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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY STUDIES series. This series describes research
performed to develop and demonstrate instrumentation, equipment
and methodology to repair or prevent environmental degradation from
point and non-point sources of pollution. This work provides the
new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
EPA REVIEW NOTICE
This report has been reviewed by the National Environmental
Research Center—Corvallis, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement
or recommendation for use.
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EPA-660/2-75-024
JUNE 1975
TAXONOMY OF KLEBSIELLA PNEUMQNIAE
ISOLATED FROM PULP/PAPER MILL WASTEWATER
Martin D. Knittel
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330
Program Element 1BB037
ROAP 21 AZX/Task No. 055
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For sale by the Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20402
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ABSTRACT
Klebsiella pneumonlae, a coliform bacterium, has been isolated from pulp
and paper wastewater effluents. It represents as much as 80% of the
total coliform bacteria present and was found able to grow in sterilized
wastewater samples.
A taxonomic comparison of isolates from the environment and from various
other sources revealed no difference among the cultures. Both the
environmental and the pathogenic cultures of K. pneumoniae exhibited the
same biochemical properties.
The deoxyribonucleic acid base (DNA) composition comparison of these
same isolates showed they all exhibited a guanine plus cylosine base
composition of 56% j^ 1.4%, and all cultures examined fell within this
range. A more detailed study of the DNA hybridization revealed that
isolates from both pulp mills and pathogenic sources had from 92 to 100%
homology to the reference culture. One pulp mill isolate had only 41%
homology, which indicates some K^ pneumoniae from pulp mills may be
phenotypically similar but genetically dissimilar from known K. pneumoniae.
It was concluded from this study that: (1) coliform bacteria with IMViC
profiles of --++ should not be disregarded since some may be K. pneumoniae,
and (2) K. pneumoniae found in clinical or from environmental sources
are biochemically and genetically related.
This report was submitted in fulfillment of ROAP 21 AZX/Task No. 055
by the Pacific Northwest Environmental Research Laboratory. Work was
completed as of June 1973.
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CONTENTS
Sections Page
I. Conclusions 1
II. Recommendations 2
III. Introduction 3
IV. Methods and Materials 5
V. Results 8
VI. Discussion 26
VII. References 29
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FIGURES
No. Page
1 Three Dimensional Plot of Total 9
Coliform Densities of a Pulp Mill
2 Three Dimensional Plot of Fecal 10
Coliform Densities of a Pulp Mill
3 Growth Curve of K. pneumom'ae in 12
Sterilized Wastewater from a Pulp
Mill
4 Bar Graph of Total Coliforms and 13
K_. pneumom'ae Densities of Various
Wastewater Sources of a Pulp Mill
5 Diagrammatic Drawing of Molecular 22
Structure of Deoxyribonucleic Acid
6 Thermal Denaturation Curve of Purified 23
Deoxyribonucleic Acid
IV
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TABLES
No. Page
1 Effluent Coliform Content from 11
Different Pulping Process
2 Cultural Reactions of Environmental 16
and Reference Cultures of K. pneumoniae
3 Differentiation of K. pneumoniae and 17
Enterobacter Species
4 Cultural Compairson of Environmental 18
K. pneumoniae to Other Species of
Klebsiella
5 Source and Serological Types of 19
Environmental K. pneumoniae
6 Tm and G + C% of DNA from Klebsiella 24
pneumoniae Isolates from Pulp Mills
7 Relative Reassociation of Human and 25
Pulp Mill Klebsiella Deoxyribonucleic
Acid
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SECTION I
CONCLUSIONS
The oriqin and health significance of K_. pneumoniae in pulp and
paper wastewater effluents remains to be determined. Several important
facts must be considered concerning the occurrence of coliform bacteria
in these wastewaters.
(a) The coliforms discharged into the water course from pulp and
paper wastewater effluents cause a degradation of the bacteriological
water quality and may act to mask the occurrence of other sources of
coliforms.
(b) The nutrient levels of these pulp mill wastewaters v/ere in
sufficient quantity to support the growth of coliforms.
(c) The K. pneumoniae isolated from pulp and paper wastewaters
were taxonomically indistinguishable from K_. pneumoniae obtained
from clinical sources.
(d) Those cultures obtained from both wastewater and clinical
sources were genetically identical by virtue of their having 92 to
100% of DMA homology. One strain tested showed only 41% homology
to reference DNA indicating that some isolates were phenotypically
identical to K. pneumoniae, but were genetically dissimilar.
(e) The question of pathogenicity of these environmental sources
has not been proven or disproven, but remains to be answered.
(f) Finally, it is concluded that until K_. pneumoniae in pulp and
paper mill wastewater effluents are shown to be non-pathogenic,
disinfections or other bacterial control methods should be practiced
on these effluents.
