AN ABSTRACT OF THE THESIS OF
JOAN MARIE LATIMER
for the
(Name)
in
MICROBIOLOGY
presented on
(Major)
MASTER OF SCIENCE
(Degree)
September 5, 1973
(Date)
Title: R FACTOR TRANSFER AMONG BACTERIA ISOLATED
FROM THE ENVIRONMENT
Redacted for privacy
Abstract approved:
(----t-cr Dr. Lyle R. Brown
R+ organisms isolated from fecal samples from swine contained
various combinations of the following r determinants: tetracycline,
kanamycin, streptomycin, ampicillin, and carbenicillin.
Of the R+ isolates identified, over 90% were grouped into
several distinct Escherichia coli biotypes; R+ Klebsiella, Salmonella
and Proteus were also encountered. Unique combinations of r determinants were found in association with specific E. coli biotypes, and
one R factor, (Tc, Amp), showed predominance in some samples
taken immediately postweaning. Identification of several isolates
carrying this R factor implicated one biotype of E. coli, (380).
R factor transfer through conjugation was tested into several
recipients, including E. coli, Klebsiella, Salmonella enteriditis, and
Proteus vulgaris, and frequencies ranging from 10 -8 to 10 -1 were
observed. Higher frequencies of transfer were noted into homolgous
species.
Compatibility tests and acridine elimination experiments were
performed on a selected number of R+ isolates.
R Factor Transfer Among Bacteria Isolated
From the Environment
by
Joan Marie Latimer
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
June 1974
APPROVED:
Redacted for privacy
Assistaessor
Microbiology
in charge of major
Redacted for privacy
Chairman Of Department of Microbiology
Redacted for privacy
Dean of Graduate School
Date thesis is presented
September 5, 1973
Typed by Il la W. Atwood for Joan Marie Latimer
ACKNOWLEDGEMENTS
I wish to take this opportunity to express my appreciation to my
major professor, Dr. Lyle R. Brown. His expert guidance and
patience have made my association with him a rewarding and memorable experience.
I am also indebted to Dr. William E. Sandine for the valuable
assistance and encouragement he has given me during this endeavor
and to the department faculty for allowing me to gain valuable teaching experience which I thoroughly enjoyed.
I want to acknowledge the assistance of those undergraduate
students who aided me in my research and to thank my fellow graduate students who have made my association with OSU a pleasant one.
Finally, I want to thank my family without whose encouragement
this endeavor would not have been possible.
This research was supported by Biomedical Sciences Support
Grant No. RR-07079 and Micro life Techniques, Sarasota, Florida.
Additional support was obtained from the Oregon State Agricultural
Experiment Station.
TABLE OF CONTENTS
INTRODUCTION
Discovery of R Factors
Distribution of R Factors
Molecular Nature of R Factors
Elimination of R Factors
Genetics of R Factors
Biochemical Mechanisms of Transferable Drug
Resistance
Role of Antibiotic Feeding in R Factor Prevalence
R+ Bacteria in Human Infections
Statement of Purpose
MATERIALS AND METHODS
Bacterial Cultures
Bacteriophage MS-2
Media
Antibiotic s
R Factor Screening Procedure
Antibiotic Sensitivity Screening Procedure
Identification System for R+ Isolates
Determination of Frequencies of R Factor Transfer
Determination of Sensitivity of R+ Isolates to MS-2
Acridine Elimination Procedure
Compatibility of Transfer Factors Among R+ Isolates
RESULTS
Incidence of Drug Resistance Among Enteric Isolates
Incidence of Transferable Drug Resistance in Swine
which were Fed Lactobacillus Concentrate
Characterization of R Factor Types
Identification of R+ Isolates
MS-2 Bacteriophage Sensitivity Studies
Generic Range of Transfer Among R+ Isolates
Acridine Elimination of R Factors from R+ Isolates
Compatibility Testing of Transfer Factors
Page
1
1
1
2
5
6
8
9
11
13
15
15
15
17
18
19
20
21
22
24
25
26
28
28
28
38
39
50
50
53
58
DISCUSSION
60
SUMMARY
67
BIBLIOGRAPHY
69
LIST OF TABLES
Page
Table
2
3
6
7
8
Sources and Antibiotic Sensitivity Characteristics
of Recipient Cultures
16
Number of Drug Resistant Organisms Among Enteric
Isolates
29
Preliminary R Factor Transfer Frequency Data
30
Incidence of R Factors Found in Swine Which Were
Fed Lactobacillus Concentrate in the Water Supply
32
Types of R Factors Found in Swine Which Were Fed
Lactobacillus Concentrate in Water
33
Incidence of R Factors Found in Swine Which Were
Bottle Fed Lactobacillus Concentrate
34
Types of R Factors Found in Swine Which Were
Bottle Fed Lactobacillus Concentrate
35
Incidence of R Factors Found in Negative Control
Animals
36
Types of R Factors Found in Negative Control
Animals
37
Total Incidence of Number and Types of R Factors
Found in Test Animals Over the Entire Sampling
Period
38
11
Characterization of R Factor Types
Group 1
40
12
Characterization of R Factor Types - Group 2
41
13
Characterization of R Factor Types - Group 3
42.
14
Characterization of R Factor Types - Group 4
43
15
Biochemical Characteristics of R+ E. coli Strains
10
16
Isolated from Swine
44
Identification of R+ Isolates - Group 1
45
LIST OF TABLES (cont.)
'Page
Table
17
Identification of R+ Isolates - Group 2
46
18
Identification of R+ Isolates - Group 3
47
19
Identification of R+ Isolates - Group 4
48
20
Distribution of R Factors Among Major E. coli
Biotypes
49
Frequency of Transfer of R Factors from R+
Isolates into E. coli W3110
51
Frequency of Transfer of R Factors from R+
Isolates into Salmonella enteriditis
52
Frequency of Transfer of R Factors from R+
Isolates into Klebsiella 190-3
54
Frequency of Transfer of R Factors from R+
Isolates into Klebsiella 104-A
55
Frequency of Transfer of R Factors from R+
Isolates into Proteus vulgaris
56
26
Acridine Elimination of R Factors
57
27
Compatibility of Transfer Factors Among R+
Isolates
59
21
22
23
24
25
R FACTOR TRANSFER AMONG BACTERIA ISOLATED
FROM THE ENVIRONMENT
INTRODUCTION
Discovery of Transferable Drug Resistance
Transferable drug resistance was first recognized in Japan in
1959 when Ochai and Akiba (47) independently showed that multiple
drug resistance could be transferred from Escherichia coli to sensitive Shigella strains through conjugation. This observation provided
an explanation for the increasing incidence of Shigella strains in Japan
which were resistant to the antibiotics streptomycin, chloramphenicol,
tetracycline, and sulfonamide following the advent of antibiotic therapy
for bacillary dysentery.
