fu@ 86S
Environmental Protection
Agency
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OH
Re&G&'cH EPA 600 2 79 O
July
Coliforms in
Drinking Water
Emanating from
Redwood
Reservoirs
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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 nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental 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.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-79-049
July 1979
COLIFORMS IN DRINKING WATER
EMANATING FROM REDWOOD RESERVOIRS
by
Ramon J. Seidler
Oregon State University
Corvallis, Oregon 97331
Grant No. R804456
Project Officer
Harry D. Nash
Drinking|Water Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U. S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the
health and welfare of the American people. Noxious air, foul water, and
spoiled land are tragic testimony to the deterioration of our natural
environment. The complexity of that environment and the interplay
between its components require a concentrated and integrated attack on
the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention,
treatment, and management of wastewater and solid and hazardous waste
pollutant discharges from municipal and community sources, for the
preservation and treatment of public drinking water supplies, and to
minimize the adverse economic, social, health, and aesthetic effects of
pollution. This publication is one of the products of that research; a
most vital communications link between the researcher and the user
community.
This project provided data to formulate recommendations on the
significance and control of 'Klebsiella pneumoniae and other coliform
organisms which are present in drinking water emanating from redwood
storage tanks.
Francis T. Mayo, Director
Municipal Environmental Research
Laboratory
iii
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ABSTRACT
Klebsiella pneumoniae and related biotypes were often encoutered in
finished drinking water supplies stored in redwood reservoirs. Biochemical
tests indicated no significant difference between isolates from drinking
water and those from clinical infections in man and animals. Approximately
17% of the Klebisella associated with redwood are fecal coliform positive
and are indistinguishable in this test from isolates of IS. coli. Multiple
resistance to antibiotics was occassionally found among Klebsiella associated
with redwood and these isolates could serve as donors in the transfer of
antibiotic resistance to _E. coli and to other Klebsiella. Comparative patho-
genicity experiments by LD5Q levels for mice illustrated that isolates of
redwood associated Klebsiella have mean LDcQ values indistinguishable from
known human and animal pathogenic isolates. The mean LDcn for JS. coli of
human clinical origin is not significantly different from Klebsiella. How-
ever, IS. coli isolates from drinking water were less virulent for mice than
either Klebsiella from any environment of IS. coli of clinical origins. Oral
ingestion by mice of Klebsiella in drinking water indicated a dramatic in-
crease in intestinal persistance when antibiotics were also administered.
Recovery of viable Klebsiella in mouse feces was detected when as little as
10 Klebsiella/lOOml was present in water used for drinking by these animals.
Based on the findings of a field survey of redwood reservoir water
supply systems, design modifications were made to control coliform contami-
nation. Several interrelated factors were found which affect water quality.
Coliforms are not found when an adequate (0.3-0.4 ppm) free chlorine
residual is maintained in the reservoir along with short retention times
(less than 7 days) and a design which promotes adequate water circulation
in the reservoir. The major factor influencing water quality is the
chlorine demand of the wooden reservoir. It is clearly not adequate to
supply incoming reservoir water with 0.3 ppm chlorine unless retention times
are very short (less than 1 day) and the reservoir has been in use for over
1 year. Under no circumstances should drinking water stored in redwood be
chlorinated only after leaving the reservoir. Two years of studies with
an experimental 3,785 liter (1,000 gal) redwood reservoir demonstrated that
colifonn-free water was consistantly obtained when these design modifications
were followed.
It was discovered that Klebsielleae are associated with the redwood
lumber used to construct the reservoirs. These coliforms are found within
the wood structure in numbers ranging from 10 to 10° per ml of expressed
liquid. A variety of laboratory experiments demonstrated that the water
extractable cyclitols comprising 4.5% redwood dry weight, provide the
nutritional basis which uniquely support the growth of Klebsielleae in the
iv
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redwood environment. These coliforms can multiply within redwood re-
servoir water which leaches out cyclitols from the staves.
This report was submitted in fulfillment of Grant No. R804456 by
Oregon State University under the sponsorship of the U. S. Environmental
Protection Agency. This report covers the period July 20, 1976, to July
19, 1978, and work was completed as of September 1, 1978.
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CONTENTS
Foreword ill
Abstract iv
Figures viii
Tables x
Acknowledgments xi
1. Introduction 1
2. Materials and Methods 4
Bacterial Cultures Assayed for Antibiotic Resistance..4
Comparative Pathogenicity Experiments 5
Fecal Coliform Response 6
Klebsiella Persistance in the Mouse Intestinal Tract..?
Experiments on a 3,785 Liter (1,000 Gal) Experimental
Redwood Reservoir 9
Colif orm Bacteria Within Wood 11
Nutritional Experiments 12
3 . Results and Discussion 14
Biochemical Properties of the Klebsiella 14
Antibiotic Resistance of the Klebsiella 18
Comparative Pathogenicity of the Klebsiella 25
Klebsiella Persistance in Mouse Intestinal Tract Following
Ingestion of Contaminated Drinking Water 28
Association of Klebsiella with Redwood Used to Construct
Reservoirs 40
Nutritional Basis for Klebsielleae Selection in Redwood
Water Storage Reservoirs 49
References 68
Appendix 72
vii
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FIGURES
Number Page
1 Typical redwood reservoir designed to store finished
drinking water 2
2 Redwood reservoir interior illustrating slime accumulation on
some staves 3
3 Interior ladder and concrete floor of reservoir illustrating
slime which has slid off of ladder 3
^ kDijQ frequency distribution in mice for Klebsiella isolated
from various environments 27
5 Percentage of mice excreting Klebsiella after ingestion of
contaminated drinking water 31
6 Mean log of Klebsiella per gram of mouse feces after ingestion
of contaminated drinking water 32
7 Percentage of mice excreting various species/biotype of
Klebsiella after ingestion of contaminated drinking water 33
8 Mean log of Klebsiella species/biotype per gram mouse feces
after ingestion of contaminated drinking water 34
9 Diagram of experimental 3,785/liter (1,000 gal) reservoir used
to test design modifications in redwood water storage
reservo.irs 35
10 Scanning electron micrograph (SEM) of interior of redwood wide
ring count sapwood (200X) 47
11 SEM of redwood wide ring count sapwood (1,OOOX) 47
12 SEM of a tracheal tube illustrating morphological types of bacteria
present (5 ,OOOX) 48
13 Diagram of the 3 cyclitols present in aqueous redwood extracts.. 50
viii
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FIGURES (continued)
Number
Page
14 Growth curves of 4 Klebseilla isolates in a defined basal salts
medium with 0.2% pinitol as sole source of carbon and
energy 56
15 Growth curves of 4 Klebsiella isolates in a defined basal salts
medium with 0.2% pinitol as sole source of carbon and
energy 56
16 Gas chromatograph (GC) profile of an aqueous redwood sawdust
extract (4g/liter water) illustrating the 3 cyclitols present
in redwood 61
17 GC profile of an aqueous redwood sawdust extract (1.67 g/liter
water) used to measure in situ utilization of cyclitols 61
18 Cyclitols present in redwood tank water on 8 day retention
period 64
19 Cyclitols present in redwood tank water held stagnant for
1 month 65
20 Cyclitols present in redwood tank water held stagnant for
2 months 65
21 Cyclitols remaining in 1.67g/liter aqueous redwood sawdust
extract after 4 days of incubation of Klebsiella MH24
(human disease origin) at 35°C 66
22 Cyclitols remaining in 1.67g/liter aqueous redwood sawdust
extract after 4 days of incubation of Klebsiella E44.5-1
(redwood sawdust origin) at 35°C 66
23 Cyclitols remaining in 1.67g/liter aqueous redwood sawdust
extract after 4 days of incubation of Klebsiella PSB-49-1
(bovine mastitis agent) at 35°C 66
ix
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TABLES
Number Page
1 Biochemical Properties of Redwood Associated Klebsiella 15
2 MPN and MF FC-Positive Klebsiella Cultures 17
3 FC Response of Environmental Klebsiella Isolates 18
4 Culture pH in m-FC Broth Test Incubated at 44.5°C Versus FC
Response 19
5 Antibiotic Resistance of Klebsiella Isolates from Various
Habitats 20
6 Drug Resistance Patterns of Multiple-Resistant Klebsiella
Isolates 21
7 Conjugal Transfer Frequencies of Drug Resistance by Selected
Klebsiella Isolates 23
8 LD50 of Klebsiella and E_. coli in Mice 26
9 LDtjQ °f ]£• pneumoniae and K. oxytoca Determined by Intraperitoneal
Injection in Mice 29
10 LDIJJQ of Environmental Klebsiella in Mice 30
11 Physical and Microbiological Parameters in the 3,785 Liter
Experimental Redwood Tank 36
12 Viable Counts at 35°C of Klebsiella Incubated in Redwood Tank
Water 39
13 Bacterial Densities in Liquid from Redwood 42
14 Bacterial Densities in Liquid from Non-Redwood Wood 44
15 Coliforms Recovered from Contact Impressions of Freshly Exposed
Wood Surfaces 45
16 Coliforms Recovered from Contact Impressions of Serial Cuts
through Redwood 46
17 Utilization of Inositol and Pinitol by Enterobacteriaceae and
Pseudomonas. ... 52
18 Utilization of Sequoyitol by Enterobacteriaceae and
Pseudomonas 54
19 Growth Response (O.D.60Q) w±th 0.2% Inositol 55
20 Growth Response in Aqueous Extracts of Redwood Sawdust in Presence
of Indigenous Flora 57
21 Growth Response in Sterile Aqueous Extracts of Redwood Sawdust.. 59
22 Growth Response in Various Concentrations of Aqueous Extracts of
Redwood Sawdust 63
23 Cyclitol Concentrations in Redwood Extracts and Experimental
Redwood Tanks 65
24 Utilization of Cyclitols in Redwood Extract by Klebsielleae 67
25 Comparison of standard plate counts with different buffers 68
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ACKNOWLEDGMENTS
This work was supported in part by Grant //R804456 from the U. S.
Environmental Protection Agency Drinking Water Research Division, Micro-
biological Treatment Branch and in part by National Tank and Pipe Co.,
Portland, Oregon, the Water Resources Research Institute at Oregon State
University and by Grant #BMS 13799 from the National Science Foundation.
Most of the experimental work was done by the following individuals:
Jan E. Morrow, Susan T. Bagley, Henry W. Talbot, Jr., Marie Carter, Phillip
Kroner, and Martin Smith. We also acknowledge support from the State of
Oregon for supplying funds for teaching assistantships for some of the
above individuals and salary support for the principal investigator.
xi
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SECTION 1
INTRODUCTION
The quality of drinking water in the United States has received much
attention in the last several years due to the research efforts facilitated
by passage of the Safe Drinking Water Act (P.L. 93-523). This Act has also
resulted in an increase of the general awareness of the public to health
problems associated with the consumption of inadequately treated or faulty
finished drinking water supplies.
Adverse publicity in public media has brought drinking water problems
into regional focus in the Pacific Northwest due to recent waterborne disease
outbreaks at a ski resort in Montana, in a community in Washington State, and
a public recreation area in Oregon. In addition to the personal discomforts
associated with gastroenteritis, the economic impact of law suits, loss of
tourism and loss of work time cost untold millions of dollars. Such water-
borne disease outbreaks, are of course, not unique to the Northwest since
over 54,000 individuals were reported ill from other outbreaks in the United
States over the period of 1961-1973 (1). Some E.P.A. officials believe that
90% of the actual outbreaks go unreported and that numerous other illnesses
are associated with the consumption of contaminated water.
The major goal of the S.D.W.A. is to assure that the public has
consistantly safe drinking water. To achieve this goal, there must be
routine and adequate water quality monitoring. In the summer of 1974 a
coliform contamination problem occurred in a rural drinking water supply
during the routine monitoring efforts of the State of Oregon Public Health
Department. Upon investigation, the origin of contamination was traced to
a 189,250 liter (50,000 gal) water storage reservoir constructed of redwood
(Fig. 1). The coliforms were isolated and identified as Klebsiella
pneumoniae and Enterobacter species. No Escherichia coli were found.
Further investigations indicated there was no obvious evidence of water
contamination from insects, rodents, or birds, and the ground water sources
(3 wells) were all pumping coliform-free water. Field surveys of similar
water systems demonstrated that this coliform contamination in finished
drinking water stored in redwood reservoirs was not an isolated situation.
Coliforms were isolated from 9 of 10 private drinking water systems and from
11 of 23 water systems in state and federal parks, and in various other
communities. It was not uncommon for total coliform counts to range from
5-200/100 ml of drinking water as tested by membrane filtration techniques.
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Figure 1. 189,250 liter (50,000 gal) redwood reservoir used to store finished
drinking water.
Inspection of the interiors of several redwood reservoirs illustrated that on
some occasions some staves were coated with a layer of slime (Figs. 2 and
3). The slime can be 0.5 cm in thickness and contains bacteria, including
coliforms, embedded in mycelial states of fungal growth.
Redwood water reservoirs are widely distributed throughout the Western
United States, and are also used in Canada, South America, and the Pacific
Islands. They are used to store the finished drinking water needs for recre-
ation areas, mobile home parks, motels, communities, and cities in these
locations.
The purposes of this study were to determine the origin(s) and cause(s)
of coliform contamination and attempt design modifications so that current as
well as newly constructed reservoirs would serve potable water. The study was
divided into three general phases. One phase involved an extensive charac-
terization of the coliform Klebsiella, comparing properties of isolates from
drinking water supplies with those that are derived from clinical disease in
man and animals. The second phase involved the testing of design modifica-
tions in a 3,785 liter (1,000 gal) experimental reservoir, constructed for
research purposes, to determine conditions which lead to coliform contamina-
tion and hopefully to their elimination. In the final phase, studies led to
the demonstration of possible coliform origins and to an elucidation of the
nutritional basis which facilitates the selective growth of Klebsiella and
Enterobacter, the coliforms most frequently encountered in these drinking
water supplies.
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Figure 2 (top). Reservoir interior illustrating slime on some staves.
Figure 3 (bottom). Slime from interior ladder on concrete floor or reservoir.
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SECTION 2
MATERIALS AND METHODS
BACTERIAL CULTURES ASSAYED FOR ANTIBIOTIC RESISTANCE
Klebsiella cultures received or isolated for this study were ornithine
decarboxylase negative, lysine decarboxylase positive, lactose positive,
citrate positive, non-motile gram negative fermentative rods. Testing of
biochemical reactions and identification procedures used are those of Edwards
and Ewing (2). Cultures were isolated by this laboratory from various
finished drinking water sources, vegetables, redwood, sawdust, woodchips and
potato processing effluents. Others were received from the Pennsylvania State
University, University of Missouri, Michigan State University, University of
Guelph Ontario, Canada, University of Texas (Houston), University of Oregon
School of Medicine, Medical University of South Carolina, St. Lukes Hospital
(Duluth, Minn.), Mercy Hospital (Pittsburgh, Pa.), B.C. Research (Vancouver,
Can.), the Environmental Protection Agency at Kingston, Rhode Island, and
the American Type Culture Collection.
Those cultures listed as Bovine Area were isolated from a variety
of areas occupied by cows, including teat ends. It is entirely possible that
those isolates from bedding and stall water may be recent contaminants from
animals that were frequently fed antibiotics both for prophylaxis and
treatment.
_E. coli strain W3110 (Nal , F ) was obtained from the laboratory of Dr.
Lyle R. Brown (Oregon State University).
Antibiotic Sensitivity Testing
Sensitivities were determined by the high concentration disk diffusion
technique as described by Bauer, et al. (3). The following antibiotic disks
were used: penicillin G (10 U, Pen), streptomycin (10 meg, Str), chlor-
amphenicol (30 meg, Chi), nitrofuradantoin (300 meg, Nitro), kanamycin (30
meg, Kn), tetracycline (30 meg, Tc), neomycin (30 mgc, Nm), ampicillin (10
meg, Am), erythromycin (15 meg, Ery) , novobiocin (30 meg, Nov), nalidixic
acid (30 meg, Nal), gentamicin (10 meg, Gent), polymyxin B (300 U, Poly),
furaltadone [30 meg, (Valsyn, Val)] and nitrofurazone (100 meg, Furacin).
Furaltadone discs were obtained from BBL, nitrofurazone from Pfizer and
the remainder were purchased from Difco. Bacto Mueller-Hinton (Difco)
plates were inoculated and after 18 to 20 h incubation at 37°C, zone
diameters were measured. Isolates were assigned as resistant or not
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resistant according to the standards in the Manual of_ Clinical Micro-
biology (4).