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SECTION II
RECOMMENDATIONS
The origin of K. pneumoniae in pulp mill wastewaters has not yet been
resolved. IK. pneumoniae can be of fecal origin and there is some evidence
to suggest this may be true in this case since fecal coliforms can also
be found in these same wastes. Proof of fecal origin would require an
indepth study tracing the origin of coliforms during the various stages
of manufacturing within the plant. The outcome of this study would
identify point sources of contamination within the plant. Once identified,
point source control might be used instead of controlling the coliforms
in the total mill effluent. Control of coliforms at the source would be
more manageable than in the treatment system.
The occurrence of coliforms, and K_. pneumoniae specifically, has been
identified by Duncan and Razzel (4) as being ubiquitous and, therefore,
their presence in pulp mill effluents are of no significance. It is
necessary in the overall understanding and control of coliforms to trace
their source in nature. The significance of K_. pneumoniae can be more
intelligently assessed if the origin is established, especially if the
origin can be established as fecal; then the probability of the presence
of other pathogenic organisms is also relatively high, and K_. pneumoniae
assumes a new priority of importance.
Additional research should be undertaken to answer the following questions:
(1) What is the source or growth site within the industrial plant
and are the organisms present of true fecal origin?
(2) Are IK. pneumoniae pathogenic and is their presence a hazard
to human health?
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SECTION III
INTRODUCTION
In 1970 a water sampling study was conducted on some rivers and streams
in the Pacific Northwest. The study found a high number of coliform
bacteria present in surface waters downstream from pulp and paper plants
(Bauer, 1970 unpublished, 17 and 18). As a result of this study, further
work was done which traced the source of the coliforms to the effluent
wastewaters of this industry. A taxonomic analysis of the coliform
cultures revealed that the majority were of an Indole, methyl red,
Voges-Proskauer, and citrate (IMViC)* profile of --++. In previous
sanitary microbiology methods, coliform with this IMViC type were discarded
as being of soil or plant origin and of no particular sanitary signi-
ficance. However, these IMViC types were further identified as Klebsiella
pneumoniae by the Enteric Bacteriology Laboratory of the U.S. Public
Health's Center for Disease Control (CDC) at Atlanta, Georgia. This
finding raised the questions as to whether the presence of K. pneumoniae
posed a health hazard, and what was the significance of coliforms and
fecal coliforms in these industrial wastewater effluents.
K. pneumoniae was originally isolated from infected lungs by Friedlander
(1882). K. pneumoniae has since been found to be the cause of other
human diseases: urinary tract infections (Edmondson and Sanford, 1967),
bacteremia (Steinhouer et al., 1966), osteomyelitis (Forman, 1963) and
meningitis (Spicak, et al, 1957). to name only a few. The point to be
stressed is K. pneumoniae is a human pathogen and can be found in the
intestinal flora of many humans (Thone, 1970). K. pneumoniae has also
been found to cause infections in cattle. Braman et al. (1973) found it
to be a causative agent in bovine mastitis.
*IMViC = four basic tests used to differentiate coliform bacteria. I = Indole,
M = methyl red, V = Voges-Proskauer, C = citrate.
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Until recently, it was difficult to separate K_. pneumoniae from E_^
aerogenes because of close physiological similarities. Ewing (1963)
developed a classification scheme that makes it possible to distinguish
between these two species but it is not widely used by sanitary bacter-
iologists at this time. Thus, identifying K. pneumoniae in pulp and
paper wastewater has been confusing because of the reclassification of
Aerobacter aerogenes to Enterobacter aerogenes and moving Enterobacter,
Klebsiella and Serratia from the Escherichieae tribe (Bergy's Manual) to
a new tribe Klebsielleae.
The objectives of this research were to resolve the taxonomy of j<^
pneumoniae found in pulp and paper wastewater effluents and to determine
if it is genetically related to K. pneumoniae obtained from human patients
with K. pneumoniae infections.
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SECTION IV
METHODS AND MATERIALS
CULTURES
K. pneumonlae were isolated from samples of pulp mill effluent by
filtering it through a sterile 0.45 y membrane filter and placing the
membrane on an Endo LES agar (Difco). After 24 hours of incubation at
35°C, colonies on the membrane were transferred to triple sugar iron
(TSI) agar slants. These cultures were incubated for 24 hours at 35°C,
and those showing an acid slant and butt with gas, but no H?S, were
inoculated into additional media for biochemical classification. All
cultures that grew on citrate, produced acetyl methylcarbinol (Voges-
Proskauer positive), were non-motile, ornithine decarboxylase negative,
indol phenol oxidase negative and gram negative rod-shaped bacteria were
identified as Klebsiella pneumom'ae. Several cultures were submitted to
the U. S. Public Health Center for Disease Control (CDC), Atlanta,
Georgia, for confirmation. Their classification confirmed the above
procedure.
Additional cultures of K. pneumoniae were obtained from the American
Type Culture Collection (ATCC), Washington, D.C., and from Dr. John
Matsen of the University of Minnesota Medical School, Minneapolis,
Minnesota.
CLASSIFICATION
The identification of the environmental isolates of K. pneumoniae and
the media and reagents used were those recommended by Edwards and Ewing
(1972).