Distribution of R Factors
Since the discovery of transferable drug resistance in Shigella
and E. coli, the presence of the drug resistance episome (R factor)
has been demonstrated throughout the world in most of the genera of
the Enterobacteriaceae (including Salmonella, Klebsiella, Enterobac-
ter, Citrobacter, Proteus, and Serratia (2). R factors have also been
shown to exist in Vibrio comma (47), Aeromonas liquefaciens (4),
Aeromonas salmonicida (5), Pseudomonas (23, 34), and Pasteurella
(17).
Molecular Nature of R Factors
Early incorporation experiments using P32 demonstrated the
effect of isotope decay on R factors in E. coli K-12, indicating that
the cellular material responsible for transferable drug resistance was
DNA (47).
Further evidence confirming the episomal nature of R factor
DNA was presented by Falkow (15) using cesium chloride density cen-
trifugation of DNA from an .R+ Proteus mirabilis. DNA from this
strain showed the presence of a distinct satellite band upon centrifugation. Loss of resistance to one or more antibiotics in the above
strain resulted in a corresponding loss in a particular density region
of the satellite DNA.
Rownd (35) also demonstrated satellite DNA in a Proteus mira-
bilis strain harboring the R factor NR1 using cesium chloride ethidium bromide density centrifugation and alkaline sucrose density
centrifugation. The R factor DNA existed in the form of covalently
closed circles. Multiple copies of the R factor DNA were present in
Proteus, but when the same R factor was transferred into E. coli or
Serratia marcescens, it was found in a ratio of one copy per cell.
Segregation of the NR1 R factor was observed in Proteus into
two distinct molecular species, one having a GC content of 58% and a
density of 1.718 grams/cm 3, and the other having a GC content of
3
52% and a density of 1.712 grams/cm 3. The latter species predomi-
nated in the absence of antibiotics.
Later work done by Rownd (36) with the same R factor showed
that the concentration of R factor DNA in Proteus varied with the
state of cellular growth, significantly increasing in stationary phase.
An extensive review by Clowes (11) summarized the present
state of knowledge of the molecular nature of bacterial plasmids,
including R factors, based on recent research efforts.
Most R factor DNA, analyzed by cesium chloride ethidium
bromide density centrifugation and alkaline sucrose density centrifugation techniques, has been shown to exist in covalently closed circu-
lar forms and in open circular forms. Catenanes of R factors have
also been isolated which are composed of several interlocking cova-
lently closed circular forms. R factor DNA ranges in density from
1.704 grams/cm 3 to 1.711 grams/cm 3, with molecular weights
ranging from 26 x 106 daltons to 78 x 106 daltons.
R factors are composed of two components, the transfer fac-
tor, designated RTF, and the individual drug resistance genes,
designated r determinants. The stability of the above components
may be host dependent as shown by Rownd, and may also be related
to plasmid type. Nisioka (32) has delineated a genetic map of the
R222 R factor as a circular molecule where chloramphenicol, streptomycin, and sulfonamide resistance are coded for within one
4
segment, and tetracycline resistance within an overlapping segment.
The remainder of the circular molecule is involved in the synthesis
of the RTF.
As discussed by Clowes (11), R factors may exist as cointe-
grates which are single macromolecular structures incorporated into
one element capable of independent replication as in the case of
R
222, or conversely, as plasmid aggregates, where the resistance
transfer factor exists separately from the r determinant and transfer occurs in a parallel fashion, independent of actual physical association between the two elements. Transfer of such elements would
involve a linear DNA strand as discussed by Ohki (33) and Rupp (37)
in the transfer of F.' episomes.
The relaxed replication (multiple copies) of R factor DNA in
Proteus noted by Rownd (36) was also observed by Kopecko (24). The
separation of molecular species led to the postulation that both the
RTF (density 1.710 grams/cm 3) and the r determinant (density
1.718 grams/cm 3) were subject to a negative control system due to
the presence of a repressor coded by the RTF, and that the RTF,
which was attached to the membrane, was under positive control.
Attachment of one strand of R factor DNA to protein was noted
by Helinski (20) who proposed that a protein-DNA complex might
play a role in R factor replication, and that the protein could be an
endonuclease needed to produce a strand specific nick. The above
5
data were in agreement with experiments done by Falkow (16) who
demonstrated that newly transferred R factor DNA became mem-
brane attached in a linear form, was subsequently released in open
circular form, and finally appeared as covalently closed circular
DNA.
Elimination of R Factors
Use of the intercalating dyes acridine orange and acriflavine to
eliminate the R222 episome, which coded for resistance to strepto-
mycin, chloramph.enicol, tetracycline, and sulfonamide, was first
demonstrated by Watanabe (46). Elimination of plasmid DNA has been
shown to result from inhibition of DNA replication. Nishimura (31)
suggested that sensitivity to such dyes may occur during the initiation
of replication; in support of this conclusion he observed that chromo-
somal replication in E. coli showed increasing sensitivity to acridine
orange only when the chromosome contained an integrated F factor.
The drug rifampin, which prevents transcription of DNA by
specifically binding to the RNA polymerase molecule, has also been
shown to eliminate episomes (7). This may indicate that transcrip-
tion must occur prior to initiation of DNA replication in R factors,
as in the bacteriophage lambda (14).
Genetics of R Factors
R factor transfer, although primarily mediated through conjugation, has also been observed to occur through transduction (49).
The genetic classification of R factors is discussed in a detailed
review by Meynell and Datta (28).
A major division of R factor types is based on the presence or
absence of the ability to inhibit the fertility of the F episome. R
factors which inhibit fertility when introduced into an F+ cell are
termed fi+ (fertility inhibition positive). Those R factors which have
no effect on the F episome are designated fi (fertility inhibition
negative)(48).