Transfer of Multiple Resistance
Conjugal transfer of multiple antibiotic resistance was attempted for
31 Klebsiella isolates using E_. coli strain W3110 as recipient and for 5 iso-
lates using Klebsiella as recipients. All chosen presumptive donors were
resistant to two or more antibiotics. Since all Klebsiella strains are
resistant to penicillin in standard doses, this antibiotic was excluded from
consideration as were novobiocin and erythromycin because they are used pri-
marily in the treatment of infections caused by gram positive organisms.
Spontaneous nalidixic acid resistant mutants (40 ug/ml) of Klebsiella
isolates with low minimum inhibitory concentrations (MICs) for Str, Tc, Chi,
Val, Nitro, Kn and/or Nm were selected for use as presumptive recipients.
MICs for antibiotics were determined for donors on MacConkey agar where
resistance to two or more antibiotics was detected. The recipient organisms
were likewise tested and concentrations between the MICs of donor and re-
cipient were used in the selective media. Preparation of donor and re-
cipient cell suspensions followed standard procedures (5). After incubation,
appropriate dilutions of the mixtures were then plated on selective media
containing nalidixic acid and combinations of antibiotics to which the
donors were resistant. Aliquots were also plated on MacConkey agar
supplemented with nalidixic acid only to determine the total number of
recipients. Frequency of transfer of a given resistance pattern was
taken as total number of transconjugants/total number of recipients.
COMPARATIVE PATHOGENICITY EXPERIMENTS
Bacterial Cultures
The origins and identification of most Klebsiella isolates have
been previously described (6). Klebsiella isolates were of the following
origins: 19 mastitis, 18 human clinical, 18 bovine-related areas and 42
from other environmental sources. In addition, IS. coli were obtained
from drinking water (6 isolates,) and from human infections (6 isolates) . Ad-
ditional bovine mastitis and other bovine-related Klebsiella (6) were ob-
tained from the University of Guelph, Guelph, Ontario, Canada. Bovine-
related isolates were divided into two groups: bovine-area (from unused and
used bedding and drinking water in stall areas) and bovine-associated (from
mouth, udder, and anal areas). Klebsiella isolates were divided into two
species, K. pneumoniae and K. oxytoca, according to the scheme of Naemura
and Seidler (7).
LD Assay
Test cultures were shaker-grown for 18 to 20 hr at 37 °C in nutrient
broth (Difco, Detroit, Mich.) with 1% added glucose. Three-fold dilutions
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for inoculation were made in steam-sterilized 5% hog gastric mucin
(Sigma, St. Louis, Mo.). Five 17-20 gm male Swiss-Webster mice were
injected i.p. with 1.0 ml of each dilution; five to six dilutions were
injected per culture. Virulence was determined by the numbers of mice
dying within 48 h after inoculation and with recoverable test organisms
from heart and liver sections at autopsy.
LDc/N Computation
Cell numbers for each inoculated dilution were transformed into
natural log values. Each LD^Q was determined using a maximum likelihood
computer program. This type of logit analysis is similar in principle
to the use of probit transformations (8).
Statistical Tests
Geometric mean LD^Q values were compared using F tests and one- and two-
tailed t tests, at an a = .05 significance level (9). All calculations were
made using natural log LDcr, values.
FECAL COLIFORM RESPONSE
Determination of Fecal Coliform (FC) Response
For membrane filtration (MF) FC tests, 24-hr shake cultures grown at
35° C were diluted to approximately 10 cells/ml. Duplicate 3-ml volumes were
filtered using 0.45 ym-pore-size membrane filters (GN-6, Gelman, Ann Arbor,
Mich.) with cultivation on m-FC medium (Difco, Detroit, Mich.) for 24 hr at
44.5 (10). Colony number and color were recorded on each plate after
incubation.
All isolates were verified by subculturing a blue colony to phenol red
lactose broth (Difco) for 24 hr at 35°C with subsequent transfer to EC
broth at 44.5°C. Tubes producing gas were considered confirmed FC. Isolates
having blue-green or light blue-gray colonies were not considered MF FC posi-
tive. These colony types had a low percent confirmation both in this and
other reported surveys (11) .
For MPN FC tests, cultures were first grown in phenol red lactose broth
at 35°C. After 24 hr, cultures were transferred to EC broth and incubated
for 24 h at 44.5°C. Tubes with growth and gas production were considered FC
positive.
pH of Cultures in m-FC Broth at 44.5°C
Selected isolates of 1C. pneumoniae and E^. coli were incubated in tubes
containing 7 ml of m-FC broth (Difco) containing 0.01% rosolic acid (Difco)
for 24 hr at 44.5°C. Direct pH measurements of the broth were made with a
combination triple-purpose electrode (no. 476022, Corning Glassware, Corning,
N.Y.). Broth color was recorded immediately upon removal from the incubator.
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Statistical Testing of Results
Results from FC tests for K.. pneumoniae and E_. coll isolates from
various sources were analyzed for statistical differences using chi-square
2x2 contingency tables (9). Differences were considered significant if
P values were less than 0.05.
KLEBSIELLA PERSISTANCE IN THE MOUSE INTESTINAL TRACT
Test Organisms
Six Klebsiella cultures were used throughout these experiments, repre-
senting pathogenic and environmental origins (three each). The K. oxytoca,
FC-positive (FC+) K. pneumoniae, and the FC-negative (FC-) 1C. pneumoniae used
were represented by two cultures each. The habitat of origin of the Klebsi-
ella cultures were: two human clinical and one from bovine mastitis, fresh
vegetable, finished drinking water, and redwood sawdust chips. The isolation
and biochemical characteristics of these cultures have been described (6).
All cultures were made resistant to 40 yg/ml nalidixic Acid (Nal) and
500 yg/ml streptomycin (Str) (both obtained from Sigma, St. Louis, Mo.) to
facilitate culture recovery from fresh mouse feces. Antibiotic (AB) resis-
tance was chromosomal and transfer via plasmids to other intestinal bacteria
was not detected. Cultures were stored on nutrient agar (Difco, Detroit,
Mich.) slants with Nal-Str until used in experiments.
Ingestion of Klebsiella in Drinking Water
Cultures were shaker-grown in brain heart infusion (BHI) broth (Difco)
with Nal-Str for 18-20 hr at 37°C and diluted to 105, 103} IQ!, or 10"1
cells/ml in sterile distilled water. 100 ml of each dilution was given to
each of eight mice as a sole source of drinking water (DW). This DW was
changed every 24 hr. Culture survival or regrowth was determined by plate
counts on Nal-Str-containing MacConkey agar (Difco). Culture regrowth in
sterile DW was found to be limited, i.e., no more than a half log increase
over original numbers within 24 hr. Mice were observed to ingest a maximum
of four ml of water in a 24 hr period. Over this period, mice given an
initial level of 10^ cells/ml DW, for example, would be ingesting three to
eight cells of Nal-Str resistant Klebsiella. After six days of continuous
exposure to Klebsiella in DW, non-sterile tap water was placed into cleaned
water bottles for the remainder for the experiments.
Test animals were individually housed in sterile, plastic cages lined
with kiln-dried wood shavings. Food pellets were supplied ad libitum.
Fecal pellets were collected immediately prior to transfering mice to fresh,
sterile cages.
Culture Recovery from Feces
One-tenth g of fresh fecal pellets were collected from each test mouse,
using flame-sterilized forceps, on days 2, 4, 6, 7, 14, 21, and 28 after ex-
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periments were begun. Fecal pellets of mice ingesting lO"1 Klebsiella/ml
DW were collected every day for up to nine days. Pellets were emulsified in
sterile tubes containing 0.9 ml of BHI broth with Nal-Str, providing a 10"1
dilution of the fecal material. Subsequent dilutions were made in the same
broth using either a 0.01 ml capacity inoculating loop or a 0.1 ml pipet.
After 18 to 20 hr incubation at 37°C, dilution tubes showing growth were
streaked onto Nal-Str-containing MacConkey agar. Plates were incubated at
37°C for 22 to 24 hr. Dilutions producing red or red-centered colonies on
this selective agar were considered presumptive for presence of the
orally-ingested Klebsiella. Standard biochemical tests confirmed the identi-
fication (2). No organisms other than the Nal-Str resistant Klebsiella
grew on this medium.
The number of Nal-Str resistant Klebsiella/g of feces was calculated as
one over the highest dilution having colonies on the MacConkey agar plates.
The detection limit for this method was one Nal-Str resistant cell/0.1 g
feces. This was experimentally determined by seeding mouse feces with known
amounts of Klebsiella.
Effect of Oral Antibiotics (AB) on Mice Ingesting Klebsiella
In these experiments, mouse DW contained 40 yg/ml Nal and 500 yg/ml Str
at all times. This AB water was supplied to the mice 48 hr prior to addition
of Klebsiella to the same drinking water. Mice were then given fresh Nal-Str
with Klebsiella water every 24 hr. After six days, cultures were removed
from DW. Fresh AB water alone was changed every 48 hr for the remainder of
the experiments. Partial decontamination of the intestinal tract was achieved
by this prior oral AB exposure. Total coliforms were reduced by 99% (10^ to
l()Vg feces using MacConkey agar) and facultatively anaerobic bacteria by
99.9% [10* to 10°/g feces using standard plate count agar (Difco) with over-
lay] . Klebsiella densities ranged from 10 to 10^ cells/ml drinking water
in these experiments. Techniques for fecal pellet collection and culture
enumeration were the same as described above.
Effect of Cyclophosphamide (CP) on Mice Ingesting Klebsiella
Test mice were injected with 200 mg/kg CP (Cytoxan, Meade Johnson Lab.,
Evansville, Ind.) three days after Klebsiella were first added to DW. Mice
ingesting 101 to 105 Klebsiella/ml DW without AB (control mice) and 10 to
10^ Klebsiella/ml DW with AB were tested in these experiments. Techniques
for fecal pellet collection and culture enumeration were the same as de-
scribed above.
The effects of comparable doses of CP on white blood cell counts and
other immune-response system functions have been previously reported (13).
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EXPERIMENTS ON A 3,785 LITER (1,000 GAL) EXPERIMENTAL REDWOOD RESERVOIR
A 3,785 liter (1,000 gal) redwood tank was constructed for use in this
investigation by National Tank and Pipe Co., Portland, Ore. It measured
1.80 m (71 in.) in height with an interior radius of .84 m (33 in.).
The 42 staves of the tank each measured 6.51 cm (2 1/16 in.) by 13.65 cm
(5 3/8 in.). A hinged plywood lid covered the top. Incoming water,
from municipal city lines, entered the tank at the top and was sprayed
evenly to all sides. A float system controlled the level of the water
and drainage was through a valve at the bottom of the tank (Fig. 9).
Various retention times for tank water were achieved with a motor timer
which controlled a solenoid valve. The timer monitored both the length of
time between drainings (Timer #1) and the length of time the drain was open
(Timer #2).
Chlorine concentrations in the incoming water were regulated by a meter-
ing pump on 303 liter (80 gal.) holding tank. The stock chlorine solution was
prepared by adding 3.79 liters (1 gal.) of commercial bleach to 53 liters
(14 gal.) of tap water (1:15 dilution). A dial on the pump was adjusted to
provide appropriate chlorine concentrations for water entering the tank.
Retention Times
It was found that water drained from the tank at the rate of 11.7 liters
(3.1 gal.) per minute. The timers were adjusted to provide retention times
based on this drain rate.
Preliminary Adjustments and Decontamination of Tank Interior
The manufacturer's recommendations were followed. In order to leach
tannins from the wood, which affect odor, color, and taste of the water,
2.27 kg (5 Ib.) of soda ash was added to a full tank and held for 24 hr.
After drainage, enough stock chlorine solution was added to bring the chlorine
concentration in the full tank to 200 ppm. Granular dry chlorine was used in
preparing the stock solution. The tank was put into service in October 1975;
however, in November 1975 it was necessary to perform a similar soda ash
treatment of 7 days duration. This second treatment greatly improved the
taste, odor, and color of the water.
Preliminary studies with the tank at 20 percent capacity were then
carried out between the first month of operation and the third month to
determine the amount of chlorine necessary to achieve an adequate free
chlorine residual (0.2 to 0.4 ppm) in the drain (outlet) water. The effect
of lengthened retention time on residual chlorine was also examined. During
this period, bacteriological examinations of the water were performed regu-
larly. No coliforms were detected during this period; total bacterial counts
averaged 350 per ml and never exceeded 2000 per ml.
During the third month, the experimental tank was filled to capacity and
set on a 1 day retention time. With this schedule, approximately one-sixth
of the tank water was drained every 4 hrs. Capacity was maintained at all
-------
times by a float system. Retention times were later increased to two, four,
eight and finally twelve days. During the sixteenth month, the chlorine
amendments were terminated in order to observe the effects of lowered
chlorine residuals on the bacteriological quality of tank water; water enter-
ing the tank then carried a free residual of 0.5 ppm, due to the chlorination
of the municipal water.
Daily observations of physical parameters of tank water
Free chlorine residuals and total chlorine residuals in the drain
water and inlet water were determined by the N,N-diethyl-para-phenylene-
diamine (DPD) method. Optical density readings and pH were also determined
for water samples.
Bacteriological examination of tank water and staves
Water leaving the tank via the drain at the bottom was periodically
examined for total coliforms and total bacterial counts, using standard
procedures (10). Total coliforms were enumerated by the membrane filtration
technique on m-Endo agar LES. Sodium thiosulfate was added to all
samples to remove any free chlorine in the water. Varying amounts of
drain water (usually 100 ml or 1000 ml) were tested. On eight occasions,
the water level was lowered and the interior surfaces of the tank were
scraped with sterile glass microscope slides. Surface material was
dispersed and tested for total coliforms by the spread plate technique
on m-Endo agar LES and Endo agar, and with phenol red lactose broth. In
addition, some non-coliform bacteria were isolated for identification.
Identification of Isolates from Staves
Media preparation and procedures used for the identification of coliform
organisms were those recommended by Edwards and Ewing (2). Final identifi-
cations were made using the API 20E System. Isolates obtained from plate
count agar were identified using 9-digit profiles of the API 20E System, or
by procedures outlined in the eighth edition of Sergey's Manual of Determi-
native Bacteriology (13,14).
Nutrient Potential of Redwood Tank Water
Samples of water from the 3,785 liter (1,000 gal) experimental tank were
collected at various times during the investigation and stored at -17°C. The
samples were thawed and filter sterilized using membrane filters. They were
inoculated with low cell densities (less than 100 cells/ml) of an 18 hr
culture of Klebsiella pneumoniae and incubated with shaking at 35°C. Plate
counts were performed at 48 hr and 96 hr. All glassware was acid washed to
remove any extraneous nutrient sources. Sodium thiosulfate was used to re-
move chlorine when necessary.
10
-------
COLIFORM BACTERIA WITHIN WOOD
Wood Samples
Samples of fresh and aged redwood were obtained from National Tank and
Pipe Company, Portland, Ore., and Simpson Lumber Company, Arcata, Cal.
Samples of other wood were obtained from mills in Oregon.
Liquid Extraction from Wood
All types of wood had outer surfaces removed using a power circular saw.
The sawblade and cutting table were surface sterilized with 95% ethanol.
Swabs of these surfaces were routinely streaked onto m-Endo agar LES (Difco,
Detroit, Mich.) to ensure absence of external contamination. The wood
samples were then cut into blocks approximately 131 cm3 (8 in3) in size.
Moisture content of the wood was determined by measuring sample weight both
before and after drying at 60°C for 7 days.
Liquid was extracted from the wood blocks using a Carver Laboratory
hydraulic press (Fred S. Carver, Inc., Summit, N.J.) operated at up to
844 kg/cm2 (12,000 psi). Blocks were placed on sterile aluminum foil
with upturned edges for liquid collection. During pressing, the blocks
were flooded with 2 ml of sterile 1:10 w/v polyvinyl pyrrolidone (PVP) to
neutralize any released phenolic compounds. Expressed liquid was transferred
to sterile tubes for bacterial testing.
Bacterial Enumeration
Dilutions of liquid expressed from wood were made in sterile, 0.01 M Tris
(pH 7.5). One-tenth ml of appropriate 10-fold dilutions were spread onto
media for viable cell counts. Total bacterial counts were made on nutrient
agar (Difco), with incubation at 30°C for 48 hr. Standard plate count pro-
cedures were not used since total bacterial counts are lowered by brief
exposure to the elevated temperature of molten agar (41). Also, the possible
loss of a selected portion of the bacterial population was of concern.