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CAPSULE DEMONSTRATION AND TYPING
Capsule production was enhanced by passing the culture into the medium
recommended by Hoogerheide (1939). After 24 hours of growth, a drop of
the culture was mixed with a drop of india ink and a thin smear made on
a slide. After the film dried, it was counter stained with basic fuchsian
dye. The capsule was a clear halo in the particles of india ink around
the red stained cell.
Capsular antigen types were determined with capsular antiserum obtained
from Difco, Detroit, Michigan. These cultures were also submiited to
the U.S. Public Health Center for Disease Control, Atlanta, Georgia, for
confirmation.
COLIFORM AND FECAL COLIFORM DETERMINATIONS
Total and fecal coliforms were estimated using the Millipore membrane
method described in Standard Methods for the Examination of Water And
Wastewater (1971).
DEOXYRIBONUCLEIC ACID BASE COMPOSITION
Deoxyribonucleic Acid (DNA) was isolated and purified from packed cell
masses of K. pneumonaie using the method of Marmur (1961) and later by a
method suggested by Anderson and Ordal (1972).
The DNA base composition was determined by observing the thermal denatur-
ation of a sample of DNA and finding the Tm values for the sample according
to Deley and Schell (1963). The percentage of guanine and cytosine
composition of the DNA sample was calculated from the Tm value using the
formula of Marmur and Doty (1962) which is:
-------
%G+C - (Tm-69.3)
0.42
where %G+C = precent guanine + cytosine content of the DNA and Tm is
midpoint of thermal denaturation curve. The value 69.3 is the mid-point
of the thermal denaturation curve of pure adenine + thymine polymer.
The value 0.42 is the slope of the empirical curve.
DEOXYRIBONUCLEIC ACID - DEOXYRIBONUCLEIC ACID HOMOLOGY DETERMINATION
Homo logy of DNA was carried out using the membrane filtration methods of
Anderson and Ordal (1972).
-------
SECTION V
RESULTS
Samples were taken on four consecutive days to determine the occurrence
and variation of coliforms in the stages of treatment of pulp and paper
wastewater. Figure 1 is a three dimensional graph of the results. The
date of sampling is plotted on the horizontal axis, the number of coliform
bacteria found are plotted on the vertical axis and the third dimension
is the place of sampling. The number of coliforms increase about two
orders of magnitude as the waste progresses from the primary influent to
the secondary effluent. The day-to-day variation of numbers of coliforms
at any sampling point remained consistent within an order of magnitude.
The intake or processing water contained less than 100 coliforms per
milliliter on any day sampled, but the primary influent waste stream
contained approximately 10,000 coliforms per milliliter. This 99.0%
increase suggests that the majority of the coliforms are coming from
areas within the pulp mill itself because the wastewater entering the
settling basin contains several thousand coliforms per milliliter.
The daily variation of fecal coliforms content is presented in Figure 2.
The response surface of the graph shows that the number of fecal coliforms
varies from day to day, but is always greater in the aeration basin.
The fecal coliform content of the intake water was low and it appeared
that the fecal coliforms originate from within the mill.
The data in Figures 1 and 2 suggest that the coliforms are growing in
the waste during the treatment process. This observation was tested by
sterilizing (autoclaved at 121°C) samples of wastewater entering the
secondary aeration basins and inoculating sub-samples with purified
cultures of K. pneumoniae and a fecal coliform originally isolated from
a sample of the secondary wastewater effluent. An unsterilized sample
of the same waste was also included. This sample would represent the
-------
CD
Secondary Effluent
Secondary Influent
Primary Effluent o>
Primary Influent
10/18 10/19 10/20
Date of Sampling
10/21
Source Water
Figure 1. Three Dimensional Plot of Total
Coliform Densities of a Pulp Mill
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Effluent Aeration Basin
Influent Aeration Basin
Effluent Settling Pond
Influent Settling Pond
10/18 10/19 10/20 10/21
Date of Sampling
Intake Water
Figure 2. Three Dimensional Plot of Fecal
Coliform Densities of a Pulp Mill
10
-------
increase or decrease in the indigenous coliform population. The results
are presented in Figure 3. The curve with the solid circles shows that
over the period of incubation the indigenous population (in the unsterilized
sample) increased by one order of magnitude. The coliforms have enough
nutrients and can compete with the other microorganisms for these nutrients.
The inocula of both the fecal coliform (open squares) and K. pneumoniae
grow more rapidly than fecal coliform culture in this waste; this may be
why it becomes the predominant coliform in this type of wastewater.
The numbers of K.pneumonaie and total coliform population were determined
in the various waste streams of the pulp mill. These data are shown in
Figure 4. K. pneumoniae constitutes approximately 70 percent of the
coliform population of these waste streams. There is a large increase
in coliform and K. pneumoniae populations during the time of retention
in the aeration basins (secondary treatment). This supports the above
observation of growth in sterilized wastewater. The presence of coliforms
and the identification of K. pneumoniae in this pulp mill effluent led
to the examination of other types of pulping processes for coliforms in
general and K. pneumoniae specifically. These data are presented in
Table 1. The lowest K. pneumoniae percentage was found in the effluent
from a defiberization plant. The highest percentage was in the effluent
from an ammonia base sulfite mill. A wider sampling of pulping processes
by Bauer (unpublished results) resulted in similar findings.