Further classification of R factors is based on the type of sex
pilus produced. One class of R factors codes for the production of
F-like pili, which are distinguishable antigenically, as well as biologically, by virtue of their sensitivity to adsorption and subsequent
infection by isometric F specific bacteriophages MS-2, M12, f2, and
Qp, and the filamentous F specific bacteriophages such as fl.
A second group of R factors, related to the bacterial colicin
(Col I) produces I-like pili which are morphologically and antigenical-
ly distinct from F-like pili, and do not adsorb F specific phages.
I-like pili are sensitive to adsorption by the filamentous I specific
phages, Ifl and If2 (52).
Lawn (25) demonstrated the existence of two additional classes
of R factors, one which produced both F and I-like pili, and a fourth
class, which produced neither F nor I-like pili. Most fi+ R factors
produce F-like pili and are thought to be closely related to F epi-
somes, while fi- R factors are primarily I-like.
The inhibition of fertility of F episomes by fi+ R factors is
presumed to be the result of a repressor produced by the R factor
which also represses F (27). Wild type fi+ and fi- R factors are
normally repressed and exhibit low frequencies of transfer. Depressed mutants can be isolated, however, whose frequencies of
transfer approach 100%.
While all R factors can coexist with F episomes, a phenomenon known as superinfection immunity is.evident when two closely
related R factors enter the same cell. Exclusion of one R factor
may be due to competition for a particular site on the cell membrane,
or due to physical restriction of the R factor DNA entering the cell.
Where incompatibility is observed, a low frequency of recombinant R
factors is noted, presumably in an effort to prevent exclusion. Conversely, compatible R factors can coexist independently in the dame
cell, without recombining to form one molecule (28).
Plasmid incompatibility has provided a valuable technique for
classifying both F-like and I-like R factors. Plasmids within an
incompatibility group are incompatible with each other. The major
compatibility groups for the F, Col, and R factors are given in
Willetts (52).
The host ranges of transfer of R factors have also been shown
to be related to the class of R factor present in the donor strain (12).
Biochemical Mechanisms of Transferable
Drug Resistince
The biochemical mechanisms of antibiotic resistance mediated
by R factors are primarily due to enzymatic inactivation of the antibiotic.
Penicillins and cephalosporins have been shown to be inactivated
by a specific 13-lactamase, whose synthesis is coded for by the r
determinant. Chloramphenicol resistance is the result of acetylation
of the antibiotic by chloramphenicol acetyltransferase, while inactivation of the aminoglycosides kanamycin and streptomycin is mediated
by acetylation with kanamycin acetyltransferase or by phosphorylation
with kanamycin monophosphotransferase, and adenylation with strepto-
mycin adenyltransferase, respectively. The enzyme kanamycin
monophosphotransferase has also been shown to effectively inactivate
the antibiotic neomycin. Conversely, the resistance to tetracycline
noted in R+ organisms is due to reduced cellular permeability (50).
Inactivation of gentamycin by R+ organisms has recently been
found to be due to the synthesis of the enzymes gentamycin adenyl-
9
transferase and gentamycin acetyltransferase, each showing specificity for one type of gentamycin (9).
Role of Antibiotic Feeding in R Factor Prevalence
Antibiotics have been widely used as animal feed supplements
since 1949. While the benefits to the animal industry in terms of
weight gains and productivity have been well documented (22), the
increase in the number of bacteria harboring transferable drug resistance episomes is becoming increasingly alarming.
Data presented by Smith (43) showed that 99% of the fecal
samples of pigs fed tetracyclines contained resistant E. coli in surveys taken in 1957, prior to the discovery of R factors. Additional
studies done at that time on marketed meat indicated that a high percentage of bacon pigs and 18% of market pigs contained E. coli resis-
tant to tetracyclines.
Walton (44) conducted a limited survey of healthy domestic
animals in England to determine the incidence of R+ organisms where
antibiotic feeding was practiced, and found that 74% of the E. coli
strains isolated from swine and calves contained transferable drug
resistance, in most cases, multiple drug resistance to tetracycline,
streptomycin, and sulfonamide.
A study done by Mitsuhashi (30) in Japan involved surveying
swine and fowl for R+ _E. coli after feeding of dairy products contain-
10
ing 0. 1% tetracycline. Results indicated that all of the swine and 38%
of the fowl excreted drug resistant E. coli, where no excretion had
occurred prior to feeding. Transferable drug resistance was carried
in 40% of the swine tested, and ZZ% of the fowl. Multiple drug resis-
tant strains were isolated at high frequencies, with the most common
resistance pattern being tetracycline, streptomycin, and sulfonamide.
The increasing incidence of R+ Salmonella typhimurium and R+
enteropathogenic E. coli in the Netherlands following the advent of
antibiotic feeding was documented by Guinee {19). Both of the above
organisms are important human pathogens.
The presence of R+ organisms in food products was shown by
Walton (45) in a survey where 40% of 400 animal carcusses tested
revealed R+ bacteria.
The prevalence of drug resistant E. coli in animal handlers and
farm animals, as well as the incidence of R+ Salmonella typhimurium
in Germany due to antibiotic feeding was noted by Weidemann (51).
Further documentation of the role of antibiotic feeding in the
emergence of multiple drug resistance mediated by R factors is
detailed in the Food and Drug Administration Task Force Report on
the Use of Antibiotics in Animal Feeds, Appendix C (54).
11
R+ Bacteria in Human Infection
The potential danger to the human population associated with the
increase in R+ bacteria as a result of antibiotic feeding is becoming
increasingly obvious.
Bensel (8) reported a fatal septicemia caused by Salmonella
pullorum, where raw eggs from an infected flock of chickens had been
ingested, providing documentation that isolates from infected animals
are capable of directly infecting humans. Anderson (3) reported an
epidemic of 600 cases of Salmonellosis due to a multiple resistant
strain of Salmonella typhimurium, phage type 29. Six deaths resulted
from infection with this organism. Phage typing confirmed the bovine
origin of the strain.
An increasing incidence of infections resulting from gram nega-
tive, multiple resistant organisms has been observed in many coun-
tries. Bergfors (10) observed that 26% of the drug resistant strains
isolated from urine specimens in a survey of Sweden harbored trans-
ferable drug resistance, and in some organisms as many as five
resistance markers were present.
