,
siderophores, or particular capsular types. For comparison, a group of
previously described K. pneumoniae human clinical isolates
was used (15).
Collection of water samples.
From November 1997 to June 1998, 208 water samples were collected in sterile 250-ml glass bottles from
196 different sampling sites at streams, lakes, and the Baltic Sea in
various geographic areas of Schleswig-Holstein, Germany. Samples were
taken 30 cm below the water surface, stored on ice for transportation,
and processed for bacteriological analysis within 4 h of
collection. The samples were classified as freshwater (conductivity,
<1,500 µS/cm), brackish water (1,500 to 15,000 µS/cm), or salt
water (>15,000 µS/cm).
Isolation of strains.
Each 250-ml sample was filtered through
a 0.45-µm-pore-size membrane (Sartorius, Göttingen, Germany).
The membranes were transferred onto Simmons citrate agar with 1%
(wt/vol) inositol and incubated for 48 h at 37°C. This medium is
highly selective but not inhibitory for the recovery of
Klebsiella (21). Presumptive Klebsiella colonies were isolated, followed by
identification according to the biochemical tests given by Ørskov
(13), which include fermentation of melezitose and
L-sorbose, gas production from lactose at 44.5°C, growth
at 10°C, pectate degradation, and utilization of
m-hydroxybenzoate and hydroxy-L-proline. A group of 207 human clinical K. pneumoniae isolates previously
obtained from human infections was used for comparison
(15).
Capsule typing.
The isolates were serotyped by the capsular
swelling method as described by Ullmann (20). Polyvalent
rabbit anticapsule sera were used for screening, and monospecific sera
were used for typing.
Hemagglutination assay.
Expression of type 1 pili
(mannose-sensitive hemagglutination [MSHA]) and type 3 pili
(mannose-resistant Klebsiella-like hemagglutination [MR/K-HA]) was examined as described previously (16),
with MSHA being assessed on guinea pig erythrocytes and MR/K-HA on
tanned ox red blood cells. Bacteria were grown statically at 48-h
intervals. Fifty microliters of bacterial suspensions (approximately
1011 bacteria/ml) and 50 µl of erythrocytes (5 × 108/ml) were mixed in porcelain tiles, rocked, and observed
for 3 min at room temperature. Agglutination was finally read after further incubation for 10 min at 4°C.
Determination of siderophore production.
For detection of
enterobactin and aerobactin production the cross-feeding bioassay of
Hantke (7) was performed as described elsewhere
(14). Nutrient agar supplemented with 2,2'-dipyridyl (200 µM) served as iron-restricted agar medium. Escherichia
coli strain H1887 (ColV
Aer
Iut+ FepA Fiu Cir
aroB) was used as the indicator strain for aerobactin
production, and strain H1939 (FepA+ Fiu
Cir FhuA FhuB
aroB) was used to indicate enterobactin production.
Aerobactin production was counterchecked using the E. coli
strain H1886, which is the Iut parent strain of H1887.
Each isolate was tested twice. The indicator strains were kindly
provided by K. Hantke, University of Tübingen, Tübingen, Germany.
Serum bactericidal assay.
The susceptibility of bacteria to
human serum was determined by the method of Hughes et al.
(9) as slightly modified previously (17).
Twenty-five microliters of bacterial suspensions (2 × 106 cells/ml) and 75 µl of normal human serum were put
into microtiter trays and incubated at 37°C. Viability was determined
immediately and after 1, 2, and 3 h of incubation by plating on
brain heart infusion agar for colony counts. Responses were graded as
highly sensitive, intermediately sensitive, or serum resistant
according to the system of Hughes and colleagues (9). Each
strain was tested three times.
Statistical analysis.
The significance of differences between
groups of bacteria was evaluated by Yates' corrected chi square test
for 2 by 2 contingency tables. Medians were compared using the
nonparametric analysis of variance test of Kruskal-Wallis and by the
Mann-Whitney test. All tests were performed using GraphPad InStat,
version 3.00 (GraphPad Software, San Diego, Calif.).
Over an 8-month period, 208 samples of natural surface water were taken
from 196 different sampling sites. One hundred ten of the water samples
(52.9%) were found to contain 123 Klebsiella strains.