TABLE 1
EFFLUENT COLIFORM CONTENT FROM DIFFERENT PULPING PROCESSES
Percent
Total Coliforms
Type of Pulping Coliforms confirmed as
Process per milliliter K. pneumoniae
Kraft
Sulfite (Ammonium)
Sulfite (Ammonium)
Defiberization
Total
6.8 x 103
1.6 x 104
3.9 x 104
3.2 x 103
Fecal
5.4 x 102
3.0 x 101
1.4 x 101
1.0 x 102
60 percent
70 percent
--
30 percent
11
-------
o
o
10
8
10
Indigenous
Coliforms^
/•
10'
10'
pneumoniae^
~.C. Positive
Fecal Conforms
0 10 20 30 40
Time (hours)
Figure 3. Growth Curve of K. pneumoniae in
Sterilized Wastewater from a Pulp
Mill
12
50
-------
ICT
CD
I io4
CD
Q.
O
QQ
10'
10'
Total Conforms
I
J K. pneumoniae
O)
Ld
Ld
Primary Secondary
Figure 4. Bar Graph of Total Coliforms and
K^ pneumoniae Densities of Various
Wastewater Sources of a Pulp Mill
13
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It was pointed out in the introduction that K. pneumoniae has an IMViC
formula of --++ and was confused with E. aerogenes (previously Aerobacter
aerogenes) because they can utilize many of the same substrates, such as
lactose, xylose, mannitol, etc. The characteristics which were previously
used to distinguish K^ pneumonia_e_ from E^ aerogenes were: lack of motility,
and production of a capsule. There were, however, some non-motile
strains of £_._ aerogenes that could be confused with K^ pneumoniae and
often the origin of the culture was the final determining factor in
identification. For instance, if isolated from water it was classified
as E. aerogenes. However, if the same culture was isolated from an
infected lung, it was classified as K. pneumoniae. The reorganization
of the classification and the inclusion of additional cultural tests by
Edwards and Ewing (1972) now makes it possible to distinguish these two
species with confidence.
Cultures representing different types of pulping operations were collected
and several chosen for a comprehensive taxonomic evaluation to determine
if they were K. pneumoniae. The classification scheme of Edwards and
Ewing (6) was followed.
The cultures all produced acetyl methyl carbinol in a buffered glucose
medium (Voges-Proskauer positive) and could utilize citrate as a sole
source of carbon. At the same time, all were negative for the methyl
red test and most were unable to produce indole from tryptophane.
According to the classification key mentioned above, this would place
the cultures in the Klebsielleae tribe. Further cultural tests showed
that these isolates were able to grow in the presence of KCN, produced
the enzyme urease, and did not produce H~S. This confirmed the placement
of the isolates in the Klebsielleae tribe.
This tribe contains the genera Enterobacter, Klebsiella, Serratia and
Pectobacterium. The genus Pectobacterium contains all of the Entero-
bacteriaceae that are pectolytic and do not grow above 35°C. The unknowns
14
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all grew well at 35-37°C, were pectolytic negative and therefore, belonged
to one of the other three genera within the tribe.
The unknown isolates were tested for their reactions in a variety of
substrates. At the same time, comparison cultures of known K. pneumoniae
obtained from the American Type Culture Collection (ATCC), Center for
Disease Control (CDC) and isolates from patients in hospitals were
included. Table 2 lists the reaction of K. pneumoniae. Comparing
various groups to the key reveals that each conforms to the expected
reaction for the classification of K. pneumoniae and where the substrate
reaction is variable (i.e., sucrose, or dulcitol) the groups also show
variability. This comparison of environmental isolates to the classifi-
cation key and to known K. pneumoniae cultures from different sources
shows that the classification of the coliforms isolated from pulp and
paper wastewaters as K. pneumoniae is valid.
The comparison of the pulp mill obtained K. pneumoniae and two species
of Enterobacter in the same substrates is shown in Table 3. The Entero-
bacter aerogenes and E. Cloacae were both obtained from the ATCC. The
three species share the same reaction to the various media employed in
their differentiation, however, K. pneumoniae can be distinguished from
the Enterobacter species by its inability to decarboxylate ornithine and
its lack of motility. All of the isolates obtained from the various
environmental sources are ornithine decarboxylate negative and are non-
motile. Therefore, they are placed in the genus Klebsiella.