The potential hazards associated with R+ opportunistic pathogens was documented by Martin (26) who showed that an R+ strain of
Klebsiella, type 22, resistant to gentamycin, streptomycin, kanamycin, neomycin, ampicillin, tetracycline, and chloramphenicol, was
12
responsible for a sudden outbreak of nosocomial infections, including
acute pyelonephritis and bacteremia. Most of the infected patients
had indwelling urinary catheters.
A survey done on clinical isolates of Klebsiella and Enterobacter by Hinshaw (21) indicated that 14. I% of the Enterobacter isolates
were R+, while 79. 1% of the Klebsiella strains harbored transferable
resistance. Clinical isolates tested were obtained over a four-month
period from urine, sputum, wounds, exudates, and blood.
Several strains of Serratia marcescens obtained from urinary
tract infections in three hospitals in New York City were shown to
harbor transferable drug resistance in a survey conducted by
Schaefler (40).
Kawakami (23) tested strains of Pseudomonas aeruginosa obtained from pathological specimens, and found that 15% contained R
factors. Smith (42) reported an incidence of 69% R+ organisms isolated from genitourinary infections in one survey.
The importance of antibiotic therapy in hospitals as a contribut-
ing factor to the emergence and dispersion of drug resistant, R+
organisms was documented in a study by Grabow (18) where 26% of
the conforms isolated from hospital waste water showed transferable
drug resistance in comparison to a figure of 4% in city sewage.
Additional documentation concerning the public health hazards
associated with antibiotic feeding is given in the Food and Drug
13
Administration Task Force Report on the Use of Antibiotics in Animal
Feeds, Appendix B (53).
Statement of Purpose
The role which antibiotic feeding has played in the emergence
of large reservoirs of drug resistant organisms has been firmly
established. The realization of the potential human health hazards
associated with such reservoirs has prompted extensive research
efforts toward finding alternatives to antibiotic feeding to promote
weight gain and to prevent disease manifestations resulting from infection with enteropathogenic E. coli.
The potential therapeutic value of the lactobacilli in human and
veterinary medicine has been reviewed by Sandine et aL (38). Prelim-
inary data (39) obtained when concentrates of viable lactobacilli were
fed to swine revealed a reduction in both the incidence of scouring in
the test swine and the number of E. coli in fecal samples. Since E.
coli isolated from scouring animals would induce colibacillosis in new-
born piglets, it was felt that the reduced scouring incidence may have
been related to a reduction in fecal coliforms. These experiments
provided an opportunity to examine the test swine for R+ organisms.
The goals of this investigation, therefore, were:
(a)
to compare the incidence of R+ bacteria in swine which
were fed lactobacilli concentrates with control animals
which received no lactobacilli
14
(b)
to characterize the types of R factors present in bacteria
isolated from the test and control animals
(c)
to identify a representative number of organisms from
among the R+ isolates
(d)
to test representative R+ isolates for sensitivity to the
bacteriophage MS-2
(e)
to determine the host range of transfer of different types
of R factors isolated from swine
(f)
to attempt elimination of R factors with acridine orange
(g)
to test compatibility among unique R factors
Animals included in the investigation received one feeding of
neomycin at birth, and did not receive any further antibiotics, unless
scouring occurred, in which case tylosin was given. Test and control
animals were housed in an environment, however, where tetracycline
(aureomycin), sulfonamides, and penicillin were routinely used.
Test swine received daily feedings of culture concentrate containing a Lactobacillus species of human origin in a concentration of
greater than 1 x 1010 organisms/ml. Dosage administration in-
creased with age, starting with 10 ml at birth, and increasing to 30
ml per day per animal at two months of age.
15
MATERIALS AND METHODS
Bacterial Cultures
The sources of the bacterial cultures which were used as R
factor recipients are listed in Table 1, together with their antibiotic
sensitivity patterns for the following antibiotics: kanamycin (K),
tetracycline (Tc), chloramphenicol (C), furadantin (Fd), nalidixic
acid (NA), streptomycin (Stm), ampicillin (Amp), carbenicillin (Cb),
and gentamycin (Gt). Resistance and sensitivity are denoted by the
letters R or S, respectively.
The nalidixic acid resistant strains listed above were spontaneous mutants of the original nalidixic acid sensitive parental strains.
E. coli CSH 63 (Cold Spring Harbor) was an Hfr strain used as
a positive control in the MS-2 bacteriophage studies.
E. coli CSH 23 (Cold Spring Harbor) was an F' lac strain used
as a positive control in the acridine orange elimination procedure.
Bacteriophage MS-2
MS-2, an RNA bacteriophage, was obtained from Dr. L. R.
Brown, Oregon State University. MS-2 was used to test representa-
tive R+ isolates for the production of F-like pili, since organisms
producing such pili would be sensitive to this F specific phage.
Table 1. Sources and Antibiotic Sensitivity Characteristics of Recipient Cultures
Source
NA
Tc
C
K
Fd
E. coli W3110
Dr. L. R. Brown
R
S
S
S
S
Klebsiella 190-3
Dr. R. J. Seidler
R
S
R
S
Klebsiella 104-A
Dr. R. J. Seidler
R
S
S
Salmonella
Dr. J. L. Fryer
S
S
Proteus vulgaris
Dr. J. L. Fryer
R
S
Aeromonas
liquefaciens
Dr. R. H. McCoy
Organism
Gt
Amp
Cb
S
S
S
S
S
R
R
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Stria
enteriditis
17
Media
Organisms to be tested for transferable drug resistance were
isolated on MacConkey agar (Difco), which is a selective medium
designed for the isolation of members of the Enterobacteriaceae.
Inhibition of growth of gram positive organisms on this medium is
due to the presence of bile salts.
R+ isolates were tested for antibiotic sensitivity patterns by
culturing overnight in Penassay broth (Difco) followed by plating on
Mueller Hinton agar (Difco) plates. Antibiotic sensitivity disks were
then dispensed onto the inoculated plates.
The taxonomic identification of R+ isolates was determined with
Enterotubes (Roche Diagnostics) and the following additional media:
Sirnmon's citrate agar, 1% Tryptone broth, GI motility medium,
Moeller decarboxylase broth supplemented with lysine, Christensen's
urea medium, phenol red broth base supplemented with 0.5% glucose,
sucrose, or lactose, triple sugar iron agar, and phenylalanine agar.