Thirteen water samples each contained two different
Klebsiella species, and two other samples contained two
different strains of the same species. In our experience, the frequency
of Klebsiella isolation from surface water depends to a
large extent on the volume investigated. In a preliminary experiment
using 1-ml samples, we were not able to isolate this bacterium from any
of the 47 water samples examined. We therefore decided to investigate
250-ml samples. Even then, the number of Klebsiella colonies
per sample was low (usually 1 to 5 CFU/250 ml). The incidence of
positive water samples was conspicuously independent of source and
season. We found no significant differences in the frequency of
isolation between freshwater, salt water, and brackish water (Table
1). Likewise, no seasonal effects could
be observed (data not shown). K. pneumoniae was most common
(n = 62; 52%), followed by K. oxytoca (n = 34; 27%) and K. planticola
(n = 27; 22%). Neither K. ornithinolytica nor K. terrigena was detected. With respect to the latter
species, this stands in striking contrast to the view that K. terrigena is an environmental species that can be isolated from
surface waters (1). The lack of K. terrigena in
our surface water samples, however, was not due to a possible
inhibiting effect of the Klebsiella-selective agar used
since in preliminary experiments we confirmed that all Klebsiella species can grow on this medium. It is
conceivable, though, that there are geographic differences in the
occurrence of K. terrigena.
Serotyping revealed K types K33 and K69 as the most common capsular
types among
K. pneumoniae and
K. oxytoca isolates
(13
to 15%) (Table
2). Both serotypes
were rarely observed (<2%)
in the group of 207 clinical
K. pneumoniae isolates investigated
for comparison purposes. In
contrast, clinical
K. pneumoniae strains
predominantly
expressed the K antigen K2 (14%), which is considered
to be a main
determinant of
Klebsiella virulence (
18).
Surprisingly,
this serotype was also the most common K type among
K. planticola isolates (11%).
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TABLE 2.
Distribution of capsule types in Klebsiella
spp. from natural surface waters and from clinical specimens
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|
Clinical
K. pneumoniae strains and isolates from surface
water were very similar with respect to the incidence of type 1 and
type 3 pili. Both groups were significantly more often fimbriated
than
K. oxytoca or
K. planticola strains (
P < 0.005) (Table
3).
However, the
statistical significance of the differences between
groups in the
production of type 3 pili was only at a
P value
of 0.07 in
some cases. MSHA expression in
K. oxytoca was very
rare
(15%).
View this table:
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TABLE 3.
Distribution of fimbriae, serum resistance properties,
and siderophores among Klebsiella spp. from natural
surface waters and from clinical specimens
|
|
The incidences of serum resistance properties differed considerably
between the groups of environmental isolates (Table
3).
About half
(53%) of the
K. oxytoca strains proved to be serum
resistant, whereas only 11% of the
K. pneumoniae and 4% of
the
K. planticola isolates were resistant (
P < 0.0001). Clinical
K. pneumoniae strains exhibited serum
resistance properties twice
as often (25%) as environmental
K. pneumoniae strains (
P < 0.05).
Siderophore production by the bacterial groups was very similar. Except
for five strains, all isolates investigated were able
to synthesize the
catechol-type siderophore enterobactin (Table
3). In contrast,
production of the hydroxamate-type siderophore
aerobactin was lacking
(environmental isolates) or very rare (clinical
K. pneumoniae strains).
To determine whether environmental species differ with respect to the
expression of virulence factors and whether they differ
in this respect
from clinical
K. pneumoniae isolates, the factors
detected
in each strain (MSHA, MR/K-HA, serum resistance, enterobactin
synthesis, and aerobactin production) were added up to get the
cumulative number of virulence factors expressed per isolate (Table
3).
The mean number of factors expressed by environmental
K. oxytoca (2.2) and
K. planticola (1.9) strains was
significantly
lower than that expressed by environmental (2.7) or
clinical (2.9)
K. pneumoniae isolates (
P < 0.01).
To sum up, a high percentage (53%) of surface water samples proved to
be positive for
Klebsiella spp., the most common species
being
K. pneumoniae. Furthermore, our data show that surface
water
isolates of
K. pneumoniae resemble clinical strains in
the expression
of virulence factors, whereas water isolates of
K. oxytoca and
K. planticola differ from clinical strains
in this respect. With
respect to the factors examined, we found no
evidence that environmental
K. pneumoniae strains are less
virulent than clinical strains.
Whether this finding has any relevance
to public health is at
present unclear and should be evaluated by
further
studies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Virology, University of Kiel, Brunswiker Str. 4, 24105 Kiel, Germany. Phone: 49-431-597-3305. Fax: 49-431-597-3296. E-mail: podschun{at}medmicrobio.uni-kiel.de.
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Applied and Environmental Microbiology, July 2001, p. 3325-3327, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3325-3327.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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