The genus Klebsiella contains three species, K. pneumoniae, K. ozaenae
and K. rhinoschleromatis Table 4 shows the tests that are used to
distinguish K. pneumoniae from the other two species. K. pneumoniae is
methyl red negative, will grow on citrate and is universally able to
decarboxylate the amino acid lysine. A comparison of the media reactions
in Table 4 will indicate that the Klebsiella sp. isolated from the pulp
15
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TABLE 2
CULTURAL REACTIONS OF ENVIRONMENTAL AND REFERENCE CULTURES
OF K. PNEUMONIAE
Percent Positive Reaction
Test Key
Indole -(+)
Methyl Red
V. P.1 +
Citrate +
H2S
Urea +
KCN +
Motility
Gelatin
Lysine +
Arginine
Ornithine
Phenylalanine
Malonate +
Glucose +
2
Lactose d
Sucrose d
Mannitol +
Dulcitol d
Pulp Mill
0
0
100
100
0
99+
100
0
0
100
0
0
0
100
100
100
100
100
50
ATCC3
25
0
100
100
0
100
100
0
0
100
0
0
0
100
100
100
75
100
50
CDC3
0
0
100
100
0
100
100
0
0
100
0
0
0
100
100
100
100
100
0
Hospital3
0
0
100
100
0
66
100
0
0
100
0
0
0
100
100
100
100
100
33
1 Voges-Proskauer reaction for production of acetyl methyl carbinol
2 d = variable reaction, most positive, some negative.
3 number of isolates from each group
pulp mill 16 total, 7 calcium base sulfite pulp mill
4 kraft pulp processes
5 defiberization pulp process
ATCC - American Type Culture Collection - 3 total
CDC Center for Disease Control - 3 total
Hospital J. Matsen, University of Minnesota, School of Medicine
Minneapolis, Minnesota - 3 total
16
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TABLE 3
DIFFERENCIATION OF K. PNEUMONIAE AND ENTEROBACTER SPECIES
Test
Indole
M.R.
V.P.
Citrate
Motility
Urea
Lysine
Arginine
Ornithine
Glucose (Gas)
Lactose
Sucrose
Mannitol
Dulcitol
K. pneumoniae
"[^environment)
Enterobacter
Aerogenes Cloacae
(ATCC) (ATCC)
17
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TABLE 4
CULTURAL COMPARISON OF ENVIRONMENTAL KLEBSIELLA PNEUMONIAE
TO OTHER SPECIES OF KLEBSIELLA
Test
K^ pneumoniae
environment
Klebsiella
Ozaenae
Klebsiella
rhinoschleromatis
Indole
Methyl Red
V.P.
Citrate
Urease
KCN
Motility
Gelatin
Lysine
Arginine
Ornithine
Phenylalanine
Malonate
Glucose
Lactose
Sucrose
Manni tol
Dulcitol
+ or -
- or +
d
D
+
(+)* or -
+ or (+)
Approximately 6 percent of all K. pneumoniae isolated will be positive
for production of Indole
9
^d = various strains given different reactions
3+ or - most positive a few may be negative
4(+) delayed positive
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TABLE 5
SOURCE AND SEROLOGICAL TYPES OF ENVIRONMENTAL K. PNEUMONIAE
Isolate IMViC
Number Formula
004 --++
008 --++
012 --++
037 --++
045 --++
050 --++
084 — ++
093 — ++
094 --++
113 --++
116 --++
118 --++
131 --++
132 --++
Place of
Isolation
Pulp Mill
River
Pulp Mill
Pulp Mill
Pulp Mill
Pulp Mill
Pulp Mill
Pulp Mill
Pulp Mill
Pulp Mill
Defiberization
Defiberization
ATCC
ATCC
Capsule Serological
Type
7
35
8
No capsule
52
8
7
7
32
60
No capsule
3
64
3
19
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mills are different from those of ozaenae and rhinoschleromatis and
match the reaction expected of a culture of K. pneumoniae. The results
of the cultural comparison in various media have shown that the lactose
positive IMViC --++ cultures isolated from pulp and paper wastewater are
indistinguishable from other K. pneumoniae cultures. When these same
cultures were serologically typed with anti-Klebsiella pneumoniae capsule
antisera, it was found that they were serologically divided into several
types. This confirmed that those cultures that had the same cultural
reactions as defined for K. pneumoniae also possessed cap'sular antigens
that would react with the K. pneumoniae capsular antisera. The source
and serological types of some of the K. pneumoniae cultures obtained
from environmental sources are presented in Table 5. There is a spread
in serological types with most appearing as the low types and predominantly
7 and 8. Clinically, the low serological types are most often isolated
from infections.
The cultures obtained from the environment were found to be nutritionally
and serologically identical to other K. pneumoniae. Another phase of
the study was to determine the guanine and cytosine base composition of
the deoxyribonucleic acid (DNA) from these pulp mill isolates of K_^
pneumoniae.
DNA is the molecule of a cell containing the genetic information governing
all activities of the cell. The DNA molecule is a polymer of four
molecules known as nucleic acid bases. The DNA molecule is composed of
two strands of these nucleic acid bases held together in a double-
stranded-helical configuration by weak hydrogen bonds between the nucleic
acid bases. Figure 5 is a schematic drawing of a molecule of DNA to
show the base pairing and strand bonding. (It is known that the nucleic
acid base guanine always pairs with the base cytosine and adenine pairs
only with thymine. The number of hydrogen bonds between guanine and
cytosine is three whereas the number between adenine and thymine is only
two.)