The procedures and diluent used in determining heterotrophic counts from
wood were at least as sensitive if not better than those of Standard
Methods (see Appendix). Total coliform counts were made using m-Endo
agar LES, with incubation at 35°C for 24 hr. In one experiment with
redwood collected directly from a California mill, both total bacteria
and total coliform counts were made at 37°C to enhance selection of
Klebsiella (15). Using non-redwood samples, most probable number (MPN)
techniques were also used to obtain confirmed total coliform counts
(10). Fecal coliform counts were made using m-FC agar (Difco), with
incubation at 44.5°C for 24 hr in a water jacketed incubator (NAPCO
#3221).
Cut Surface Contact Impressions
Outside surfaces of wood samples were removed as described above and im-
pressions were made by bringing a freshly exposed surface into contact with
m-Endo agar LES. With two samples of fresh redwood, six to nine successive
11
-------
.6 cm (1/4 in.) sections were cut through each block, after outer surface re-
moval. An impression of each successively exposed surface was made on m-Endo
agar LES. All plates were examined for growth and green-sheened colonies
after 24 and 48 hr incubation at 35°C. Total coliform counts were based only
on the number of typical colonies appearing after 24 hr.
Coliform Identification
Confirmed coliform isolates from all wood experiments were tested bio-
chemically for identification. A green-sheened colony from m-Endo agar LES
was considered to be a confirmed coliform if it produced gas in lactose broth,
within 24 hrs at 35°C. Presumptive speciation was made using Triple Sugar
Iron Agar, urea, and Simmons' citrate agars, lysine and ornithine decarboxy-
lase broths, and motility tests. Further identification and characterization
was made using standard biochemical tests (2) and the API 20E system
(Analytab Products, Inc., Plainview, NY). Klebsiella isolates were
speciated according to Naemura et^ al_. (L.G. Naemura, S.T. Bagley, and R.J.
Seidler. Abstr. Annu. Meet. Am. Soc. Microbiol. 1977. I 149, p. 179) and
(7).
Electron Photomicrographs of Wood
3
Samples 0.2 to 0.5 cm were cut from redwood blocks, processed through
solutions of ethanol and trichlorotrifluroethane in increasing concentra-
tions, and dried by the critical point method of Cohen, et al. (16). After
drying, the cubes were split parallel to the longitudinal axis of the wood
fibers to expose fresh internal surfaces. These pieces were fastened to
aluminum planchets and coated with approximately 200 A° of 60:40 gold/pal-
ladium. Samples were observed at 15 Kv in an ISI Mini-SEM MSM-2 scanning
electron microscope and images recorded on Type 55 Polaroid film.
NUTRITIONAL EXPERIMENTS
Aqueous Redwood Extracts
Aqueous extracts of redwood sawdust used in these experiments were pre-
pared in the following manner: 1.67g or 4g of redwood heartwood sawdust was
placed into 1 liter of sterile double distilled water and allowed to
stand at room temperature for 4 days. At this time, sterile 0.1% sodium
hydroxide was used to neutralize the extract. The extract was sterilized
by membrane filtration (0.22 urn) and pipetted into sterile flasks for
the growth experiments. (Preliminary studies showed that the aqueous
extract prepared from 1.67g/liter sawdust resulted in optimum growth of
inoculated organisms capable of utilizing cyclitols). The 4g/liter
sawdust extract was used only for quantitating cyclitols in redwood
sawdust extracts. Non-sterile extracts were not membrane filtered.
Preparation of Samples for Gas Chromatography
In all cases 0.5 ml samples of redwood extract or tank water were pro-
cessed. Quebrachitol (a gift from Dr. Laurens Anderson, Dept. Biochemistry,
12
-------
University of Wisconsin) a cyclitol not present in redwood, was used as an
internal quantitation standard. 0.05 ml of a 1 mg/ml aqueous solution of
quebrachitol was added to all samples before dry heat evaporation. In the
case of the more concentrated sawdust extract (4g/liter) it was necessary to
remove contamination from phenolic compounds. This was achieved by lead
acetate precipitation (17).
Dry samples were silylated with .2 ml of Tri Sil Z (Pierce Chemical Co.,
Rockford, 111.) for 3 hr at room temperature and 2 yl was injected into the
gas chromatograph.
A Hewlett-Packard Model 5710A gas chromatograph with a flame-ionization
detector was used in these experiments. This instrument was equipped with a
6 ft. 3% OV-17 stationary phase on a Gas Chrom Q support (100-12 mesh) for
analysis of redwood extracts. The oven temperature used was 160 C, with a
carrier gas (helium) flow of 20ml/min. Injection port temperature was
260°C; detector temperature was 300°C. Peaks were recorded with a Hewlett-
Packard Model 7123A strip chart recorder. The FID control was set with a
range of 10 and attenuation of 2 for all analyses.
Quantitative analyses were determined by first preparing a tracing on
uniform thickness bond paper. The peaks were cut out and weighed on a Metier
analytical balance and the mass compared to that of the internal standard.
Preparation of Bacterial Cultures for Nutritional Experiments
Cultures were grown overnight in Difco penassay broth at 28°C. Cultures
were diluted to 10~6 through sterile 0.01M Tris buffer, pH 7.5 and inoculated
into the sawdust extract to achieve an initial density of about 10^ cells/ml.
Tris buffer diluent provided viable counts indistinguishable from those ob-
tained when Standard Methods buffer was used (see Appendix). Appropriate
media controls were used to assess growth in distilled water or basal salts
medium without a carbon source. Cultures examined for utilization of
cyclitols in a defined medium were propagated in basal salts medium in the
presence of a growth limiting amount of glucose (0.02%).
Basal Salts Medium
A defined medium was used to test for cyclitol utilization as a sole
source of carbon and energy. The medium contains the following ingredients in
g/1: Tris-buffer to PH7, lOg; NH4C1, 2g; KC1, 2g; KH^, Ig; Na^, O.lg
(solution A). Solution B contains in g/100 ml: CaCl2'2H20, 0.05g; MgCl2,
0.02g. To prepare 1 liter of medium, combine 900 ml sterile solution A, 10 ml
solution B, and add 100 ml of the test carbon source to achieve 0.2% of the
latter.
13
-------
SECTION 3
RESULTS AND DISCUSSION
BIOCHMEMICAL PROPERTIES OF THE KLEBSIELLA
Table 1 summarizes the biochemical test responses of redwood associated
Klebsiella and those published by Edwards and Ewing (2). All isolates con-
form to the current taxonomic criteria for 1C. pneumoniae (2,4). The only
significant deviation in incidence of positive reactions was found in
the tests for indole production and urease activity. A significantly
greater incidence of indole production was observed among the clinical
(human-derived) and redwood associated isolates compared to the frequency
compiled by Edwards and Ewing. The environmental group also had a smaller
number of urease-positive isolates.
The percent variability in urease reaction between sources is similar
to data reported by Henriksen (18) in which 96% of his pathogenic and fecal
Klebsiella strains were urease positive and 69% of the water strains were
positive. Positive urease activity has been suggested by Buttiaux (19) and
Mossel (20) as indicative of Klebsiella strains of fecal origin. All FC-
positive isolates in this study were urease positive. However, all but one
of the FC-negative pathogenic strains and 68% of the environmental FC-
negative strains were also urease positive. Thus, in using the current
concept and techniques for FC detection, urease activity alone would not be
sufficient to indicate that a Klebsiella isolate is of fecal origin.
The indole-positive isolates, which appear otherwise to be K. pneumoniae
by routine biochemical tests, resemble the "oxytoca" group as proposed by
Lautrop (21), Stenzel et al., (22), and Von Reisen (23). Others have il-
lustrated that indole-producing Klebsiella are present in significantly higher
frequencies in human feces and clinical material than might be indicated from
the compilations of Edwards and Ewing. Thus, regardless of indole and
urease responses, all redwood associated isolates obtained in the present
studies are indistinguishable from what others have also referred to as
Klebsiella and such isolates are described from human/animal infections and
from human fecal material.
The field surveys on the bacteriological quality of finished drinking
water emanating from redwood tanks illustrated that Klebsiella is usually not
accompanied by the presence of _E. coli (24). Because of the ubiquity of
Klebsiella in other botanical environments apparently free from obvious fecal
pollution, the immediate health importance of Klebsiella has been under-
estimated (11,25). Identification of Klebsiella has also been confused with
14
-------
TABLE 1. BIOCHEMICAL REACTIONS OF REDWOOD ASSOCIATED KLEBSIELLA ISOLATES
COMPARED TO DATA GIVEN BY EDWARDS AND EWING (3)
Test of substrate Redwood Clinical
Associated (Edwards and Ewing)
(% +) Signb % +
Indole
Methyl red
Voges Proskauer
Simmons' citrate
H2S (TSI)
Urease
KCN
Motility
Gelatin
Lysine decarboxylase
Arginine dihydrolase
Ornithine decarboxylase
Phenylalanine deaminase
Malonate
Glucose: Acid
Gas
Lactose: Acid
Gas
Sucrose
Mannitol
Dulcitol
Inositol: Acid
Gas
Arabinose
Rhamnose
Glycerol
33
14
92
100
18
75
71
0
5
100
0
0
0
80
100
94
100
100
100
100
69
100
80
100
83
100
6.0
- or + 13.3
+ 91.1
+ 97.7
0
+ 94.5
+ 97.7
0
3.3
+ 97.2
0
0
0
+ 92.5
+ 97.0
+ 96.5
+ 98.2
+ 98.0
+ 98.9
+ 100.0
-or + 31.5
+ 97.9
+ 91.9
+ 99.9
+ 99.3
+ 93.0
alsolates obtained from field and laboratory redwood reservoirs and from red-
wood sawdust chips.
bThe sign indicates: +, 90% or more positive in 1 or 2 days; -, 90% or more
negative; - or +, majority negative.
15
-------
that of Enterobacter (Aerobacter), and Klebsiella has been grouped as a
total colifonn. However, the fecal coliform (FC) elevated temperature
test is considered a most relevant test for examining the fecal origin
of coliform bacteria (26). Klebsiella is carried in the intestinal
tract of some 30 to 40% of humans and animals and some environmentally-
derived Klebsiella isolates have given a positive response in this test
(11,19,27-30). The applicability of the FC test response in the present
studies seemed relevant except for some important missing information.
That is, no one had examined the incidence of the FC test response among
known pathogenic isolates of Klebsiella. Tables 2-4 summarize results
of a comparative analysis on the FC test responses of 191 Klebsiella of
pathogenic and environmental sources.
Using standard MPN techniques (10), approximately 85% of the human
and animal pathogenic isolates were FC positive as judged by gas production
from lactose when incubated at 44.5°C compared to approximately 17% of
the environmentally-derived isolates. A breakdown of the FC responses
of environmental Klebsiella is provided in Table 3.
Isolates obtained from small experimental 65 liter redwood tanks
(24), from redwood sawdust and wood chips, from finished public drinking
water supplies, and from fresh vegetables account for most of the FC-
positive results using both MPN and membrane filter (MF) technique. The
high percent confirmation of MF FC-positive Klebsiella from all sources
indicates they are true fecal coliforms and conform to the operational
definition of a coliform of fecal origin (10,26). Although the original
source of the FC-positive redwood associated Klebsiella has not and
cannot be determined with certainty (origins from insects, birds, animals,
or humans) their presence is made more significant in view of the 85%
FC-positive incidence among Klebsiella isolates of known clinical origins
and strongly supports their sanitary significance.
The presence of high Klebsiella numbers in botanical environments
in general results from their regrowth (31); (see later sections). This
regrowth further complicates any interpretation of their sanitary signifi-
cance in drinking water supplies emanating from redwood reservoirs.
Thus, the occurrence of FC-positive Klebsiella is indicative of fecal
pollution at some point in time which may have been recent or much
earlier. The presence of specific cell densities of Klebsiella in the
absence of E. coli does not imply the presence of other pathogenic
enteric bacteria. The deterioration of environmental quality is based
on the opportunistic pathogenic nature of Klebsiella per se and a recogni-
tion of the nature by which exposure and subsequent clinical manifestation
of infection are generally separated in time.
Studies have supported the contention that indole producing Klebsiella
pneumoniae should be considered as a separate taxon (1C. oxytoca or
Oxytocum) and not, as in the present context, as FC negative It. pneumoniae
(7,21-23). Such taxonomic considerations, which the data support, have
a relevant impact on the FC surveys. For example, the incidence of FC
positive Klebsiella among the known pathogenic isolates changes signifi-
cantly since all but one of the FC negative cultures was indole producing.
16
-------
TABLE 2. MPN AND MF FC-POSITIVE KLEBSIELLA CULTURES
Origin
Klebsiella pneu-
moniae
Human clinical
Mastitis
ATCC
Environmental
No. of iso-
lates
46
22
3
120
Positive
response *
MPN
82.6
86.3
66.7
16.7
FC
MF
71.7
86.3
66.7
15.8
% Confir-
mation
93.6
100.0
100.0
89.5
a
Numbers indicate the
EC broth.
TABLE 3. FC
percentage of MF
FC-positive isolates
RESPONSE OF ENVIRONMENTAL KLEBSIELLA
confirmed in
ISOLATES
Origin
Redwood lab tanks
Public drinking water
No. of iso-
lates
11
43
Private drinking water 21
Redwood sawdust and
chips
Vegetables
Industrial effluents
15
19
11
Positive
response
MPN
27.3
16.3
0.0
33.3
21.1
9.1
FC
MF
27.3
16.3
4.8
33.3
15.8
9.1
% Confir-
mat ion
100 . Oa
85.7
0.0
100.0
100.0
100.0
See footnote, a, Table 2.
^Includes pulp and paper mill (five), textile mill (four), and potato
processing effluent (two).
17
-------
Thus, nearly 100% of the 1C. pneumoniae sensu stricto of clinical origins
are fecal coliforms. One-third of the 120 environmentally-derived
Klebsiella were indole producing. Thus, the actual incidence of FC
positive 1C. pneumoniae from the environment was at least 19 of 80 or
some 24%. The sanitary significance of the oxytoca group should not be
discounted either, even if FC negative, since 34% of Klebsiella fecal
isolates and 17% of human clinical isolates (15% in the present survey)
have been reported as indole-producing.
In the present survey, Klebseilla fecal coliform positive colony
color and morphology ranged.from typical flat and dark blue with or
without precipitated bile, to raised, mucoid, dark blue, to raised with
blue centers and cream-colored edges. Also, some 40% of the fecal
coliform positive cultures exhibited flat, dark blue colonies with some
orange coloration. Because of this diversity in reaction on mFC agar,
experiments were conducted to determine whether pH in mFC broth (with
rosalic acid) could be used to correlate with the patterns of FC response
(MPN and MF) and observed colony morphology. The results from 42 Klebiella
able to grow at 44.5°C (aerogenic or anaerogenic) and 12 FC positive E_.
coli are illustrated in Table 4.
A distinct response pattern was indeed recorded correlating final
pH, colony morphology, and responses with MF and MPN techniques. Thus,
the Klebsiella and _E. coli which were MF+,MPN± had pH < 5.3; MF(-), MPN+
isolates had a pH range of 5.3 to 5.8, while MF(-), MPN(-) isolates had
a pH greater than 5.8.
Klebsiella isolates with typical dark blue colonies on mFC agar or
blue-orange colonies produced the lowest pH (<5.3). Klebsiella cultures
which were MF(-), MPN+ had blue-centered colonies with cream colored
edges or were light blue-gray or blue-green in appearance. MF(-),
MPN(-) cultures produced pink or cream colored colonies on mFC agar.
ANTIBIOTIC RESISTANCE OF THE KLEBSIELLA
In order to further characterize Klebsiella from finished drinking
water, other related environments, and from clinical infections, some
183 isolates were tested for resistance to 11 antibiotics. Some isolates
found to be resistant to 2 or more antibacterials ("multiple resistant"
in this study) were also tested for their ability to transfer resistance
to E_. coli and to other Klebsiella isolates. Results of these experiments
are presented in Tables 5 to 7.
Table 5 summarizes the percent of isolates resistant to antibiotics.
Although antibiotic resistance was recorded for isolates associated with
water stored in redwood reservoirs, with industrial effluents and with
other botanical material, as might be expected the greatest incidence of
resistant isolates was observed among the human and animal clinical
specimens. The high incidence of resistance to Pen and Amp would be
expected for gram-negative bacteria and this observation carries no
relevant health significance.