All of the above media were obtained from Difco Laboratories. The
identification of some isolates necessitated the use of the following,
media: Moeller decarboxylase broth supplemented with ornithine
hydrochloride, MRVP broth, malonate broth, and phenol red broth
supplemented with O. 5% adonitol, ino sitol, mannitol, rhamno se, or
raffinose. Confirmation of the identification of certain isolates also
18
required the use of polyvalent Salmonella and Shigella antisera. The
additional media and antisera were also obtained from Difco Labora-
tories.
The generic range of transfer of R+ isolates was tested on
MacConkey agar supplemented with antibiotics when E. coli, Klebsi-
ella, Aeromonas, or Proteus were recipients. Transfers into Salmonella were tested on Simmon's citrate agar supplemented with
antibiotics.
MS-2 bacteriophage sensitivity testing was done on R agar
(29). Test organisms were grown in R broth, were subsequently
inoculated into R overlay agar along with the MS-2 phage, and then
plated on R agar.
Acridine elimination of R factors from R+ isolates was tested
in LB broth (29) with subsequent plating to MacConkey agar.
Antibiotic s
Tetracycline hydrochloride (Nutritional Biochemical Corporation) was added to MacConkey agar and Simmon's citrate agar from a
stock solution containing 2 mg/ml to give a final concentration of 20
µg /ml. Streptomycin sulfate, B grade, (Calbiochem) was added to
MacConkey agar and Simmon's citrate agar from a stock solution containing 2.5 mg/ml to give a final concentration of 25 Fig/ml.
Nalidixic acid was obtained from Bectin, Dickinson, and
19
Company. Due to the low solubility of this antibiotic, it was necessary to prepare the stock solution of 1.0 mg/ml in the following manner: 1.0 ml of a 0.1 N NaOH solution was added to 10 mg of nalidixic
acid. A minimal salts solution (1) was added as a buffer to the nalidixic acid solution in a volume of 9.0 mls. The stock solution was
then added to. MacCon.key agar to give a final concentration of 20 p.g/
ml.
Acridine orange (Allied Chemical Corporation) was added to LB
broth (29) from a stock solution containing 100 µg /ml to give a final
concentration of 50 p.g/ml.
Antibiotic sensitivity disks (Difco) containing the following anti-
biotics were used in the antibiotic screening procedure: tetracycline
(5 and 30 p.g), streptomycin (2 and 10 p.g), kanamycin (5 and 30 p.g),
chloramphenicol (5 and 30 lig), furadantin (50 and 300 p.g), ampicillin
(2 and 10 p.g), carbenicillin (50 p.g), and gentamycin (10 p.g).
R Factor Screening Procedure
Fecal samples taken from young swine were plated on MacCon-
key agar plates. After overnight incubation at 37° C, twenty isolated
colonies from each sample were reinoculated onto a MacConkey agar
premarked master plate. The master plates were incubated overnight at 37°C, and were subsequently replica plated, using a stab
replicator, to MacConkey agar plates supplemented with either 20
20
µg /ml of tetracycline hydrochloride, or 25 µg /ml of streptomycin
sulfate. After overnight incubation at 37°C, colonies were scored
for both tetracycline and streptomycin resistance.
Those colonies showing resistance to tetracycline and/or strep-
tomycin were separately tested for drug resistance transfer into E.
coli W3110. The recipient was grown in static culture in Penassay
broth at 37°C, and prior to testing, 0.5 ml samples were dispensed
into tubes which were then placed in replicator blocks in patterns
matching the master plate grid pattern. Sterile Penassay broth was
added in a volume of 0.5 ml to each tube, and test organisms were
inoculated directly from the master drug plates into the recipient
tubes using a stab replicator. The conjugal mixtures were then incubated overnight at 37°C.
Transfer of drug resistance was confirmed bysplating directly
on MacConkey agar supplemented with nalidixic acid and either tetra-
cycline or streptomycin, again using a stab replicator. The presence
of nalidixic acid insured elimination of the donors. Control plates
were inoculated to test for nalidixic acid resistance in the donor population and streptomycin or tetracycline resistance in E. coli W3110.
Antibiotic Sensitivity Screening Procedure
Potential donors obtained from the R factor screening procedare were inoculated into Penassay broth, and were grown overnight
21
at 37 °C. After overnight incubation, broth cultures were diluted with
sterile Penassay broth to match a turbidity standard prepared by
adding 0.5 ml of a 1% BaC1 solution to 99.5 ml of a
2
H2SO4 solu-
tion. Diluted cultures were streaked in three planes onto the surface
of two Mueller Hinton agar plates according to the procedure of
Bauer (6). Antibiotic sensitivity disks were dispensed on the plates
using a Difco sensitivity disk dispenser, and the plates were incubated
overnight at 37°C. Zones of inhibition indicating sensitivity to the
test antibiotics were measured in millimeters.
Identification System for R+ Isolates
The Enterotube system which was initially employed to identify
R+ isolates consisted of a prepared, multimedia tube containing eight
different media. An enclosed inoculating needle allowed rapid inoculation of an unknown organism by pulling the needle through the entire
enclosed compartment. The following tests were included in the sys-
tem: glucose fermentation, lysine decarboxylation, ornithine decarboxylation, production of I-12S, production of indole, fermentation of
lactose, deamination of phenylalanine, fermentation of dulcitol,
hydrolysis of urea, and utilization of citrate as a sole source of carbon. Isolates were keyed out on the basis of characteristic reaction
patterns.
Due to the presence of intermediate biochemical types among
22
the R+ isolates, it was necessary to adopt the ASCP Numerical Coding and Identification System for the Enterobacteriaceae. This system, developed by Dito (13), was based on a coding system in which
positive reactions observed in the following tests were given a numerical value: citrate utilization-512; indole production-256; hydrolysis
of urea-128; motility-64; lysine decarboxylation-32; glucose fermen-
tation-16; lactose fermentation-8; sucrose fermentation-4; production
of H S-2; and deamination of phenylalanine-1. Each organism tested
2
was given a code number which represented the summation of all of
the positive results obtained in the initial test system. This code
number was consulted in the key, and was either assigned a species
identification, or was placed in a group of organisms. Where a group
designation was given, further tests were required which separated
the unknown organism from other organisms in the same group. This
system provided a means of numerically coding different biotypes of
the same bacterial species, which could not be achieved with the
Enterotube system.