20
-------
When a purified sample of DNA is heated, the double stranded molecule
will separate into single strands. As this happens, there is a hyper-
chromatic shift in the optical density taken at 260 nm. The amount of
energy required to separate guanine and cytosine is greater than that
required to separate adenine and thymine. Therefore, the richer the DNA
in guanine and cytosine the more energy (heat) is required to cause the
strands to separate. When the change in optical density of a solution
of DNA is plotted as a function of temperature, a sigmoid curve is
obtained as shown in Figure 6. The mid-point of this curve is the
temperature at which one-half of the DNA is in the single stranded
state. This mid-point value is the Tm value of the DNA. Because the Tm
value of DNA is dependent upon the G+C percent, it can be used to calculate
the average guanine plus cytosine percentage (G+C percent). (See formula,
Methods and Materials Section.)
The DNA of similar bacteria have a similar G+C percent. Therefore, the
DNA of all bacteria of a like species should share a similar G+C percent.
DNA was extracted and purified from a number of K. pneumoniae cultures
obtained from various environmental sources and culture collections and
%G+C base composition determined as described above. The data are shown
in Table 6. Several cultures were isolated or obtained from the same
source and values averaged. (The values appearing as +_ behind the
values are the statistical variation around the mean within a 95% confi-
dence limit). All fall within a +_0.5°C which is well within the error
of the method. The overall G+C percent of all groups, calculated from
the Tm value, is 56.4 percent +_ 1.4 percent and is comparable to the
values of 55 to 57 that have been reported for K. pneumoniae (Hill (10)
and Starr and Mandel (20)).
Regardless of the origin of the culture, the results show if, it meets
the nutritional criteria of the classification scheme, there is no
difference in the DNA base composition, thus the cultures are related
21
-------
DNA Structure
P04
P04
deoxyribose
31
Adenine Thymine
I >^~~
Hydrogen Bonds
eoxyribose
3'
P04
Guanine
deoxyribose
P04
Cytosine
i
deoxyribose
P04
Thymine Adenine
deoxyribose
P04
•
deoxyribose
P04
Figure 5. Diagrammatic Drawing of Molecular
Structure of Deoxyribonucleic Acid
P04
22
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o
CO
OJ
o
c
o
_Q
O
CO
JQ
o>
1.3
1.2
•§ «-0
o:
0.9
A_A ||8 =92.2
0—0 131 = 93.0
D—D 141 = 93.5
AAA
A o
oo
AA-A' /
DDd
n-nn-o
80 90
Temperature (°C)
100
Figure 6. Thermal Denaturation Curve of Purified
Deoxyribonucleic Acid.
23
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and are K. pneumonias. The Tin value and G+C percent of DNA and E^
aerogenes (Table 6) have been found to be 92.1C and 54.3 percent, respec-
tively. These values are lower than those reported here for K. pneumoniae
(Starr and Mandel, 20), however similar to those reported in the literature
(Hill, 10). The similarity of G+C percent shows the two species are
closely related, which is borne out by the similarity in their nutrition
and physiology. However, the difference in the Tm values also shows
that K. pneumoniae and E. aerogenes are separate and distinct species of
bacteria.
TABLE 6
Tm AND G+C% OF DNA FROM KLEBSIELLA PNEUMONIAE ISOLATES
FROM PULP MILLS
Source
Rivers
Pulp Mill A
Pulp Mill B
Pulp Mill C
ATCCa
Human
Average all groups K.
Ent. aerogenes
Average
Tm (C°)
92.7 + 0.2
92.3 +_ 0.5
93.1 + 0.5
92.4 + 0.2
93.1 +_ 0.4
92.9 + 0.5
pneumoniae 93.1 + 0.6
92.1 + 0.6
G+C%
57.7 + 0.7
57.3 + 0.5
56.5 + 1.0
54.9 +_ 0.5
56.6 + 0.9
56.2 + 1.0
56.4 + 1.4
54.3% +1.0
aAmerican Type Culture Collection, used as a reference
Culture obtained from hospital infection, used as a reference.
24
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The G+C base composition similarity between the environmental K. pneumoniae
and the other K. pneumoniae cultures revealed that all were related. A
more precise test of their relatedness is the determination of DNA
homology among the various cultures. Using the technique of Anderson
and Ordal (1), the percent of DNA duplex formation between the culture
obtained from an infection and cultures from the environment and ATCC
was tested.
The results (Table 7) revealed that the DNA from human K. pneumoniae
bound to the DNA of cultures from a pulp mill and ATCC to the same
degree as it bound to its homologous DNA. These data are further supported
by similar findings of Knittel and Seidler (13). They used an optical
method of DNA-DNA renaturation. The standard DNA was the ATCC type
strain, 13882 of K. pneumoniae. Seven pulp mill isolates of K. pneumoniae
were tested and 6 of the 7 showed from 81 to 90% of its DNA in common
with the type species. One of the 7 showed only 32% of its DNA was
similar to the type strain, pointing out that there may be some K^
pneumoniae that are phenotypical ly the same as the type strains of K^
pneumoniae, but are genetically unrelated. That is, they could be
biotypes of K. pneumoniae.