18
-------
TABLE 4. CULTURE pH IN m-FC BROTH TEST INCUBATED AT 44.5°C VERSUS FC RESPONSE
V£>
FC response
MF MPN
Klebsiella pneumonias
Clinical + +
+ (-)a
(-) +
(-) (-)
Mastitis + +
Environmental + +
+ (-)
(-) +
(-) (-)
Escherichia coli
Human clinical + +
Environmental + +
No. of iso
lates
9
3
5
4
8
6
1
3
3
7
5
pH range (% occurring)
<5.3
66.7
100.0
0.0
0.0
100.0
66.7
100.0
0.0
0.0
71.4
100.0
5.3-5.8
33.3
0.0
100.0
0.0
0.0
33.3
0.0
100.0
0.0
28.6
0.0
>5.8
0.0
0.0
0.0
100.0
0.0
0.0
100.0
0.0
100.0
0.0
0.0
Parenthesis indicate growth but a FC-negative reaction.
-------
TABLE 5. ANTIBIOTIC RESISTANCE OF KLEBSIELLA ISOLATES FROM VARIOUS HABITATS
PERCENT RESISTANT
NUMBER
ORIGIN TESTED PEN STR CHL NITRO KN TC NOV NM ERY AMP NAL GENT POLY VAL FURACIN
Mastitis 29 100 37.9 6.7 6.7 3.5 17.2 100 3.5 100 72.4 000 24.1 0
Human
Medical 57 100 17.5 1.8 40.5 8.8 24.6 100 10.5 96.4 98.3 3.5 1.8 0 19.3 0
Total
Medical 86 100 24.4 3.5 30.2 7.0 20.9 100 8.1 97.6 90.7 2.3 1.2 0 23.3 0
NJ
0 Bovine
Area 23 91.3 47.8 4.4 0 0 30.4 100 0 100 52.2 000 17.4 0
Water 27 100 7.4 0 0 7.4 0 100 0 100 66.7 0 0 0 0 0
Vegetable 19 100 0 0 10.5 0 0 100 0 100 78.9 0 0 0 0 0
Pulp Mill 12 100 00 8.3 0 0 100 0 100 91.7 0 0 0 0 0
Other 16 100 12.5 0 0 0 12.5 100 0 100 81.3 0 0 0 18.8 0
Total
Environment 97 98.9 15.5 1.0 3.1 2.1 9.3 100 0 100 72.2 0 0 0 7.2 0
-------
Within the clinical group of cultures, those of human origins were
resistant to Nitro, Kn, and Nm in substantially greater percentages than
the mastitis isolates. The opposite was observed for Str. The in-
cidence of Val resistance among the human isolates (19.3%) was very
similar to that of the mastitis isolates (24.1%) even though the drug is
presumably not available for human use.
Among the remainder of the isolates from various environments
(excluding bovine area), resistance to Str, Nitro, Kn, Tc, and Val were
recorded. Resistance to two antibiotics was recorded from four of 27
Klebsiella isolated from redwood tanks. It must be indicated that 3 of
these 4 isolates came from one small experimental laboratory redwood
tank originally filled with finished municipal drinking water. No
coliforms were ever detected in this municipal water which carried a
total chlorine residual (OTO technique) of 0.3-0.5ppm. The tank water
contained no free or combined chlorine. Two of these isolates were
resistant to Amp, Str while the third was resistant to Amp, Kn. The
remaining Klebsiella isolate (Amp, Kn) was isolated directly from the
finished drinking water stored within a redwood reservoir on a public
water supply system.
Table 6 presents the drug resistance patterns of Klebsiella resistant
to two or more antibiotics. It is apparent that those isolates with re-
sistance to three or more drugs were almost exclusively from clinical
origins. The two exceptions were isolates from textile mill effluents.
A total of 20 different multiple-resistance patterns were found among the
human medical isolates. Of the 27 "water" isolates, 22 were obtained
from finished drinking water emanating from public or privately owned red-
wood tanks and only 1 (Amp, Kn) was resistant to 2 antibiotics. Other
Klebsiella from botanical environments were found resistant to two or
more antibiotics. These included 3 from redwood chips, 1 from pulp mill
effluent, 2 from textile mill effluents and 2 from the surfaces of fresh
vegetables.
TABLE 6. DRUG RESISTANCE PATTERNS OF MULTIPLE-RESISTANT KLEBSIELLA ISOLATES
IT) x-s
CO
CM
RESISTANCE PATTERN
amp, str
amp, val
CM
W
•H
4J
•H
•U
CO
cd
*g^
3
4
Medical
s
cd
g
3
1
2
cd
0) <— N
<; CM
CM
rH rH
0) rH
rH rH
.O -H
cd S
4J
o) a.
00 rH
0) 3
> PH
-
-
I
M
G)
rC
jj
0
-
3
(continued)
21
-------
TABLE 6. (continued)
in
RESISTANCE PATTERN
amp, nitro
amp, tc
amp, kn
str, tc
str, chl
amp, str, tc
amp, nitro, val
amp, nitro, str
amp, str, chl
amp, str, val
amp, tc, val
amp, nitro, nal
amp, nitro, tc
amp, tc, chl
amp, str, tc, val
amp, nitro, nm, val
amp, str, tc, nitro
amp, tc, nm, kn
amp, str, tc, nm, val
amp, str, tc, nitro, val
amp, str, tc, kn, nm,
amp, str, tc, kn, nm,val
amp, str, tc, nitro, kn, nm, val
amp, str, tc, nitro, kn, nm, gent,
TOTAL MULTIPLE RESISTANT
s-*.
cy*
CM
co
•H
4J
•H
4J
co
cd
-
-
-
2
1
1
2
1
1
-
-
-
-
-
1
-
-
-
-
-
-
-
1
val -
17
•*-* en
CN
j-^ XM^
cd
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3 - -
2 - -
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1 - -
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1 - -
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1 - -
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1 - -
37 11 4
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215
Numbers in brackets are number of
strains tested.
Resistance to pen, nov and ery were omitted.
22
-------
Evidence of R-plasmid mediated drug resistance, in the form of conjugal
transfer frequencies of selected markers, is presented in Table 7. Potential
donors were selected which were resistant to 2 or more antibiotics. Data
are illustrated from selected experiments with _E. coli Nalr W3110 and various
Nal Klebsiella as recipients.
TABLE 7
. CONJUGAL TRANSFER FREQUENCIES OF DRUG RESISTANCE BY SELECTED
KLEBSIELLA ISOLATES WITH E. COLI Nalr W3110 OR OTHER Nal^
KLEBSIELLA
AS RECIPIENTS
ISOLATE
C-2
C-l
BC 1738
E44.5-2
VI 3
V5
V125
MH 10
SL 5
ORIGIN
Redwood
Redwood
Pulp mill
Redwood
MARKERS
DETECTED
E. COLI W3110
amp, val
amp , val
amp , nitro
amp , val
Redwood amp , str
tank water
Redwood amp, str
tank water
Potato
Celery
Human
sputum
amp , nitro
amp, nitro
amp, str, tc
nm,kn
MARKERS
TRANSFERRED
AS RECIPIENT:
amp
val
amp , val
amp
val
amp , val
-
amp
val
amp, val
str
str
nitro
nitro
str
amp
tc
kn
nm
FREQUENCY OF TRANSFER:
TRANSCONJUGANTS /
RECIPIENT
6.9 x 10~
2.9 x 10
4.2 x 10
8.5 x 10~
8.1 x 10~
2.4 x 10
-
1.2 x W~6,
6.0 x 10 :
1.6 x 10
6.1 x 10~4
4.0 x 10~A
1.6 x 10~6
-4
5.5 x 10 *
2.0 x 10~
1.4 x 10 j
2. 6 x 10~2
2.1 x 10 2
2.1 x 10
(continued)
23
-------
TABLE 7. (continued)
ISOLATE
UT 465
UG 14
UG 28
UG 6
UG 16
MARKERS
ORIGIN DETECTED
E. COLI W3110
Human amp , str , fur
blood tc
Bovine str, tc
Clinical
" str, tc, chl
unused str, tc
bedding
unused amp, str
bedding
MARKERS
TRANSFERRED
AS RECIPIENT:
amp
str
fur
amp,nitro
str,nitro
str
tc
str, tc
str
chl
str, chl
str
tc
str, tc
—
tc
str, tc
FREQUENCY OF TRANSFER:
TRANSCONJUGANTS/
RECIPIENT
5.1 x 10~^
2.6 x 10 '
1.2 x 10 .
2.3 x 10
2.5 x 10
1.0 x 10~
9.8 x 10 7
4.3 x 10 °
2.9 x 10~^
1.2 x 10 ^
1.2 x 10
2.7 x 10~?
5.8 x 10 ,
1.4 x 10
-
49 -v 1 fi
• £. A, _LVJ —,
6.6 x 10
KLEBSIELLA AS RECIPIENTS:
KLEBSIELLA
RECIPIENT
ORIGIN DONOR ORIGIN
MARKER
SELECTED
FREQUENCY OF TRANSFER:
TRANSCONJUGANTS/
RECIPIENT
V-104A Vegetable UG 14 Bov. Clinical str 7.6 x 10
" " UG 28 Bov. Clinical chl 5.9 x 10
" " UG 34 Bov. Anal val 4.6 x 10
MH 15 Salad UG 28 Bov. Clinical chl 2.9 x 10
" " UG 34 Bovine Anal val 6.2 x 10
11 " UT 465 Human, blood nitro 3.2 x 10
" SL 5 Human, sputum kn 1.2 x 10
nm 1.0 x 10
(continued)
24
-4
-4
-4
-6
-5
-3
-3
-3
-------
TABLE 7. (continued)
KLEBSIELLA
RECIPIENT ORIGIN
DONOR ORIGIN
MARKER FREQUENCY OF TRANSFER
SELECTED TRANSCONJUGANTS/
RECIPIENT
KLEBSIELLA AS RECIPIENTS;
PS 401LR Bovine UG 28 Bov. Clinical chl
Mastitis
" SL 5 Human, sputum kn
nm
1.1 x 10
-2
3.9 x 10
1.1 x 10
-5
-4
—9 —8
Overall, the transfer of markers ranged from 10 to 10 trans-
conjugants per recipient. In all, 31 potential Klebsiella donors were tested
for transfer of antibiotic resistance and transconjugants were detected for
28 cultures.
Selected isolates that supported measurable transfer into _E_. coli were
chosen as donors to various Klebsiella of environmental and medical origins.
Depending upon the markers selected, Klebsiella isolates had various degrees
of success as conjugal recipients. In the case of Str, Tc and the Kn-Km
markers, transfer was a more frequent event when E^. coli was the recipient.
In the case of Val and Nitro, the frequencies were of the same magnitude, and
Chl was usually mobilized more frequently into Klebsiella than into E_. coli.
These data illustrate that R-plasmid bearing Klebsiella isolates from a
variety of "natural" origins are able to mobilize drug resistance to either
an IS. coli or another Klebsiella recipient with frequencies similar to iso-
lates of clinical origins. If these Klebsiella were to become involved in
colonizing compromised hosts, such cultures would have an obvious selective
advantage accompanying antibiotic therapy.
COMPARATIVE PATHOGENICITY OF THE KLEBSIELLA
The major point of these studies was to compare and contrast in a con-
trolled system the virulence properties themselves among a set of Klebsiella
isolates derived from various habitats and differing in known phenotypic
properties. In all cases of mouse death following injection, the inoculated
Klebsiella was recovered from several organs and was also present in the
blood stream. Death was therefore confirmed to be due to the inoculated
organism and was the result of the relative invasiveness of the test culture
and the ability of the culture to overcome the normal host defenses.
Mean LD^Q values for 97 Klebsiella and for 12 E_. coli isolates are
-'*•' • • - - " ~~
illustrated in Table 8.
The mean LD5Q values for Klebsiella of all origins
25
-------
TABLE 8. LD5Q OF KLEBSIELLA AND _E. COLI ISOLATES DETERMINED BY INTRAPERJ.'rONEAL INJECTION IN MICE
ro
NUMBER
CULTURE SOURCE OF ISOLATES
Klebsiella spp. Mastitis
Clinical
Environment al
E. coli Clinical
Finished Drinking
Water
19
18
60
6
6
MEAN LD503
(cells per ml)
1.5 x 104
4.6 x 104
4.2 x 104
2.8 x 104
5
2.5 x 10
RANGE
(cells per ml)
3.5 x 101 to 4.9 x 105
5.3 x 103 to 6.0 x 105
1.0 x 103 to 7.9 x 105
4.5 x 103 to 2.9 x 105
4 6
6.2 x 10 to 2.2 x 10
Geometric mean based on natural log values.
-------
ranged from 1.5 x 10^ to 4.6 x 104 cells. Statistical tests indicate no
significant difference (a = .05) in the mean values for Klebsiella derived
from mastitis, clinical, or environmental origins. The mean LD5Q for E.
coli of human clinical origin is not significantly different from mean~~values
for Klebsiella from any source. On the other hand, E_. coli from finished
drinking water had a significantly higher mean LD5Q than both the clinical
E. coli and all Klebsiella. In a study also conducted in this laboratory,
the LDjjQ values for other gram-negative bacteria (Enterobacter, Erwinia,
Salmonella, Aeromonas, Pseudomonas and Spirillum) were 100- to 1,000-fold
higher, indicating a significantly lower virulence for mice than Klebsiella
(32).
Although the Klebsiella LD50 values have a 2-log range for clinical and
environmental isolates and a 4-log range for mastitis cultures, the central
tendency for all groups is within the same log 4.0 to 4.9 range (Figure 4).
Mastitis Klebsjella
-H
Clinical
2-
8-
7-
6-
5-
3-
2-
1-
I I I I I I I I
I I I I I I I I I I
Environmental Klebsjella
n_
• 1 1 1 1 iii
0 114 1 1|9 I 2l3 2l7 I 3l2 33 4
i —
i
4
5
]
T
1
—
4
I 1
1 1
9
5
3
5
_J
3, 83
log LDM
Figure 4. ^50 frequency distribution in mice for Klebsiella isolated from
various environments.
27
-------
The mean LD5Q values for various groups of the Klebsiella isolates
(based on FC response and indole production, It. oxytoca) are presented in
Table 9. Although the calculated mean LD50 values range from 1.1 x 10^ to
6.0 x ICn cells and sample size varied from 3 to 23 cultures, statistical
tests again indicate no significant difference in the mean LD^Q values for
any of the various groups.
The environmentally derived Klebsiella were sub-divided by habitat of
origin to possibly ascertain whether any differences in mean LD5Q values
could be detected (Table 10). Two interesting correlations were revealed.
First, there is no significant difference between the mean LD^Q values of
Klebsiella isolated from finished drinking water, from bovine associated en-
vironments, and from bovine-areas compared to those Klebsiella of clinical
origins. Second, the finished drinking water-derived Klebsiella have a
significantly different mean LD50 (more virulent) than the drinking water
derived _E. coli but are indistinguishable in virulence from the clinical 15.
coli. Klebsiella and E_. coli isolated from finished drinking water were
generally obtained from the same water samples (24).
The overall conclusion of this comparative survey illustrates that a
Klebsiella from any source is equally pathogenic in the animal test system.
Unlike the Klebsiella isolates, there is a difference in virulence between
IJ. coli from different sources. This indicates that E_. coli, which can also
be an opportunistic human pathogen (33), may have diminished virulence upon
entry into the environment. A similar observation has also been made for
Salmonella (32). Klebsiella entering the environment may have no such de-
crease in virulence as a recent st3dy with three Klebsiella of pathogenic
origin (31) showed no change in LD values after 100 generations of growth
in sterile pulp mill effluent.
The sanitary significance of FC-negative Klebsiella may be enhanced due
to the finding that no difference in mouse pathogenicity was observed between
the FC-positive and FC-negative isolates, regardless of habitat of origin.
It must also be remembered that the indole positive (K. oxytoca) isolates of
Klebsiella are also found as human and animal pathogens (27,30) and are
present in the feces of warm-blooded animals (30,35). In addition, the
importance of environmentally-derived Klebsiella is based on their op-
portunistic pathogenicity and is now underscored by the fact that drinking
water-derived FC positive and FC negative Klebsiella have statistically
lower LDcri values than drinking water-derived FC positive E. coli.
DU — ___
KLEBSIELLA PERSISTANCE IN MOUSE INTESTINAL TRACT FOLLOWING INGESTION OF
CONTAMINATED DRINKING WATER
Klebsiella within the intestinal tract can be a direct cause of no-
socomially acquired infection (36,37) and intestinal persistance or coloni-
zation results from ingestion of contaminated foods and liquids (33,36). The
previous section on Klebsiella LD5Q levels illustrated that, given the chance
to enter the intraperitoneal cavity, Klebsiella from any environment or of
28
-------
TABLE 9.