Determination of Frequencies of R Factor Transfer
Potential R + isolates obtained from the screening procedure
were separately tested in conjugal systems to determine frequencies
of R factor transfer into E. coli W3110, Klebsiella 190-3, Klebsiella
104-A, Salmonella enteriditis, Aeromonas liquefaciens, and Proteus
23
vulgaris. Recipient cultures were grown in Penassay broth as pre-
viously indicated, and after incubation, were dispensed in sterile test
tubes in volumes of 0.5 ml. Test organisms were grown overnight on
MacConkey agar supplemented with either tetracycline or streptomy-
cin, and were then inoculated directly into the recipient tubes using a
sterile inoculating loop. Sterile Penassay broth was added to each
conjugal mixture in a volume of 0.5 ml, and the tubes were then incubated overnight at 37°C.
Following incubation, the conjugal mixtures were diluted in
sterile Penassay broth in ten fold dilutions ranging from 10-1 to 106.
Sample volumes of 0.1 ml from each dilution were plated on Mac Con-
key agar supplemented with nalidixic acid plus either tetracycline or
streptomycin to determine the number of recipients which had
received the test R factor, and on MacConkey agar supplemented
with nalidixic acid to determine the total number of recipients. A
total count of donors and recipients was determined by plating on plain
MacConkey agar. Controls were done to determine the frequency of
nalidixic acid resistant donors and tetracycline or streptomycin resistant recipients. The frequency of transfer of an R factor was calculated by dividing the number of recipients which had received the R
factor by the total number of recipients present, as indicated below:
of Resistant Recipients
Frequency of transfer = Number
Total Number of Recipients
24
If the number of drug resistant donors or recipients present on
the control plates was significant, the frequency of transfer was corrected by subtracting the frequency of nalidixic acid resistant donors
or tetracycline or streptomycin resistant recipients from the initial
frequency value.
The method of determination of the frequency of transfer of R
factors into Salmonella enteriditis was similar to the procedure
described above, with the exception of the selective medium employed.
The number of recipients which had received the test R factor was
determined by plating on Simmon's citrate agar supplemented with
either tetracycline or streptomycin, and the total number of recipients present was determined by plating on plain citrate agar. This
method of selection was devised because nalidixic acid resistant mutants of Salmonella enteriditis did not grow in a dispersed manner in
broth culture, exhibited rough colony morphology, and therefore could
not be quantitated in R factor transfer experiments.
Determination of Sensitivity of R+ Isolates
to MS-2 Bacteriophage
R+ isolates to be tested for sensitivity to the male specific RNA
bacteriophage MS-2 were grown overnight at 37°C in R broth. An
MS-2 phage lysate containing 6 x 107 PFU/ml was diluted 1:100 in R
broth. R overlay agar was dispensed in tubes in volumes of 2.5 ml
25
held at 45 °C, which were supplemented with either tetracycline or
streptomycin.
Test organisms were dispensed in 0.1 ml volumes into each of
two overlay tubes. A volume of 0.1 ml of the 1:100 dilution of MS-2
phage was added to one tube, and the tubes were then poured over R
agar plates. The second tube served as a seed control for each test
organism.
E. coli CSH 63, an Hfr strain, was treated in the same manner
as the test organisms, and served as a positive control. The overlay
tubes for this organism, however, were not supplemented with antibiotics.
All of the test plates were incubated overnight at 37°C, and
following incubation, were scored for the presence or absence of
plaques, indicating sensitivity or resistance to MS-2, respectively.
Acridine Elimination Procedure
R+ isolates to be tested for elimination of R factors with
acridine orange were inoculated into LB broth (29) and were incubated
overnight at 37°C. Following incubation, the test cultures were
diluted to 105 organisms/ml, in a volume of 10 mis of LB broth.
E. coli CSH 23, and F' lac strain, was used as a positive control,
and was tested under the same conditions.
Acridine orange was added to each test flask at a concentration
26
of 50 p.g/ml. The cultures were incubated on a shaker for 12 hours at
37°C. After 12 hours of incubation, the cultures were diluted in LB
broth and plated on MacConkey agar. The plates were incubated
overnight at 37 °C, and were then replica plated from the plain MacConkey agar plates to MacConkey agar supplemented with either tetra-
cycline or streptomycin, using the velveteen replication technique.
The replica plates were incubated overnight at 37°C, and the frequency of elimination of the R factors tested was determined by sub-
tracting the number of colonies present on the replica plates from the
number of colonies present on the plain MacConkey agar plates, and
dividing this value by the total number of colonies.
The frequency of elimination of the F' lac episome from the
positive control culture was determined by dividing the number of lactose positive colonies present on MacConkey agar by the total number
of colonies present. Those colonies from which the episome had been
eliminated by acridine orange treatment appeared to be lactose negative on MacConkey agar.
Compatibility of Transfer Factors Among R+ Isolates
Nalidixic acid resistant mutants of R+ isolates were obtained to
be used as recipients in the compatibility testing procedure. Donors
were then selected whose R factors contained determinants different
from those of the recipients.
27
Donors and recipients were mixed in the same volumes as in the
R factor transfer studies, and the tubes were subsequently incubated
overnight at 37 o C. Following incubation, dilutions of the conjugal
mixtures were plated on MacConkey agar supplemented with nalidixic
acid and tetracycline or streptomycin. Colonies of the nalidixic acid
resistant recipients were tested to determine their total antibiotic
sensitivity patterns, in order to ascertain whether or not both donor
and recipient R factors were present, or if exclusion of one R factor
had occurred.
28
RESULTS
Incidence of Drug Resistance Among Enteric Isolates
Preliminary data showing the incidence of drug resistant organisms in the Oregon State University swine herd were obtained by plating randomly selected fecal samples on MacConkey agar supplemented
with tetracycline and streptomycin. The results of a representative
number of bacterial counts are listed in Table 2. It may be seen that
the number of drug resistant coliforms comprised a significant proportion of the total coliform count in each sample.