TABLE 7
RELATIVE REASSOCIATION OF HUMAN AND PULP
MILL KLEBSIELLA DEOXYRIBONUCLEIC ACID
Origin
Human
Human
Pulp Mill
Pulp Mill
Pulp Mill
Strain
Number
141a
131
116
006
94
%
Reassociation
100
100
100
92
41
areference deoxyribonucleic acid
25
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SECTION VI
DISCUSSION
The coliforms in the wastewater from pulp and paper mills are composed
of as much as 80 percent K. pneumoniae. There seems to be little relation-
ship between the type of pulping operation and the occurrence of K_._
pneumoniae or coliforms in general.
The coliforms grow during the treatment operation. If samples are taken
of the wastewater on consecutive days, there is a 10 and sometimes 100-
fold increase in coliforms by the time the waste has passed through the
secondary treatment system. When samples of secondary influent wastewater
are sterilized and inoculated with either fecal coliform or K. pneumoniae
there is growth of cells during the incubation period. At the same
time, the indigenous population of coliforms in an unsterilized sample
also shows an increase in numbers (Figure 2). The significance of these
observations is that there are sufficient sources of carbon and nitrogen
available in the wastewater to support the growth of coliform bacteria.
Therefore, it is possible that other intestinal borne pathogenic bacteria
could also grow.
Additional observation indicates that the major source of coliforms
appears to be from within the mill. The intake water to the mill contains
some coliforms, <100/100 while the wastewater effluent from the mill
into the primary treatment ponds contains several thousand per 100 ml of
sample. The exact location of these intra-mill sources are unknown.
As pointed out earlier, as much as 80 percent of the coliforms are j^
pneumoniae. This percentage remains fairly constant at all sampling
points at a particular mill. The reason for this is unclear at this
time and further research work is necessary.
26
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An extensive cultural comparison of several of the gram negative rod
shaped bacteria from these pulp mill wastewaters has confirmed they are
K. pneumoniae. This observation is further supported by the comparison
of the G+C percent of the DNA from these same cultures, and the same
relationship holds if the cultures are compared to known stock cultures
from clinical infections or ATCC cultures. Regardless of where the
culture was obtained it showed the same reactions in the various media
used for classification and conformed to the criteria outlined for the
classification of K. pneumoniae. The results of the DNA-DNA Duplex
experiments provide a firm basis that the K^ pneumoniae bacteria in the
pulp mill are the same as those found in human infections.
There is some doubt as to the sanitary significance of total coliform
counts on wastewater because the method counts a number of lactose
(19)
fermenting bacteria that may not indicate fecal originv . The presence
of K. pneumoniae in these waters presents a possible public health
hazard, especially when it represents such a high percentage of the
total coliform count. A review of the literature (12), has shown that
there has not been a case of K. pneumoniae infection that can be traced
to a waterborne source. The present study, however, confirmed that the
K. pneumoniae isolated from environmental sources is indistinguishable
from K. pneumoniae obtained from clinical infections, or from stock
cultures of known K. pneumoniae, and all meet the criteria for the
classification of K. pneumoniae.
The presence of a potential pathogenic bacterium in pulp and paper mill
wastewaters leads to the question if it is a hazard to public health.
Whereas this study has confirmed that K. pneumoniae found in this environment
is the same, both culturally and genetically, as those found in clinical
infections, the study does not answer the question of its pathogenicity
and public health hazard. It does say that the potential to cause
infection is present. When the total number of K. pneumoniae discharged
27
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1 5
per day is calculated, it is found to be 2.1 x 10 K. pneumoniae per
day. With this number of K. pneumoniae being discharged to a receiving
stream each day the risk to public health should be high even with an
organism of low pathogenic capability. At the same time the bacteriological
quality of the receiving stream is also being degraded below the point
of discharge. This would make it difficult to determine other sources
of coliform contaminations. When masked by this number of K. pneumoniae,
this potential public health hazard and degradation of bacteriological
water quality should be controlled at its source by disinfection or
other means.
28
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SECTION VII
REFERENCES
1. Anderson, R. S. and E. J. Ordal. 1972. Deoxyribonucleic Acid
Relationships Among Varine Vibrios. J. Bact. 109:696-706.
2. Braman, S. K., R. J. Eberhart, M. A. Asbury, and G. J. Hermann.
1973. Capsular Types of Klebsiella pneumoniae associated with
Bovine Mastitis. Amer. Vet. Med. Assoc. 162:190-1111.
3. Deley, J. and J. Schell. 1963. Deoxyribonucleic,Acid Composition
of Acetic Acid Bacteria. J. Gen. Microbiology 33:243-253.
4. Duncan, D. W. and W. E. Razzel. 1972. Klebsiella Biotypes Among
Coliforms Isolated from Forest Environments and Farm Produce, App.
Microbiology 24:933-938.
5. Edmondsen, E. B. and J. P. Sanford. 1972. The Klebsiella-
Enterobacter (Aerobacter)-Serratia group, A Clinical and Bacter-
iological Evaluation. Medicine 46:223.