K>
VO
JV
NUMBER
CULTURE SOURCE OF ISOLATES
h
FC Positive K.
pneumoniae Mastitis
Clinical
Environmental
FC Negative K.
pneumoniae Mastitis
Clinical
Environmental
K. oxytoca Mastitis
Clinical
Environmental
11
11
14
3
3
23
5
4
23
MEAN LD5Qa
(cells per ml)
>
I,
1.6 x 10
3.9 x 104
5.5 x 104
/
1.5 x 10
6.0 x 104
4.5 x 104
1.1 x 104
5.8 x 104
3.7 x 104
RANGE
(cells per ml)
1 15
3.5 x 10 to 4.9 x 10
5.8 x 103 to 6.0 x 105
1.0 x 103 to 7.9 x 105
3 4
6.5 x 10 to 2.5 x 10
1.6 x 104 to 3.3 x 105
1.3 x 103 to 7.5 x 105
4.6 x 103 to 2.4 x 104
5.3 x 103 to 5.1 x 105
3.4 x 103 to 6.3 x 105
aGeometric mean based on natural log values.
Fecal Coliform.
CK. oxytoca are, by definition, FC negative (21,22).
-------
TABLE 10. LD5Q OF ENVIRONMENTAL KLEBSIELLA ISOLATES DETEEMINED BY
INTRAPERITONEAL INJECTION IN MICE
SOURCE
Finished Drinking water
(Redwood reservoirs)
Vegetables
Redwood sawdust and chips
Bovine-associated
Bovine-area
NUMBER
OF ISOLATES
28
8
6
9
9
MEAN LD5Q
(cells per ml)
5.2 x 104
1.4 x 105
9.1 x 104
1.5 x 104
1.3 x 104
RANGE
(cells per ml)
3.8 x 103 to 6.9 x 105
4.1 x 103 to 7.9 x 105
6.0 x 104 to 4.2 x 105
1.0 x 103 to 1.3 x 105
1.6 x 103 to 1.3 x 105
Geometric mean based on natural log values.
Isolates from potatoes (5), carrots (2), and Swiss chard (1).
Isolates from bovine mouth (5), anal area (1), and udder (3).
Isolates from unused t
in bovine stall areas.
Isolates from unused bedding (2), used bedding (2), and drinking water (5)
any biotype has no significant difference in levels of virulence for mice.
The purpose of these studies was to determine what levels of ingested
Klebsiella are necessary to persist in the mouse intestinal tract and how
such orally administered levels compare with known concentrations of
Klebsiella in drinking water and other environments. In this manner it
becomes possible to compare the ability of known levels of pathogenic
isolates to survive/persist in the intestines compared to environmentally-
derived isolates whose prior ecological history is unknown. Fig. 5
summarizes the percentage of mice excreting Klebsiella after oral ingestion
while Fig. 6 illustrates the average density of Klebsiella in fecal
pellets.
Percent animal carriers reach a constant amd maximum value by day
2 which persists until the contaminated water is replaced with municipal
water on day 6. The maximum percent animal carriers varied with the
Klebsiella densities per ml of water (10^, 101, and 10~1) and resulted in
percent animal carriers of 80%, 63%, and 8%, respectively. It should be
noted that mice provided 10"1 Klebsiella/ml (10/100 ml) were only ingesting
about one viable Klebsiella per 24 hr taking into consideration some
Klebsiella regrowth in the water. Nevertheless it was still possible to
detect Klebsiella in some two of 48 control mice and nine of 48 mice ad-
ministered antibiotics.
With every dose level studied, there was a pronounced increase in
percent animal carriers and fecal cell densities accompanying antibiotic
30
-------
treatment. When the drinking water contained Nal (40 ug/ml) and Str
(500 ug/ml) the respective animal carrier rates increased to 95%, 78%,
and 19% for the three-dose levels (Fig. 5).
Fecal densities for Klebsiella provided at 10 and 10 /ml of drinking
water are presented in Fig. 6. With no antibiotic stress,- Klebsiella are
lost from the intestine within 4 days after removal of contaminated drinking
water containing 101 cells/ml. On the other hand Klebsiella persist at an
average density of 300/gm feces 8 days after removal from contaminated drink-
ing water when antibiotics are continuously supplied along with a single CP
injection (given on day 3). In similar experiments, fecal densities are
correspondingly greater at the 10 Klebsiella/ml dose level.
100
2 4 67 10 14 21 28
DAYS AFTER EXPOSURE TO Klebsiella IN DRINKING WATER
Figure 5. Percentage of mice excreting Klebsiella after ingestion of con-
taminated drinking water. Symbols represent levels of Klebsiella/ml drinking
water: A, A, 10J; 0, 0, 101; , , 10-1. Open symbols with antibiotics,
closed, without antibiotics in drinking water; dashed lines CP injected day
3, solid lines, no CP. Klebsiella present in drinking water through day 6.
31
-------
2 4 67 10 14 21 28
DAYS AFTER EXPOSURE TO Klebsfello IN DRINKING WATER
Figure 6. Mean log of Klebsiella per gram of mouse feces after ingestion
of contaminated drinking water. Levels of Klebsiella/ml drinking water:
A, A, 103; 0, 0,
10J
Other symbols as in Figure 5.
Klebsiella recovered from feces of even control mice (no stress)
were in excess of the orally ingested number. For example, based on actual
viable counts in the drinking water and volume of water consumed, mice
provided titie 10-^- cells/ml actually ingested approximately 320 total
cells over the first 4 day period. On day 4, the level recovered equalled
320 cells, or every Klebsiella ingested was recovered. After art additional
48 hr some 240 cells were ingested and 400 Klebsiella were recovered in one
sample. With oral antibiotics and ingestion of the same number of Klebsiella,
20,000 Klebsiella were recovered per gm feces on both days and 4 and 6 demon-
strating considerable multiplication of Klebsielal in the intestinal tract.
A distinction among Klebsiella in terms of both percent animal carriers
(Fig. 7) and fecal densities (Fig. 8) was observed when cultures were divided
by fecal coliform response and K. oxytoca species. Although each of the 3
groups contained only 2 Klebsiella isolates (one clinical, one environmental)
the data was clear in that fecal coliform positive isolates colonized the
greatest percent of animals and achieved the highest cell densxties/gm feces.
Fecal coliform negative Klebsiella performed the poorest in both respects
while the indole producing K. oxytoca were intermediate.
32
-------
100 -
I I I
FC+ K. pneumoniae
K. oxytoca
^
M >
11 iTsi i
I 11
FC - K. pneumoniae
i i i
2467 10 14 21 280246710 14 21 280246710 14 21
DAYS AFTER EXPOSURE TO I01 Klebsiella / ml DRINKING WATER
28
Figure 7. Percentage of mice excreting various species/biotypes of .
Klebsiella after ingestion of contaminated drinking water containing 10
cells/ml of FC+ K. pneumoniae, 1C. oxytoca, or FC- K. pneumoniae. A, A, with
antibiotics, 0, 0, without antibiotics in drinking water. Open symbols, CP
injected on day 3, closed symbols, no CP. Klebsiella present in drinking
water through day 6.
The results (Figs. 5 and 6) illustrate that ingestion of 10 or greater
Klebsiella/ml over a short period can result in temporary intestinal carriage
at levels up to 100-times the ingested dose. The results of these studies
using mice probably should nq't be extrapolated to the human population
although some experimental data from humans is relevant. In one experiment
it was found that approximately 105 Klebsiella ingested by humans would re-
sult in a temporary fecal persistance for 3-4 days at levels of 10 /gm.
These observations are compatable with the results of other studies in this
laboratory using a single oral administration of Klebsiella to mice.
Fecal coliform response and species, not habitat of origin, had a
significant effect on the ability of a Klebsiella to persist in the mouse
intestinal tract (Figs. 7 and 8). The fecal coliform positive Klebsiella
were more effective than K. oxytoca and the latter were more effective than
33
-------
I I 11 I I I
FC + K. pneumoniae
0246710 14 21 280246710 14 21 280246710 14 21
DAYS AFTER EXPOSURE TO I01 Klebsiella / ml DRINKING WATER
28
Figure 8. Mean log of Klebsiella species/biotype per gram mouse feces after
ingestion of contaminated drinking water. Symbols as in Figure 7.
fecal coliform negative cultures. While oral antibiotics significantly
increased the persistence levels of fecal coliform negative cultures, levels
attained were still below those for the other two groups. These results have
important implications concerning the ecological distributions of
the various types of Klebsiella. Greater than 85% of the known pathogenic
Klebsiella tested for fecal coliform response have been positive in this
test. K. oxytoca accounted for some 14% of the pathogenic cultures and the
fecal coliform negative specimens for about 1%. Isolates of Klebsiella from
human and animal feces are also in roughly the same proportions (7,30,38).
Among the environmentally-derived Klebsiella isolates, the reverse order
has been found with the fecal coliform negative Klebsiella predominating.
Fecal coliform positive Klebsiella from clinical origins will regrow
in botanical environments, including drinking water emanating from redwood
reservoirs, pulp mill effluents, surfaces of fresh vegetables, and in aqueous
extracts of sawdust (31,39). Since fecal coliform positive Klebsiella repre-
sent the majority of Klebsiella responsible for infections and they are also
the type most likely to persist in the intestinal tract, their isolation
should be regarded as the most potentially significant health hazard. The
34
-------
indole positive, fecal coliform negative K. oxytoca should also be similarly
viewed. They are the primary agents of infections in animals and humans and
are also carried in feces. The significance of these Klebsiella might be
overlooked in various environmental samples since they would appear as total
and not fecal coliforms.
THE 3,785 LITER (1,000 GAL) EXPERIMENTAL RESERVOIR
ADJUSTABLE FLOAT
CHLORINE METERING
PUMP-
CHLORINE SOLUTION
TANK
MUNICIPAL
WATER—*~
SUPPLY
TIMER #1
TIMER
TO DRAIN
SOLENOID VALVE
Figure 9. Diagram of experimental 3,785 liter (1,000 gal) reservoir used to
test design modifications in redwood water storage reservoirs.
Based on findings drawn from the field surveys in Oregon design modi-
fications were made on a 3,785 liter experimental reservoxr which
true Sflr research purposes (Fig. 9). An automated chlorine meterxng
rt^rars:^; ^ SE - -
35
-------
TABLE 11. PHYSICAL AND MICROBIOLOGICAL PARAMETERS IN THE 3,785 LITER
EXPERIMENTAL REDWOOD TANK
OJ
ON
Month of
operation
1
1
1
2
2
3
3
4
5
5
5
6
7
8
9
10
11
Retention
time (days)
2a
4a
8a
8a
12a
12a
1
1
1
2
4
4
8
8
8
8
8
Number of
days sampled
7
9
6
13
11
6
20
26
5
4
21
14
19
28
24
27
28
Free
Chlorine
at inlet
(ppm)
1.7
4.9
11.5
23.3
18.2
30.0
8.6
2.1
1.9
3.0
3.9
5.1
8.2
8.6
5.8
5.8
4.2
Free
Chlorine
at outlet
(ppm)
0.3
0.1
0.2
0.5
0.1
0.1
3.7
0.9
0.2
0.4
0.7
0.4
0.3
0.5
0.2
0.5
0.6
PH
7.0
7.1
7.4
7.0
7.0
7.0
7.1
7.1
7.0
7.1
7.1
7.0
6.8
7.0
7.0
7.1
7.3
OD600
.009
.018
.020
.009
.012
.008
.001
.001
.001
.003
.001
.001
.001
.000
.001
.000
.000
Average total
count /ml
225
1040
123
15
3
>1000
>1000
>2000
>2000
10
95
<10
25
<10
<10
Stave
scraping
NCDb
NCD
NCD
NCD
NCD
NCD
(continued)
-------
TABLE 11. (continued)
LO
Month of
operation
12
12
13
14
15
16
17
18
19
20
Retention
time (days)
8
12
12
12
12
12
12
12
12
12
Number of
days sampled
19
7
28
23
23
19
7
7
6
3
Free
Chlorine
at inlet
(ppm)
3.3
3.9
3.0
2.7
1.5
0.5
0.5
0.5
0.5
0.5
Free
Chlorine
at outlet
(ppm)
0.4
0.3
0.4
0.5
0.4
0.1
0.2
<0.1
<0.1
<0.1
PH
7.2
7.2
7.2
7.3
7.2
7.1
7.2
7.2
7.0
7.0
°D600
.000
.000
.000
.000
.000
.000
.000
.000
.000
.000
Average total Stave
count /ml scraping
40 NCD
20
1n Klebsiella pneu-
<1U moniae FCC-
21 NCD
2060
1090
860
10
48 NCD
Tank operating at 20 percent capacity,
NCD - no coliforms detected.
"FC~; fecal coliform negative.
-------
Total bacterial counts in the outlet water ranged from 2,000/ml to
less than 10 per ml during the study period. The counts were generally in
the 10-50/ml range when an adequate free chloring residual of 0.2-0.4 ppm
was maintained. Chlorine additions from the metering pump were terminated
in the 16th month and municipal water with 0.3-0.5 ppm became the only source
of chlorine. This resulted in a lowering in the chlorine residual at the
outlet and a temporary increase in total counts to 2,000/ml, which decreased
to less than 50/ml by the 19th mo. of operation. Non-coliform isolates were
identified and found to consist of: Pseudomonas spp., P_. fluorescens,
Moraxella, Xanthomonas, Bacillus, Micrococcus, and one Staphylococcus
epidermidis.
Coliforms were detected in only one of the eight experiments in which
the interior surfaces of the staves were scraped and examined. Only five
of the eighteen staves sampled yielded coliforms which were identified as
fecal colifonn negative _K. pneumoniae. Subsequent stave scrapings failed to
detect any coliforms. At no point were coliforms detected in water emanating
from the tank, even when volumes of 1,000 ml were filtered and assayed by the
MF technique.
Two factors were confirmed to be important in maintaining a free chlorine
residual in the water of the redwood reservoir. As the tank aged, the
chlorine demand decreased (Table 11). Thus, from the 7th to the 15th month
(8 to 12 day retention), the amount of chlorine needed to maintain a free
residual of 0.4 ppm decreased from 8.2 ppm to 1.5 ppm. Retention time also
has an important influence on chlorine residuals. In the 4th month of
operation (1 day retention), 2.1 ppm at the inlet maintained a residual of
0.9 ppm, but 5.1 ppm was needed on a 4 day retention to maintain a lower
residual of 0.4 ppm. Also, on an 8 day retention, 8.2 ppm in the inlet water
was required to maintain 0.3 ppm in the outlet water.
The presence of colored material (tannins) in the reservoir water
(Optical density measurements, Table 11) also indicates the presence of a
variety of organic matter. To test whether this material contains growth
promoting substances for coliforms, a strain of 1C. pneumoniae was inoculated
into dechlorinated filter sterilized samples of tank water. Of 15 water
samples tested, five supported growth of the Klebsiella to levels of 10^ to
107/ml (Table 12). Since all glassware was acid washed, the only source of
nutrients was from the redwood used to construct the tank. No correlation
was found between those samples that supported growth and those that did not.
These studies illustrate that, with routine adjustments of design, red-
wood water reservoirs can be operated without coliform contamination.
However, there are certain factors that must be taken into consideration,
which, if not adhered to, can result in high levels of coliform contamination
as recorded in the field surveys. These factors include maintaining an
adequate chlorine residual in the reservoir and throughout the distribution
system, maintaining short retention times of water in the reservoir, and
a design which promotes adequate water circulation within the reservoir.