Organisms exhibiting resistance to either tetracycline or streptomycin in the preliminary samples were subsequently tested for the
presence of transferable drug resistance using E. coli W3110, Salmonella enteriditis, Klebsiella 190-3, and Klebsiella 104-A as recipients. The frequencies and ranges of transfer of several R+ isolates
are shown in Table 3; these data indicate a wide range of transfer frequencies among different genera.
Incidence of Transferable Drug Resistance in Swine
which were Fed Lactobacillus Concentrate
The occurrence of R+ organisms among enteric isolates was
determined for all test and control animals over a three month period.
The percent R+ isolates was calculated by dividing the number of
R+
29
Table 2. Number of Drug Resistant Organisms among Enteric
Isolates
Sample
3P1
3P5
3P6
3P8
3P9
3P11
3P16
4P6
4P7
4P11
4P15
3-1
3-2
3-3
3-4
3-5
7-1
7-5
8-1
8-7
Number of Organisms/Gram
Tc Resistant
Total
4.5 x
1.4 x
4.9 x
5.0 x
1.5 x
9.1 x
8.1 x
109
108
109
109
109
108
108
1.6 x 108
1.2 x 107
6.0 x 107
7.5 x
3.3 x
108
108
1.5 x 108
6.5 x 106
3.4 x 107
1.1 x 108
1.2 x 108
1.5 x 108
2.1 x 108
2.9 x 107
2.8 x 109
1.1 x 108
4.0 x 109
4.7 x 109
8.7 x 108
7.3 x 108
2.8 x 108
1.6 x 108
7.0 x 106
3.0 x 106
1.7 x 107
1.5 x 108
7.2 x 107
6.1 x 106
1.9 x 107
1.1 x 108
1.1 x 108
1.1 x 108
1.0 x 108
2.2 x 107
Stm Resistant
3.5 x
7.0 x
109
4.4x
109
4.5 x
1.3 x
7.2 x
109
7.1x
108
107
109
108
1.5 x 108
5.2 x 106
4.0 x 10 6
1.1 x 108
6.1 x 107
3.8 x 107
4.5 x 106
1.7 x 107
1.4 x 107
6.4 x 107
2.0 x 107
8.5 x 107
2.4 x 107
Table 3. Preliminary R Factor Transfer Frequency Data
Donor
Markers
Transferred
E. coli
W3110
Recipients
Salmonella
Klebsiella
enteriditis
190-3
Klebsiella
104A
Salmonella
12-B
K, Stm, Amp, Cb
E. coli
2-8
NT*
1.5 x 10-5
2.3 x 10-6
2.3 x 10-3
3.9x 10-4
Tc, Stm, Amp, Cb
5.3 x 10-5
2.4x
Tc, Stm
3.8 x 10-2
NT
1.0 x 10-5
NT
Tc, K, Strn, Amp, Cb
9.2 x 10-3
NT
9.3 x 10 -7
NT
10-8
E. coli
3-17
E. coli
14-1
Symbols:
* NT = not tested
- = no transfer indicated
31
organisms encountered in a sample by the total number of organisms
tested, where the number of isolates tested in each case was 20.
Table 4 is a summary table showing the marked fluctuation in the
incidence of R+ isolates in swine which were fed Lactobacillus con-
centrate in water. Sample numbers represent the four sample periods in which samples were taken over a three month period. The
first samples were taken at approximately two weeks of age.
The types of R factors present in the swine which were fed
Lactobacillus concentrate in water are listed in Table 5. As is evi-
dent, the spectrum of R factor types encountered encompassed resistances to many of the antibiotics tested.
The percent R+ isolates and types of R factors found in swine
which were bottle fed Lactobacillus concentrate are shown in Tables
6 and 7, respectively. Comparable data for the negative control animals are given in Tables 8 and 9. While the bottle fed swine and negative control animals also demonstrated considerable variation in the
incidence of R+ isolates as well as R factor types, it is important to
note the significant increase in R+ organisms at the time the third
samples were taken, three dayspostweaning.
The total incidence of number and types of R factors found in
test and control animals over the entire sampling period is given in
Table 10. A prevalence of three R factor types, (K,Stm), (Tc, Strn),
and (Tc, Amp), is apparent from the data shown.
32
Table 4. Incidence of R. Factors Found in Swine which were Fed
Lactobacillus Concentrate in the Water Supply
Sample Number
Swine
Number
1
2
3
1443-7
10%
40%
35%
15%
1443-2
0%
75%
0%
0%
1444-3
20%
50%
20%
15%
1444-8
10%
0%
10%
0%
1447-4
25%
0%
35%
15%
1447-5
80%
47%
9%
10%
Table 5. Types of R Factors Found in Swine Fed Lactobacillus Concentrate in Water
Sample Number
Swine
Number
1443-7
Tc, K, Strn, Amp, Cb
Tc, K, Strn
3
4
Tc, Stm, Amp, Cb
Tc, Stm, Amp
Tc, Stm
Tc, K, Stm
Tc, Stm
Tc, Strn, Amp, Cb
Tc, Stm, Amp, Cb
Tc, K, Strn
Tc, Strn, Amp, Cb
Tc, K, Stm
Tc, K, Strn, Amp
2
1
Tc, Stm
Stm
Stm
K, Strn , Amp, Cb
Tc, Stm, Amp, Cb
Tc, Stm
Stm, Amp, Cb
1443-2
1444-3
1444-8
1447-4
1447-5
*
Tc, K, Stria, Amp
Tc, K, Stm
Tc, Sim, Amp, Cb
Tc, K, Stm
Tc , Amp
Tc, Stm
Tc, Stm, Amp, Cb
Tc, Stm
Tc, K, Stm, Amp
Tc, K, Strn, Amp
K, Stm, Amp
K, Stm
- (No R Factors Detected)
Tc, Strn, Amp, Cb