6. Edwards, P- R. and W. H. Ewing. 1972. Identification of Entero-
bacteriaceae. Third Edition. Burgess Publishing Co., Minneapolis,
Minnesota. 362 pp.
7. Ewing, W. H. 1963. An Outline of Nomenclature for the Family
Enterobacteriaceae. Int. Bull. Bact. Nomen. Tas. 13:95-110.
8. Farman, J. 1963. Friedlander's Osteomyelitis (Report on three
cases). South African Med. J. 37:351.
9. Friedlander, C. 1882. Uber Die Schizomycetan bei der acuten
Fribrosen Pneumonia. Arch. Pat. Anat. Physio!. Klin. Med. 87:319.
29
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10. Hill, L. R. 1966. An Index to Deoxyribonucleic Acid Base Compo-
sitions of Bacterial Species. J. Gen. Microbiol. 44:419-437.
11. Hoogerheide, J. C. 1939. Studies on Capsule Formation. 1. The
Conditions under Which Klebsiella pneumoniae (Friedlander's
Bacterium) Forms Capsules. J. Bact. 38:367-371.
12. National Council for Air and Stream Improvement, Inc. 1972.
Klebsiella pneumoniae infection. A Review with Reference to the
Water-Borne Epidemiological Significance of _K. pneumoniae Presence
in the Natural Environment. Tech. Bull. 254. 260 Madison Ave.
New York, New York.
13. Knittel, M. D. and R. J. Seidler. 1974. Biochemical and Genetic
Comparison of Klebsiella pneumoniae from Pulp Mills and Clinical
Sources. American Society for Microbiology. Proceedings, p. 41.
14. Marmur, J. 1961. A Procedure for Isolation of Deoxyribonucleic
Acid from Microorganisms. J. Molec. Biol. 3:208-218.
15. Marmur, J. and P. Doty. 1962. Determination of Base Composition
of Deoxyribonucleic Acid from Its Thermal Denaturation Temperature.
J. Molec. Biol. 5:109-118.
16. Ordal, E. and P. Mayers. 1973. University of Washington, Department
of Microbiology, Seattle, Washington. Personal communication and
Unpublished data.
17. National Council for Air and Stream Improvement, Inc. 1971.
Recent Field Studies of Sanitary Water Quality in Receiving Waters.
Technical Bulletin No. 246. 260 Madison Avenue, New York, New
York. 70 pp.
18. Spicak, A. P., G. M. Eisenberg, W. Weiss and H. F. Flippin.
1957. Klebsiella Meningitis. American Journal of Medicine 22:865.
30
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19. American Public Health Association. 1971. Standard Methods
for Examination of Water and Wastewater, 13th Edition, 1015 18th
Street, Washington, D.C. pp. 678-685.
20. Starr, M. P. and M. Mandel. 1969. DNA Base Composition and
Taxonomy of Phytopathogenic and Other Enterobacteria. J. Gen.
Microbiol. 56:113-123.
21. Steinhouer, B. W., T. C. Eickhoff, J. W. Kislak and M. Finland.
1966. The Klebsiella-Enterobacter-Serratia Division, Clinical
and Edpidemiological Characteristics. Ann. Int. Med. 65:1180.
22. Thone, B. T. 1970. Klebsiella in Faeces. Lancet 2:1033.
31
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA~660/2-75-024
4. TITLE AND SUBTITLE
Taxonomy of Klebsiella pneumoniae Isolated from
Pulp/Paper Mill Wastewater
3. RECIPIENT'S ACCESSI ON-NO.
5. REPORT DATE
June 1975
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Martin D. Knittel
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Pacific NW Environmental Research Laboratory
200 SW 35th Street
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NO.
1BB037
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Industrial Waste Treatment Research Program
Pacific NW Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97330
13. TYPE OF REPORT AND PERIOD COVERED
Final, 9/71 to 6/73
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
K. pneumojriae isolated from pulp mills was compared to clinical isolates of
K_. pneumoniae. Cultures found to be identical in biochemical reaction in various
media and conformed to the recognized schemes of classification of K. pneumoniae.
Nucleic acid base composition comparison of these isolates showed that all exhibited
a G+C% base composition of 56%;+1.4%, and all cultures examined fell within this
range. A study of the heterologous binding capability between DNAs of these
cultures revealed that isolates from both the environment and pathogenic reference
had a base sequence from 80 to 100% in common. This confirms the earlier results
that pulp mill isolates are the same as clinical isolates.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Klebsiella pneumoniae
Pulp mill waste waters
Taxonomy of K^_ pneumoniae
DNA Base Ratio:Homology
Microbiology
B. DISTRIBUTION STATEMEN1
Distribution unlimited
19. SECURITY CLASS (This Report)
21. NO. OF PAGES
31
20. SECURITY CLASS (This page)
22. PRICE
EPA Form 2220-1 (9-73)
if U S GOVERNMENT PRINTING OFFICE I975—698-985 /IO REGION 10
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