Even though the experimental 3,785 liter reservoir was constructed and
maintained under the best known conditions, K. pneumoniae was found on one
occasion to be colonizing the staves. This observation, coupled with their
38
-------
TABLE 12. VIABLE COUNTS AT 35C OF KELBSIELLA INCUBATED IN REDWOOD TANK WATER
Date sample collected
2/26/75
4/5/75
10/24/75
11/13/75
12/18/75
2/7/76
6/30/76
7/29/76
9/15/76
10/10/76
11/19/76
1/11/77
2/10/77
3/10/77
5/18/77
municipal
water
double distilled
water
0 hr
9.0 x 101
7.0 x 101
4.5 x 101
2.3 x 10°
6.0 x 101
5.5 x 101
2.3 x 10°
2.3 x 10°
7.0 x 101
8.0 x 101
4.0 x 101
2.3 x 10°
8.0 x 101
9.5 x 101
6.0 x 101
7.5 x 101
9.5 x 101
48 hr
1 x 101
1 x 101
3.0 x 102
1 x 101
1 x 101
1 x 101
3.7 x 105
1.5 x 106
1 x 101
1.3 x 107
5x10°
2.5 x 106
4.2 x 106
1 x 101
1 x 101
1 x 101
1 x 101
96 hr
1 x 101
1 x 101
3.0 x 102
1 x 101
1 x 101
1 x 101
1.6 x 106
1.5 x 106
1 x 101
9.4 x 106
1 x 101
1.8 x 106
5.0 x 106
1 x 101
1 x 101
1 x 101
1 x 101
demonstrated ability to regrow in the tank water, clearly enhances the
necessity to maintain a free chlorine residual throughout the system at all
times.
The amount of chlorine required to maintain a free residual in the out-
let water actually depends upon a combination of 3 interrelated phenomena.
The combination of a new reservoir and lengthy retention time (7 days or
more) and poor water circulation each individually aggravate the chlorine
demand. In order to maintain a free residual at the outlet with adequate
water mixing within the experimental reservoir, it required approximately
1 ppm free chlorine in the inlet water for each day of water retention during
the interval of 3-10 months. The introduction of water with an initial
chlorine residual of 0.3 ppm is therefore clearly inadequate.
39
-------
During the first 3 months of operation it was necessary to use 15-20 ppm
and more chlorine to maintain a residual on an 8 to 12-day retention period.
Therefore, if possible, it would be highly beneficial to install and bring
on line a new reservoir several months prior to the actual serving of finished
drinking water to the consumer. Prior to the preservice period, it is
recommended that the manufacturer's guide for a 24 h soda ash soak to leach
tannins and other water soluble material be extended to 7 days. Optical
density readings taken during the second treatment of 7 days with the ex-
perimental reservoir showed that considerable additional material was leached
from the wood. After this treatment, however, the odor and color of the
water became quite acceptable and optical density reading demonstrated no
significant difference compared with finished municipal water.
During the preservice period after the soda ash treatment, the reservoir
should not contain unchlorinated, stagnant water, but rather, should operate
in some intermittant fashion to drain out nutrients which continue to become
leached from the wood. Also a chlorine residual should be maintained to
prevent slime accumulation on the stave surfaces. If slime does accumulate,
it should be scrubbed from the wooden surfaces so that it cannot slough off
and enter the distribution system at later times.
Finally, all redwood reservoirs should utilize a plumbing arrangement
at the top of the tank for inlet water that sprays the water evenly to all
sides. The outlet fixture should be located at or near the tank bottom.
This promotes mixing and the spraying of water onto the upper staves, pro-
motes aging of wood and leaches out nutrients from areas that might not be-
come immersed below the water line. By adjusting the level of floats, the
volume of water in the reservoir can be matched with the daily anticipated
usage of water such that retention times can be maintained to a minimum.
In summary, redwood tanks can be a satisfactory means to store water
for drinking purposes, providing a few routine guidelines are followed and
a monitoring schedule of chlorine residuals and bacteriological sampling is
maintained. This is especially important for new redwood tanks, where the
likelihood of coliform contamination is most severe. When properly managed,
however, redwood water systems can be expected to provide potable drinking
water throughout the life of the tank.
ASSOCIATION OF KLEBISELLA WITH REDWOOD USED TO CONSTRUCT RESERVOIRS
In the previous section, evidence was presented to document the presence
of Klebsiella and Enterobacter emanating from wooden reservoirs containing
finished drinking water. In this section, evidence will be presented to
demonstrate that the origin of these coliforms can be from the wood used to
construct reservoirs. Evidence has been obtained in field surveys which
demonstrates that E. coli is also occasionally present in the finished drink-
ing water but its origin should be considered to be due to more recent fecal
contamination of the water distribution system and not from the redwood
per se.
40
-------
Table 13 illustrates the results of 3 experiments which examines the
bacterial content of liquid aseptically extracted from redwood heartwood
(HW) and sapwood (SW). The results indicate that not only are high levels
of standard plate count bacteria present, but-high levels of total coliforms
as well. Levels of total coliforms ranged from 3.2 x 102 to 1.4 x 10°/ml
of liquid extracted. High total coliform counts also correspond with high
levels of total standard plate counts.
All coliforms identified were of the Klebsiella and Enterobacter genera.
E.. agglomerans was the only coliform identified from the redwood SW tested in
Experiment 1. Samples in Experiment 2 were incubated at higher temperatures
to reduce the numbers of Enterobacter and enhance the isolation of Klebsiella.
Klebsiella species were in fact recovered from all samples having detectable
total coliforms. In Experiment 3 Klebsiella were also isolated from redwood
samples although incubation was at 35°C. No fecal coliforms were detected
from any wood sample by plating onto mFC agar.
Liquid expressed from other types of wood was also examined for bac-
terial content (Table 14). With the exception of cedar, all woods tested
were SW and had levels of standard plate counts comparable to those found
from redwood. All wood except cedar also had recoverable total coliforms
but counts were 10- to 100-times less than in the redwood extracts. Coli-
forms identified were essentially the same as those from redwood, with the
additional isolation of Citrobacter freundii.
Contact impressions of freshly exposed wood surfaces (Table 15) gave
results parallel to the total coliform counts in liquid expressed from
the same wood samples (Table 13). All wood types from which total coliforms
were obtained via pressing also had total coliforms present on the inner
surfaces.
Table 16 presents the results of contact impressions made from suc-
cessive serial cuts through two redwood SW samples having confluent coli-
form growth (Table 15). The results generally demonstrate that coliforms
are present throughout the entire depths of the block.
Scanning electron micrographs (SEM) of wide ring count SW sections
(Figs. 10 to 12) taken at increasing magnifications clearly demonstrate the
presence of several types of bacteria within the wood structure. Further
evidence that the bacteria defected in these studies are actually within the
wood itself is the fact that no bacteria were detected on the surface
sterilized saw blade, table top, or other equipment after surface sterili-
zation.
The three types of coliforms identified in these studies from all
types of wood, Klebsiella, Enterobacter. and £. freundii, have few bio-
chemical test differences from previously reported studies (2,4). The only
test reaction with possible significance was the increase in numbers of
Enterobacter spp. able to ferment inositol. Forty-eight percent of E.
agglomerans and 33% of IS. cloacae fermented inositol, as compared to
reference values of 18% and 22% (2), respectively, for the two species.
All of the Klebsiella isolates fermented inositol.
41
-------
TABLE 13. BACTERIAL DENSITIES IN LIQUID EXPRESSED FROM REDWOOD AND COLIFORMS IDENTIFIED
NS
Type of wood
Experiment 1.
Sinker HW
Heavy Stain HW
No. of
samples
Bacterial Counts per ml
Total Bacteria*
Total Coliform0 Coliforms Identified
Cutwood samples 1-2 months old
2
1
Young Growth HW 2
Wide Ring Count
Heavy SW
Experiment 2.
Trim green HW
HW
HW and SW
HW and SW chips
HW and SW
HW and SW
SW
Experiment 3.
Bottom log, HW
Bottom log. SW
SW 2
2
Freshly cut wood
1
2
1
2
1
1
1
Freshly cut wood
3
3
3.3 x 105
1.3 x 103
NG
6
4.5 x 10°
1.7 x 106
from saw mill
3.5 x 104
1.7 x 104
5.0 x 105
1.3 x 105
1.0 x 105
1.30 x 105
NG
samples from a log
5.0 x 102
1.4 x 107
NG
NT
NG
TNTC Enterobacter agglmerans
3
2.0 x 10 E. agglomerans
NG
NG
4.0 x 10 Klebsiella pneumoniae
3
2.0 x 10 K. pneumoniae
2
5.0 x 10 K. pneumoniae, K. oxytoca
4
3.0 x 10 K. pneumoniae, E. cloacae,
E. agglomerans
NG
pile.
NG
1.4 x 10 E. agglomerans
(continued)
-------
TABLE 13. (continued)
No. of
Type of wood samples
3rd cut, HW 3
3rd cut, SW 3
Top log, HW 3
Top log, SW 3
Bacterial Counts per ml
Total Bacteriaa Total Coliform Coliforms Identified
NG NG
6 4
1.3 x 10 4.5 x 10 K. pneumoniae, E.
agglomerans, E. sp .
1.0 x 102 NG E. sp.
3 2
5.7 x 10 3.2 x 10 K. pneumoniae, E.
agglomerans
HW-Heartwood; SW-Sapwood; NG-No growth; NT-Not tested; TNTC-Too numerous to count.
a
Average results after 48 hr of incubation on nutrient agar at 30°C, except for experiment 2 which
was incubated at 37°C.
Average results after 24 hr of incubation on m-Endo agar LES at 35°C, except for experiment 2, which
was incubated at 37°C.
-------
TABLE 14. BACTERIAL DENSITIES IN LIQUID EXPRESSED FROM NON-REDWOOD AND COLIFORMS IDENTIFIED
Type of wood
Cedar
Cottonwood SW
No. of
samples
2
2
Bacterial Counts per ml
Total Bacteria3
<1.0 x 10
3.8 x 105
Total Coliformb
MPN° m-Endod
<2.0 0
6.5 x 101 0
Coliforms Identified
Enterobacter agglomerans
E. agglomerans, Citrobacter
freundii
Douglas fir,
freshly cut SW 3
Douglas fir,
racked SW 2
4.5 x 10
7.0 x 10'
0.9
1.3
0 E. agglomerans
0 Klebsiella pneumoniae,
Hemlock SW 2 2.0 x 106 2.0
E. agglomerans
5 K. pneumoniae, E. agglomerans
C. freundii6
SW-Sapwood; MPN-Most probable number.
aResults after 48 hr incubation at 30°C on nutrient agar.
No fecal coliform were detected.
CAverage of confirmed counts after 24 hr incubation at 35°C on EMB agar.
Average results using m-Endo agar LES after 24 hr incubation at 35°C.
"Isolated as a green-sheened colony on m-Endo agar LES after 48 hr incubation.
-------
TABLE 15. COLIFORMS RECOVERED FROM CONTACT IMPRESSIONS OF FRESHLY EXPOSED WOOD SURFACES
Type of Wood
Redwood, HSW
Redwood, WSW
Cedar
Cottonwood, SW
Douglas fir, freshly cut SW
Douglas fir, racked SW
Hemlock, SW
No. of
Samples
2
2
2
2
6
4
2
No. of Coliforms
24 hr 48 hr
TNTC
TNTC
NG
TNTC
TNTC
NG
NG 9
E_. agglomerans
NG
NG I1
10
b
Coliforms Identified
Enterobacter agglomerans
E_. agglomerans
Citrobacter freundii
E_. agglomerans, £. freundii
C. freundii
HSW-Heavy sapwood; WSW-Wide range count sapwood; SW-Sapwood; TNTC-Too numerous to count; NG-No growth.
Reported as average number of green-sheened colonies appearing on m-Endo agar LES at 35°C.
Number of colonies on only impression plate showing growth.
-------
TABLE 16. COLIFORMS RECOVERED FROM CONTACT IMPRESSIONS OF SERIAL CUTS THROUGH REDWOOD'
No. of Surface
Type of Redwood Samples Examined
Heavy Sapwood 2 upper surface
2C
3
4
center
6
7
8
lower surface
Wide Ring Count Sapwood 2 upper. surface
2
center
4
5
lower surface
No. of Coliforms
24 hr 48
TNTC
12
10
NG
NG
NG
15
30
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
hr
TNTC
150
150
100
100
100
75
75
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC-Too numerous to count; NG-No growth.
al
bT
Q
aBlock roughly 8 in. were cut in 1/4 in. slices.
Reported as average number of green-sheened colonies appearing on m-Endo agar LES at 35°C.
°Number refers to position of cut section from upper surface.
In all cases, E. agglomerans was the only coliform identified and was present in all of the wood sections.
-------
Figure 10 (top). Scanning electron micrograph (SEM) of interior of redwood
wide ring count sapwood (200X). Trachael tubes with round bordered pits are
shown. Arrow indicates field enlarged for Figure 2.
Figure 11 (bottom).
present (l.OOOX).
SEM of the interior of a trachael tube showing bacteria
47
-------
Figure 12. Morphology of individual bacteria within a trachael tube (5.000X)
Although large numbers of total coliforms were isolated from redwood
lumber, and to a lesser degree from other woods, none of the isolates were
fecal coliform positive. It has been previously illustrated that fecal coli-
form positive Klebsiella have been isolated from redwood sawdust, from small,
experimental redwood tanks, and from public drinking water emanating from
redwood reservoirs. While the absence of fecal coliform positive isolates
might lead some to believe these coliforms in lumber are of non-fecal
origins, the comments concerning the health significance of different
Klebsiella biotypes/species mentioned earlier, must also be considered.
In summary, coliforms in drinking water emanating from redwood
reservoirs arise from at least two general types of contamination. This
includes the colonization of redwood lumber before tank assembly and
contamination arising from unsatisfactory source or distribution water
after tank assembly.
Evidence of wood contamination prior to tank assembly was demonstrated
by the presence of large numbers of coliforms present inside of freshly
cut redwood. This contamination could have been caused by the activity
of wood-boring insects, birds, and the exposure of lumber to the elements
before and after processing at the mill. Because of the unique growth
promoting water soluble nutrients in redwood (see next section), any low
48
-------
level of contamination by Klebsiella during storage of staves out of
doors or during tank assembly is also likely to allow colonization of
stave surfaces (Figs. 2 and 3). Colonization is facilitated by an
ineffective chlorine residual.
Fecal coliform positive K. pneumoniae and fecal coliform negative
K. oxytoca have been isolated from drinking water emanating from redwood
reservoirs (6). These species were characterized in depth in this study
and were found to be indistinguishable from isolates derived as primary
pathogens from humans and animals. However, since regrowth of Klebsiella
occurs in the redwood environment, their presence should not imply that
enteric pathogens as Salmonella are also present. The greatest potential
health risk therefore involves the simultaneous presence of both Klebsiella
and JJ. coli, indicating that fecal contamination has been recent. SinTe"
it is not practical to identify coliforms to species during routine
water quality monitoring, coliforms emanating from redwood reservoir
water should be monitored and controlled as in any other potable water
supply system.
NUTRITIONAL BASIS FOR KLEBSIELLEAE SELECTION IN REDWOOD WATER STORAGE
RESERVOIRS
The presence of Klebsielleae in high cell densities in lumber and in
drinking water supplies emanating from redwood reservoirs begs the
question of the mechanism which supports their presence and promotes their
selective proliferation in this unique botanical environment. The report
of Anderson (40) illustrates that seven classes of chemicals are water
extractable from redwood. In the present studies, two of the classes of
compounds were found to be utilized by Klebsielleae as a source of fermentable
carbon. These compounds are cyclitols and free sugars.
It was reported that 4.5% of the aqueous extracts of dry redwood were
cyclitols and only 0.1% were free sugars (40). The sugars present are L-
arabinose, D-glucose, D-fructose, L-rhamnose, sucrose, and raffinose.
A review of the biochemical reactions of the Enterobacteriaceae indicates
that representatives of all the genera are able to utilize one or more of
these sugars as an energy source (2). Thus, carbohydrates could not be
assumed to represent the selective basis for Klebsiella and Enterobacter
multiplication in redwood environments.
The cyclitols present in redwood extracts consist of pinitol (60%),
sequoyitol (30%), and inositol (10%) [Fig. 13, references 17,40)]. A review
of the biochemical reactions of the Enterobacteriaceae indicates that
inositol is a distinct differential substrate fermented almost solely by
members of the Klebsielleae (2). Information on the fermentation of the
other cyclitols by Enterobacteriaceae was not found in the literature.
Several approaches were used to determine the nutritional significance
of cyclitols in selectively supporting the multiplication of Klebsiella and
Enterobacter in drinking water present in redwood reservoirs. These studies
49
-------
H OH
I i
CH30
H HO
H] -fH
OH H OH H
SEQUOYITOL meso-INOSITOL
Figure 13. Cyclitols present in aqueous redwood extracts.
included surveys of the fermentability of cyclitols by various genera of
enteric bacteria, turbidimetric measurement of growth in defined media con-
taining a cyclitol as the sole carbon and energy source, measuring growth
in aqueous extracts of redwood sawdust, and finally, the iri situ measurements
of cyclitol utilization in aqueous redwood extracts as determined by gas
chromatography.
The data in Tables 17 and 18 illustrate the genera of various enteric
bacteria which produce acid in phenol red broth base containing a cyclitol
as the fermentable substrate. Some 180 isolates of the Tribe Klebsielleae
were tested for inositol fermentation and only 1 isolate of Enterobacter
cloacae was negative. Over 80% of the Klebsiella isolates from both clinical
and non-clinical environments also fermented pinitol while fewer isolates of
Enterobacter and neither of the Serratia possessed this capacity. None of
the IS. coli, Aeromonas, Shigella, and Proteus isolates fermented any of the
cyclitols tested. A smaller number of isolates was tested with sequoyitol
because of the small amount of this chemical available for testing. Never-
theless, 37/40 members of the Klebsielleae also fermented this cyclitol.
Some isolates of Salmonella and Yersinia enterocolitica also fermented 1
or more cyclitols and thus there exists the possibility that enteric patho-
gens may be capable of colonizing the redwood environment as well. Results
of further examinations in the other test systems, however, illustrate that
this is not to be expected in the case of Salmonella. Also, exhaustive
attempts to isolate Salmonella by conventional enrichment procedures from
redwood sawdust, lumber, and from the 3,785 liter (1,000 gal) experimental
tank failed. Isolation of Yersinia was not attempted.
In order to confirm that cyclitols can serve as sole sources of carbon
and energy, selected enteric bacteria were grown in a defined medium with
either 0.2% inositol or pinitol. Growth at 35°C was monitored by measuring
culture turbidity. Table 19 indicates that all tested Klebsiella. Entero-
50
-------
bacter, Serratia, and Erwinia exhibited growth to moderately high levels of
turbidity. The _E. coli, _P. aeruginosa, and Citrobacter clearly did not grow
while the Yersinia isolate exhibited less than 2 generations of growth.
Figs. 14 and 15 demonstrate the growth kinetics of 4 selected Klebsiella
while using inositol or pinitol as a carbon and energy source. The growth
rate and final cell yield are seen to vary from isolate to isolate, but
clearly all Klebsiella tested can utilize either substrate. Control flasks
without added cyclitols had insufficient turbidity to measure.
Tables 20 and 21 summarize results of a survey in which various species
of enteric bacteria and 1?. aeruginosa were tested for multiplication in non-
sterile (indigenous flora) and sterile aqueous extracts of sawdust (1.67g
sawdust/1 water). Twelve of the 14 Klebsiella tested in the sterile extract
increased from 102 - 1Q4 times over the level of inoculation. Nine of the 14
same isolates increased IQl - 1Q3 times in the presence of indigenous
bacteria associated with the sawdust. Other representatives of the Klebsi-
ella and Erwinia also multiplied extensively in the extracts. P_. aeruginosa,
JE. coli, Citrobacter, and Yersinia enterocolitica also multiplied in the
sterile extract but, except for modest growth by Yersinia, did not grow
in the presence of the indigenous flora. The growth by the latter genus
in the sterile extract probably reflects utilization of the low level of
carbohydrates present which are likely to also be rapidly metabolized by
the indigenous flora.
Except for one significant exception, Salmonella, growth in non-
sterile sawdust extracts coincides well with fermentation of cyclitols
in phenol red broth base. The lack of Salmonella growth in extracts and
the ability to ferment cyclitols in broth reflects the sensitivity of
this enteric pathogen to some growth inhibitory substances in aqueous
sawdust extracts. Klebsiella and Erwinia are resistant to such inhibition.
In the final experimental series a gas chromatograph was utilized to
detect cyclitols in redwood reservoir water and to demonstrate the in situ
utilization of these nutrients by Klebsiella and Enterobacter in aqueous
redwood extracts.
Fig. 16 demonstrates a typical gas chromatograph profile and retention
times for components contained in a 4 g/1 aqueous redwood sawdust extract.
All cyclitols present in redwood are detected in the relative proportions
reported previously (40), and are sufficiently resolved so as to permit
quantitative estimates of their concentration.
In all growth experiments an extract was prepared from 1.67 g sawdust/1
distilled water even though inositol could not be detected in this more
dilute preparation (Fig. 17).
This extract was a compromise which reflected a sawdust extract
cyclitol concentration comparable to what was observed in water stored
in redwood reservoirs and which was neither growth inhibitory due to
high polyphenolic concentrations nor provided sufficient extraneous
contaminants to indiscriminatly allow the growth of non-cyclitol using
51
-------
TABLE 17. UTILIZATION OF INOSITOL AND PINITOL BY ENTEROBACTERIACEAE AND PSEUDOMONAS
Ul
Positive On
Phenol Red Inositol (1%)
Number Tested
Escherichia coli
Shigella
Salmonella
Citrobacter
Klebsiella pneumoniae (ATCC)
Klebsiella pneumoniae (environ.)
Klebsiella pneumoniae (clinical)
Klebsiella pneumoniae (mastitis)
Klebsiella (ATCC-oxytoca)
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter liquefaciens
Enterobacter hafniae
Enterobacter cloacae
Serratia marcescens
Proteus vulgaris
Erwinia carotovora
Erwinia (other)
26
3
5
2
3
60
43
30
2
10
21
2
1
8
2
2
2
5
Number Positive
0
0
2
1
3
60
43
30
not tested
10
21
2
1
7
2
0
1
1
Positive On
Phenol Red Pinitol (1%)
% Number Positive %
0
0
40
50
100
100
100
100
100
100
100
100
88
100
0
50
20
0
0
2
0
3
52
39
23
2
9
11
2
0
1
0
0
0
0
0
0
40
0
100
87
91
77
100
90
52
100
0
13
0
0
0
0
(continued)
-------
TABLE 17. (continued)
Positive On Positive On
Pseudomonas
Yersinia enterocolitica
Yersinia pseudotuberculosis
Number Tested
3
7
2
Phenol Red Inositol
Number Positive
1
7
0
(1%)
33
100
0
Phenol Red Pinitol
Number Positive
0
0
0
(1*)
0
0
0
Oi
OJ
-------
TABLE 18. UTILIZATION OF SEQUOYITOL BY ENTEROBACTERIACEAE
AND PSEUDOMONAS
Number Tested
Escherichia coli
Shigella
Salmonella
Citrobacter
Klebsiella pneumoniae (ATCC)
Klebsiella pneumoniae (environmental)
Klebsiella pneumoniae (clinical)
Klebsiella pneumoniae (mastitis)
Klebsiella (ATCC-oxytoca)
Enterobacter aerogenes
Enterobacter cloacae
Serratia liquefaciens
Serratia marcescens
Proteus
Erwinia
Yersinia enterocolitica
Pseud omonas
4
2
4
1
3
18
7
5
2
1
1
1
2
1
4
4
1
Number Positive
0
0
2
0
3
17
7
5
2
1
0
1
1
0
0
1
0
% Positive
0
0
50
0
100
94
100
100
100
100
0
100
50
0
0
25
0
54
-------
TABLE 19. GROWTH RESPONSE (O.D., ) WITH 0.2% INOSITOL PLUS BASAL
SALTS MEDIUM
Culture O.D., A at 0 hr O.D.,-... at 13 hr O.D.,.- at 20 hr
bUU oOO out)
Yersinia enterocolitic .009 .021 .032
Enterobacter agglomerans .009 .039 .112
(Erwinia herbicola)
Erwinia carotovora .010 .040 .109
Klebsiella ATCC13182 .010 .320 .396
Ps. aeruginosa .015 .015 .012
Serratia marcescens .012 .050 .105
Enterobacter aerogenes .012 .368 .568
_E. coll .009 .011 .009
Citrobacter freundii .009 .005 .001
Serratia liquefaciens .009 .200 .288
(formerly Enterobacter)
55
-------
1.0
0.5
o
o
to
Q
O 0.10
0.05
0.01
i I I I I
- 0.2% INOSITOL
i I I i
J I
• KLEBSIELLA SL-I
A KLEBSIELLA VIII
o KLEBSIELLA PC2
• KLEBSIELLA BCI827
I I I I I I
6 8
TIME (HOURS)
10
12
14
1.0
0.5
o
o
(O
Q 0.10
o
Q 0.05
0.01
Ul I | I T I I
0.2% PINITOL
i r
\ r
• KLEBSIELLA SL-I
A KLEBSIELLA VIM
o KLEBSIELLA PC»
• KLEBSIELLA BCI827
I I
I
I I
I i
468
TIME (HOURS)
10
12
I -
I i
14
Figure 14 (top). Growth of Klebsiella in defined basal salts medium with
inositol as sole carbon and energy source.
Figure 15 (bottom). Growth of Klebsiella in a defined basal salts medium
with pinitol as sole carbon and energy source.
56
-------
TABLE 20. GROWTH RESPONSE IN AQUEOUS EXTRACTS OF REDWOOD SAWDUST IN PRESENCE OF INDIGENOUS FLORA
Culture Standard Plate Count per ml, Days After Inoculation
Klebsiella pneumoniae
PSB49-1
PS401LR
MSU1684
SL-1
UT1500
U010994
U011065
MH31
JH2
H
pc2n
K2
13882
13182
13883
Enterobacter aerogenes 13048
Enterobacter cloacae
Serratia llquefaclens 14460
Serratia marcescens
Enterobacter agglomerans
Origin
bovine masitits
bovine mastitis
bovine mastitis
human medical
human medical
human medical
human medical
human medical
drinking water
drinking water
redwood
ATCC
ATCC
ATCC
ATCC
OSU
ATCC
OSU
0
3.9 x 102
2.6 x 102
1.1 x 102
3.0 x 102
4.5 x 102
3.1 x 102
1.6 x 102
3.9 x 102
6.0 x 101
4.9 x 102
3.0 x 102
o
4.3 x 10
1.8 x 102
1.6 x 102
1.9 x 102
3.3 x 102
6.5 x 102
2.7 x 102
1.8 x 102
2
4.3 x 104
9.4 x 104
1.1 x 102
4.7 x 103
2.1 x 103
5.6 x 102
1.8 x 102
6.0 x 102
1.8 x 103
1.5 x 104
3.4 x 102
0
7.4 x 10
3.4 x 103
5.5 x 102
3.7 x 102
3.7 x 102
7.0 x 103
3.8 x 102
3.2 x 103
6
1.4 x 105
3.0 x 105
3.0 x 101
3.4 x 104
3.3 x 103
3.6 x 103
1.7 x 102
5.3 x 102
3.0 x 104
7.0 x 105
4.1 x 102
5
3.2 x 10
1.6 x 104
9.2 x 102
1.1 x 103
2.1 x 102
7.3 x 103
4.1 x 102
3.1 x 105
(continued)
-------
TABLE 20. (continued)
Standard Plate Count per ml, Days After Inoculation
00
Culture
Erwinia carotovora
Yersinia enterocolitica
Pseudomonas aeruginosa
Citrobacter freundii
Escherichia coli DS5-1
Shigella dysenteriae
Salmonella typhi
Salmonella enteritidis
Proteus vulgaris
Indigenous flora (uninoculated)
Water control (PSB49-1)
Origin 0
3.0 x 102
3.0 x 102
5.4 x 102
2
5.9 x 10
4.5 x 102
3.2 x 102
1.6 x 102
6.6 x 10
3.1 x 102
4.6 x 102
5.4 x 102
2
1.0 x 105
1.1 x 10
2.2 x 102
2
5.5 x 10
5.0 x 102
-------
TABLE 21. GROWTH RESPONSE IN STERILE EXTRACTS OF REDWOOD SAWDUST
Cn
vo
Culture
Klebsiella pneumoniae
PSB-49-1
PS 401LR
MSU 1684
SL 1
UT 1500
UO 10994
UO 11065
MH 31
JH42
PC2II
K2
13882
13182
13883
Enterobacter aerogenes 13048
Enterobacter cloacae
Enterobacter agglomerans
Serratia liquefaciens 14460
Serratia marcescens
Standard Plate
0
9
4.0 x 10
2
6.4 x 10
1.8 x 102
7.4 x 102
4.1 x 102
6.6 x 102
6.6 x 102
7.3 x 102
4.9 x 102
1.1 x 103
5.2 x 102
8.8 x 102
6.4 x 102
5.4 x 102
1.0 x 103
3.6 x 102
1.7 x 102
3.1 x 102
2.5 x 102
count per
2^
3
6.7 x 10
4
2.1 x 10
9.0 x 101
4.7 x 103
3.0 x 104
5.8 x 102
9.5 x 102
1.8 x 104
1.3 x 104
4.3 x 104
6.7 x 103
1.0 x 103
8.8 x 102
1.3 x 103
1.2 x 103
6.1 x 102
1.2 x 102
1.7 x 103
3.1 x 102
ml, days after inoculation
6
6
1.1 x 10
6
, 1.1 x 10
2.0 x 101
1.1 x 106
1.5 x 106
8.7 x 104
6.8 x 104
1.2 x 106
5.6 x 105
1.3 x 106
3.4 x 106
1.1 x 106
1.1 x 106
2.1 x 104
1.5 x 105
8.0 x 102
3.7 x 105
1.2 x 106
8.9 x 102
(continued)
-------
TABLE 21. (continued)
Culture
Erwinia carotovora
Yersinia enterocolitica 867
Pseudomonas aeruginosa
Citrobacter freundii
Escherichia coli DS5-1
Shigella dysenteriae
Salmonella enteritidis ser.
paratyphi B
Standard Plate count per ml, days after inoculation
r\ o £
1.1 x 10;
5.5 x 10'
1.5 x 10J
9.5 x 10"
5.6 x 10'
1.5 x 10'
8.5 x 10'
1.6 x 10~
2.3 x 10
3.1 x 10
1.8 x 10
6.6 x
7.6 x
1.1 x
3.0 x 101
1.2 x 10'
£
2.5 x 10*
2.4 x 10
5.9 x 10*
1.1 x 10^
3.3 x 10-
1.0 x 10]
1.0 x 103
1.0 x 10
Procedure for these experiments was the same as with the non-sterile redwood (Table 20), except the
redwood extract was filter sterilized before it was placed in the flasks, and the cultures were plated
on plate count agar without antibotics.
-------
AQUEOUS REDWOOD
EXTRACT 4g/1000 ml
QUEBRACHITOL
(INTERNAL STANDARD - 0.5 ug)
INOSITOL
(MAJOR PEAK)
QUEBRACHITOL
(MINOR PEAK)
INOSITOL
(MINOR PEAK)
AQUEOUS REDWOOD
EXTRACT l.67g/IOOOml
SEQUOVITOL
TIME
°t02" Il'l5"
TIME (MINUTES)
Figure 16. Gas chromatograph (GC) profile of an aqueous redwood sawdust extract illustrating the
relative abundance of the 3 cyclitols present.
Figure 17. GC profile of an aqueous redwood sawdust extract at the optimum concentration used to
measure in situ utilization of cyclitols (see Figs. 21-23).
-------
species (Table 22a). Figures 18, 19 and 20 show the relative proportion
of cyclitols present in water stored in small experimental redwood tanks
housed in the laboratory. Tank 2 was adjusted to an 8-day retention
time and was on line about 2 months (about 8 retention turnovers) when
the determinations were made. Tank 3 was sampled on two occasions, the
first 1 month and the second 2 months after addition of finished municipal
drinking water. The water in this tank was static. The profiles illustrate
the continuing accumulation of cyclitols which are leached out of the
wooden staves. The concentrations of these cyclitols, which can support
growth of Klebsielleae, are presented in Table 23.
Bacterial studies on the tank water at the time of sample preparation
showed tank 3 contained about 103 total coliforms/ml and some 106 total
bacterial/ml. Tank 2 had no detectable coliforms (by MF). However,
these filters were massively contaminated with non-coliform colonies
which could have masked coliform detection and enumeration in both tank
samples.
Cyclitols can still be detected in the tank water probably because
cyclitols are leached out faster than coliforms can use them under the
given temperature .and overall nutritional limitations of the milieu.
Elevated cyclitol concentrations are more likely to be found in newer
redwood reservoirs during the first year or so of operation. Tank 2,
which has water continuously entering and leaving, probably established a
flora and nutrient concentration much like that operating in a chemostat.
Figs. 21-23 illustrate the cyclitol profiles after 4 days of incu-
bation of the indicated cultures in the 1.67 g/1,000 ml sawdust extract.
The profiles demonstrate that during growth to levels of 105 to 106
cells/ ml there is an obvious decline in both the sequoyitol and pinitol
peak heights (control is Fig. 17). Table 24 summarizes the quantitative
utilization of these cyclitols during the incubation of Klebsiella and an
E_. agglomerans isolate which was also examined. The Klebsiella utilized
60-90% of the available pinitol and 90-100% of the sequoyitol while the
E^. agglomerans used 94-100% of both cyclitols. These results confirm
that under conditions which simulate those found in redwood reservoirs,
the Klebsiella and Enterobacter are capable of multiplication on cyclitols
as a source of carbon and energy. Cyclitols leach from the staves due
to their extraction by water. These observations can account for the
presence of high numbers of Klebsielleae in redwood lumber, on staves,
and in the drinking water stored in redwood reservoirs. This confirms
that necessity for maintaining adequate chlorine residuals, short retention
times, and adequate mixing of finished drinking water stored in redwood
reservoirs.
62
-------
TABLE 22. GROWTH RESPONSE IN VARIOUS CONCENTRATIONS OF AQUEOUS EXTRACTS OF REDWOOD SAWDUST
Grams of redwood used
per liter of water
U)
Standard plate count per ml,
days after inoculation
0
Klebsiella pneumbniae
PSB49-1 0
.10
.21
.42
.835
1.67
8.35
Escherichia coli DS5-1 0
.10
.21
.42
.835
1.67
8.35
9
5.5 x 10
5.4 x 10,
3.5 x 10,
4.0 x 10,
4.2 x 10,
6.2 x 10,
4.2 x 10
6.0 x 10,
6.3 x 10,
5.8 x 10
4.5 x 10,
5.8 x 10,
6.2 x 10,
4.6 x 10
9
4.5 x 10,
5.1 x 10,
3.7 x 10,
7.5 x 10,
1.0 x 10,
2.1 x 10
6.8 x 10
4.9 x 10 J
5.6 x 10,
5.8 x 10,
5.0 x 10,
4.1 x 10,
7.0 x 10,
1.8 x 10
9
4.0 x 10,
3.3 x 10
1.3 x 10 ;:
8.2 x 10^
5.3 x 105
8.4 x IQr
1.3 x 10
2.1 x 10,
3.4 x 10,
3.6 x 10,
1.8 x 10
9.0 x 10:
6.8 x 107
6.0 x 10
-------
UJ
V)
o
a.
w
UJ
cc
tr
o
UJ
o
TANK 2
SEQUOYITOL
TIME
Figure 18. Cyclitols present in redwood tank water on 8 day retention
period.
64
-------
TANK 3
TIME TIME
Figure 19 (left). Cyclitols present in redwood tank water held stagnant for
1 month.
Figure 20 (right). Cyclitols present when water is stagnant for 2 months.
TABLE 23. CYCLITOL CONCENTRATIONS IN REDWOOD EXTRACTS AND EXPERIMENTAL
REDWOOD TANKS
SAMPLE
1.67g sawdust/1000ml
4.00g sawdust/1000ml
Tank 3 3/10/78
Tank 3 3/14/78
Tank 2 3/14/78
ug/ml pinitol ug/ml sequoyitol ug/ml inositol
30.38 10.24 ND
60.49 19.51 8.36
28.58 10.38 1.95
56.61 18.58 0.35
26.51 5.61 ND
65
-------
KLEBSIELLA E44.5-I
TIME
TIME
KLEBSIELLA PSB-49-1
SEQUOYITOL
TIME
Figures 21-23. Cyclitols remaining in 1.67 g/liter agueous redwood sawdust
extract after 4 days of incubation of Klebsiella at 35°C. MH24, human
origin; E44.5-1, redwood sawdust origin; PSB-49-1, bovine mastitis origin.
66
-------
TABLE 24. UTILIZATION OF CYCLITOLS IN REDWOOD EXTRACT BY KLEBSIELLEAE
ON
Pinitol
yg/ml in
extract
Culture at inoculation
—
E. agglomerans
(Erwinia herbicola
ICPB EH103) 30.4
Klebsiella
E44.5-1 (redwood) 30.4
MH 24 (vegetable) 30.4
BC 1959 (pulp mill) 30.4
PSB-49-1 (mastitis) 30.4
4 days
1.8
9.1
6.5
12.6
3.0
Sequoyitol
yg/ml in
extract
"/ 7
/O '0
utilzation at inoculation 4 days utilization
94 10.2 ND 100
70 10.2 ND 100
79 10.2 ND 100
59 10.2 0.8 92
90 10.2 ND 100
ND = not detected.
-------
REFERENCES
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Environmental Protection Agency, Office of Public Affairs, Washington,
D.C., 1976. 9 pp.
2. Edwards, P. R., and W. H. Ewing. Identification of Enterobacteriaceae,
3rd ed. Burgess Publishing Co., Minneapolis, 1972.
3. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. Antibiotic
Susceptibility Testing by a Standardized Single Disk Method. Amer. J.
Clin. Pathol. 45:493-496, 1966.
4. Blair, J. E., E. H. Lennette, and J. P. Truant. Manual of Clinical
Microbiology. Williams and Wilkins, Baltimore, Maryland, 1970.
5. Hinshaw, V., J. Punch, M. J. Allison, and H. P. Dalton. Frequency of
R-Factor Mediated Multiple Drug Resistance in Klebsiella and Aerobacter.
Appl. Microbiol. 17:214-218, 1969.
6. Bagley, S. T., and R. J. Seidler. Significance of Fecal Coliform-
Positive Klebsiella. Appl. Environ. Microbiol. 33:1141-1148, 1977.
7. Naemura, L. G., and R. J. Seidler. Significance of Low-Temperature
Growth Associated with the Fecal Coliform Response, Indole Production,
and Pectin Liquefaction in Klebsiella. Appl. Environ. Microbiol.
35:392-396, 1978.
8. Armitage, P. Statistical Methods in Medical Research. Blackwell
Scientific Publications, Oxford, 1971.
9. Snedecor, G. W., and W. F. Cochran. Statistical Methods, 6th ed. The
Iowa State University P/ess, Ames, 1967.
10. American Public Health Association. Standard Methods for the
Examination of Water and Wastewater, 14th ed. American Public
Health Association, Inc., New York, 1975.
11. Bordner, R. J., and B. J. Carroll. Proceedings of Seminar on the
Significance of Fecal Coliform in Industrial Wastes. No. TR-3
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Center, U. S. Environmental Protection Agency, Denver, Colo., 1972.
68
-------
12. DeWys, W. D., A. Goldin, and N. Mantel. Hematopoietic Recovery after
Large Doses of Cyclophosphamide: Correlation of Proliferative State
with Sensitivity. Cancer Res. 31:1692-1697, 1970.
13. Baird-Parker, A. C. Family Micrococcaceae. In: Sergey's Manual of
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eds. The Williams and Wilkins Co., Baltimore, Maryland, 1974.
14. Gibson, T., and R. E. Gordon. Genus Bacillus. In: Bergey's Manual of
Determinative Bacteriology, 8th ed. R. E. Buchanan and N. E. Gibbons,
eds. The Williams and Wilkins Co., Baltimore, Maryland, 1974.
15. Brown, C., and R. J. Seidler. Potential Pathogens in the Environment:
Klebsiella Pneumoniae, a Taxonomic and Ecological Enigma. Appl.
Microbiol. 25:900-904, 1973.
16. Cohen, A. L., D. P. Marlow, and G. E. Garner. A Rapid Critical Point
Method Using Fluorocarbons ("Freons") as Intermediate and Transitional
Fluids. J. Microscopie 7:331-342, 1968.
17. Anderson, A. B., R. Riffer, and A. Wong. Chemistry of the Genus
Sequoia - VI. On the cyclitols Present in Heartwood of Sequoia
sempevirens. Phytochem. 7:1867-1870, 1968.
18. Henriksen, S. D. Studies on the Klebsiella Group (Kaufman) II.
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19. Buttiaux, R. 1959. The Value of the Association Escherichia-group A
Streptococci in the Diagnosis of Contamination in Foods. J. Appl.
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20. Mossel, D. A. An Ecological Investigation on the Usefulness of Two
Specific Modifications of Eijkman's Test as One of the Methods Used
for the Detection of Fecal Contamination of Foods. J. Appl. Bacteriol.
25:20-27, 1962.
21. Lautrop, H. Gelatin-Liquefying Klebsiella Strains (Bacterium oxytocum
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22. Stenzel, W., H. Burger, and W. Mannheim. Zur systematik und
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Gung der Sogenannten Oxytocum-typen. Zentralbl. Bakteriol. Parasxtenkd.
Infektionskr. Hyg. Abt. 1 Orig. Reihe A 219:193-203, 1972.
23. Von Reisen, V. L. Pectinolytic Indole-Positive Strains of Klebsiella
pneumoniae. Int. J. Syst. Bacteriol. 26:143-145, 1976.
24. Seidler, R. J., J. E. Morrow, and S. T. Bagley. Klebsielleae in
Drinking Water Emanating from Redwood Tanks. Appl. Environ. Microbiol.
33:823-905, 1977.
69
-------
25. Eickhoff, T. C. Klebsiella pneumoniae Infection: a Review with
Reference to the Water-Borne Epidemiologic Significance of 1C.
pneumoniae Presence in the Natural Environment. National Council
of the Paper Industry for Air and Stream Improvement, Inc., Technical
Bulletin No. 254, New York, N.Y., 1972.
26. Geldreich, E. E. Sanitary Significance of Fecal Coliforms in the
Environment. U. S. Department of the Interior, Federal Water
Pollution Control Administration Publication WP 20-3. U. S. Government
Printing Office, Washington, B.C., 1966.
27. Davis, T. J., and J. M. Matsen. Prevalence and Characteristics of
Klebsiella Species. Relation to Association with a Hospital Environ-
ment. J. Infect. Dis. 130:402-405, 1974.
28. Duncan, D. W., and W. E. Razzell. Klebsiella Biotypes among Coliforms
Isolated from Forest Environments and Farm Produce. Appl. Microbiol.
24:933-938, 1972.
29. Dufour, A. P., and V. J. Cabelli. Characteristics of Klebsiella from
Textile Finishing Plant Effluents. J. Water. Poll. Control Fed.
48:872-879, 1976.
30. Bagley, S. T., and R. J. Seidler. Significance of Fecal Coliform-
positive Klebsiella. Appl. Environ. Microbiol. 33:1141-1148, 1977.
31. Knittel, M. D., R. J. Seidler, C. Eby, and L. M. Cabe. Colonization
of the Botanical Environment by Klebsiella Isolates of Pathogenic
Origin. Appl. Environ. Microbiol. 34:557-563, 1977.
32. Bagley, S. T., and R. J. Seidler. Comparative Pathogenicity of Environ-
mental and Clinical Klebsiella. Health Lab Sci. In Press, 1978.
33. Shooter, R. A., M. C. Faiers, E. M. Cooke, A. L. Breaden, and S. M.
O'Farrell. Isolation of Eshcerichia coli, Pseudomonas aeroginosa, and
Klebsiella from Food in Hospitals, Canteens, and Schools. Lancet
275:390-392, 1971.
34. Dondero, N. C., C. T. Thomas, M. Khare, J. F. Timoney, and G. M.
Fukui. Salmonella in surface waters of central New York State. Appl.
Environ. Microbiol. 33;791-801, 1977.
35. Martin, W. J., P. K. W. Yu, and J. A. Washington. Epidemiological
Significance of Klebsiella pneumoniae - a 3 Month Study. Mayo Clin.
Proc. 46:785-793, 1971.
36. Montgomerie, J. X., D. E. M. Taylor, P. B. Doak, J. D. K. North, and
W. J. Martin. Klebsiella in Faecal Flora of Renal-Transplant Patients.
Lancet 2:787-792, 1970.
70
-------
37. Selden, R. , S. Lee, W. L. L. Wang, J. V. Bennett, and T. C. Eickhoff.
Nosocomial Klebsiella Infections: Intestinal Colonization as Reservoir.
Ann. Intern. Med. 74:657-664, 1971.
38. Klein, D. J., J. A. Spindler, J. M. Matsen. Relationship of Indole
Production and Antibiotic Susceptibility in the Klebsiella Bacillus.
J. Clin. Microbiol. 2:425-429, 1975.
39. Talbot, Jr., H. W. , J. E. Morrow, and R. J. Seidler. Control of
Coliform Bacteria in Finished Drinking Water Stored in Redwood Tanks.
J. A. W. W. A. In Press, 1979.
40. Anderson, A. B. The Influence of Extractives on Tree Properties.
1. California Redwood (Sequoia sempevirens). J. Inst. Wood Sci.
8:14-34, 1961.
41. Klein, D. A., and S. Wu. Stress: a Factor to be Considered in
Heterotrophic Microorganism Enumeration from Aquatic Environments.
Appl. Microbiol. 27:429-431, 1974.
71
-------
APPENDIX
COMPARISON OF STANDARD PLATE COUNT OBTAINED WITH STANDARD METHODS AND
TRIS BUFFERS AS DILUENTS
The purpose of these experiments was to compare the efficiency in
the recovery of viable counts of various Klebsiella cultures following
dilutions through standard methods phosphate buffer (SM) and Tris-
hydroxymethyl aminoethane (Tris) buffer. The influence of nutritional
and temperature stress was also monitored by holding the cells in the
diluent on ice during plating and then at 40°C for 2 hr. The results
presented in the table below illustrate that Tris buffer diluent yields
counts which are higher (although not significantly) than those obtained
with SM buffer.
TABLE A-l. COMPARISON OF STANDARD PLATE COUNTS WITH DIFFERENT BUFFERS
Klebsiella Culture
d
TJ me ATCC
(hr) SMB
0 73G '
77
87
Average 79
2 69
84
81
Average 78
13883
TB°
76
64
69
69
87
78
100
88
PSB-49-1
SMB TB
97
85
120
100
96
99
98
98
122
106
136
121
112
117
139
123
B14
SMB TB
37
36
33
35
35
50
32
39
48
38
38
41
33
65
40
46
Cultures were shaken in Nutrient Broth with 1% added glucose for 20 hr at
37°C.
Standard Methods Phosphate Buffer, sterile.
0.01 M Tris Buffer pH 7.5, sterile.
(continued)
72
-------
TABLE A-l. (continued)
d
Triplicate plate counts were made at 0 and 2 hours after culture dilutions.
All samples and dilutions were kept on ice during actual plating times and
in a 40°C incubator between platings.
_ o
All counts represent number of colonies on the 10 Standard Plate Count
Agar plates. Statistical analyses of all averages, using Student's t at
a = 0.05, indicate no significant differences between any of the averages,
regardless of time sampled or buffer used. All counts are times 10
cells/ml.
73
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
i. REPORT NO.
EPA-6QQ72-79-049
2.
3. RECIPIENT'S ACCESSION'NO.
4. TITLE AND SUBTITLE
COLIFORMS IN DRINKING WATER EMANATING FROM
REDWOOD RESERVOIRS
5. REPORT DATE
July 1979 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ramon J. Seidler
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Oregon State University
Corvallis, Oregon 97331
(Department of Microbiology)
10. PROGRAM ELEMENT NO.
ICC 824J SOS 2; Task 25
11. CONTRACT/GRANT NO.
R 804456
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory—Gin, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final July 1976 July 1978
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Harry D. Nash (513) 684-7232
16. ABSTRACT
Coliforms of the genus Klebsiella were found in many finished drinking
water supplies which used reservoirs constructed from redwood. Coliform
contamination was associated with slime which accumulated on some stave surfaces
below the water line on the tank interior. Based on the results of field surveys,
design modifications were made on an experimental 1,000 gallon reservoir in order
to control coliform contamination. Coliforms were not found when free chlorine
residuals of 0.3 to 0.4 ppm were maintained in the tank water. Poor water mixing
and retention times of 7 days or longer made it difficult to maintain a free
residual due to the chlorine demand of the wooden reservoir. Minimum retention
times (less than 7 days) and adequate water mixing (no common inlet/outlet) can be
achieved by proper choice of reservoir size and regulation of float levels. In the
absence of free chlorine residuals, Klebsiella are able to multiply on water soluble
nutrients leached from the redwood. Cyclitols, comprising 4.5% redwood dry weight,
are the nutrients which uniquely support the growth of Klebsiella and Enterobacter
in the redwood environment. Information in this report will be of interest to
technically oriented personnel in water supply and treatment and environmental
microbiologists.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Water tanks
Water Storage
Coliform bacteria
Chlorination
Water Treatment
Potable water
Klebsiella
coliform contamination
redwood tanks
cyclitol nutrient
13B
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
86
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
74
« U.S. GOYERNUEXTPSIKTfflGOmCt 1979-657-060/5340
------- |