Tc, K, Strn
Tc, Stm
Tc, Stm, Amp
Tc, Stm, Amp, Cb
Tc, K, Stm
Tc, K, Stm, Amp
34
Table 6. Incidence of R Factors Found in Swine which were Bottle
Fed Lactobacillus Concentrate
Sample Number
Swine
Number
1
1442-2
2
3
4
0%
40%
35%
40%
1442-3
0%
29%
68%
65%
1445-9
0%
15%
60%
70%
1445-11
35%
0%
55%
0%
1446-5
80%
20%
70%
0%
1446-6
95%
5%
10%
0%
Table 7. Types of R Factors Found in Swine which were Bottle Fed Lactobacillus Concentrate
Sample Number
Swine
Number
Tc, Stm
Tc, K, Strn , Amp
1442-2
3
2
1
Tc,Amp
1445-9
Tc, Strn , Amp
Tc, K, Stm
Tc, Amp
Tc, Stm
Tc, Stm
Tc, Amp, Cb
Tc, Stm
Tc, Stm
Tc, Stm
Tc, K, Stm, Amp, Cb
Tc, Strn , Amp, Cb
Tc, Stm, Amp
Tc, Amp
Tc, K, Strn, Amp, Cb
K, Stm
1445-11
Tc
1446-6
Stm, Amp, Cb
T c , Strn , Amp , Cb
1442-3
1446-5
4
Tc, K, Stm, Amp, Cb
Tc, K, Stm
Tc, K, Stm, Amp, Cb
Tc, Stm
Tc, Stm
Tc, K, Stm, Amp, Cb
Tc, Stm
K, Stm
Stm, Amp, Cb
Tc, K, Stm, Amp, Cb
Tc, K, Stm, Amp
Tc, K, Stm
K, Stm
36
Table 8. Incidence of R Factors Found in Negative Control Animals
Sample Number
Swine
Number
1
1442-1
1442-4
1445-2
2
3
4
0%
10%
100%
15%
5%
5%
85%
0%
0%
47%
5%
NS
1445-8
0%
0%
60%
0%
1446-2
100%
0%
35%
0%
1446-7
100%
15%
6%
Table 9. Types of R Factors Found in Negative Control Animals
Sample Number
Swine
Number
1442-1
1442-4
Tc, K, Stm
Tc, Stm
Tc, Amp
Tc, Stm, Amp, Cb
Tc, Amp
Tc, Stm
1445-2
Tc, Amp
Tc, Stm, Amp
Tc, Stm, Amp, Cb
Tc, Amp, Cb
1445-8
Tc, Amp
Tc, Stm, Amp, Cb
Tc, Stm
1446-2
1446-7
Tc, Stm
Stm
K, Stm
Tc, K, Stm, Amp, Cb
Tc, Stm
K, Stm
4
3
1
Tc, Stm, Amp, Cb
Tc, Stm, Cb
Tc, Stm
Tc, Amp, Cb
Tc, Stm, Amp, Cb
Tc, Stm
38
Total Incidence of Number and Types of R Factors Found
in Test Animals Over the Entire Sampling Period
Table 10.
Sample Number
R
Factor
2
3
4
2
Tc, K, Strn
15
19
4
Tc, K, Stm, Amp, Cb
11
4
1
Tc, K, Sim, Amp
13
1
K, Strn
63
1
Stm, Amp, Cb
1
Tc, Strn
1
Stm
7
14
42
18
5
K, Strn, Amp, Cb
1
Tc, Stm , Amp, Cb
6
22
5
66
Tc, Amp
T c , Strn , Amp
5
Tc, Amp, Cb
1
Total
30
104
64
146
1
50
39
Characterization of R Factor Types
R+ isolates from all of the test animals were characterized
with regard to their antibiotic sensitivity patterns. The antibiotic
sensitivity patterns of a representative number of those organisms
tested are given in Tables 11, 12, 13, and 14. Resistance or sensi-
tivity are denoted by the letters R or S, respectively, where resistance was determined by a zone of inhibition around the antibiotic disk
of 6 mm or less, and sensitivity was indicated where the zone of inhibition exceeded 7 mm.
Identification of R+ Isolates
The taxonomic identification of a representative number of R+
isolates was accomplished utilizing the ASCP Numerical Coding and
Identification System for the Enterobacteriaceae, as previously indicated. The majority of the isolates tested were identified as E. coli
strains, which fell into several different biochemical types. The
reaction patterns and numerical codes for these types are delineated
in Table 15. Tables 16, 17, 18, and 19 are summary tables designat-
ing the biochemical code, species identification, and resistance pat-
terns of the above R+ isolates. A clustering of unique R factors
with certain biotypes of E. coli was observed and is shown in Table
20.
40
Table 11. Characterization of R Factor Types - Group 1 *
Antibiotic Concentration in µg
R4-
Tc
Isolates
5
30
K
C
5 30
5
30
Fd
50 300
Stm
2
10
Amp
2
10
Cb Gt
50
10
S
S
A14443S5
RR
SS
RR
SS
RR
RS
A14443S16
RR
SS
RR
SS
RR
SS
A14443T18
RR
SS
RR
SS
RR
SS
A14451156
SS
SS
RR
SS
RR
SS
A144511S11
RR
SS
RR
SS
RR
RR
R
S
A14465S1
SS
SS
SS
SS
RR
RR
R
S
A14465518
RR
SS
RR
SS
RR
RR
R
S
A14466S1
RS
SS
RR
SS
RR
RS
S
A14466S3
RR
SS
RR
SS
RR
SS
A14466S6
RR
SS
RR
SS
RR
RR
A1446751
RR
SS
SS
SS
RR
S
A14474S10
RR
SS RR
SS
RR
RR
S
S
S
* Group 1 includes isolates obtained during the first sampling period.
41
Table 12. Characterization of R Factor Types - Group 2 *
Antibiotic Concentration in 1.1.g
R+
Tc
C
K
5 30
5 30
5 30
C14422S6
RR
SS RR
SS
RR
C14422S13
SS
RR
SS
C14423S2
RR
RS
C14423S6
S
C14432S2
RS
C1443253
S
S
C14432S5
S
S
C14432S8
RR
C14432S11
S
C14432S17
RR
C14432S18
S
C14432S19
RR
C1443756
S
C14437S10
SS
C14443S13
C14459S3
RS
RS
RR
C14465S2
RR
SS RR
C14465S5
RR
RR
SS
SS
RR
RR
SS
Isolates
C14443S14
C14475S1
C14475S12
C14475T16
S
S
S
S
Fd
SS RR
SS RR
SS
SS
SS
SS
SS
Gt
10
50
10
SS
S
S
RR
RR
S
S
RR
RS
RR
RR
SS
S
S
SS
S
S
S
S
S
S
2
10
2
S
S
S
S
S
S
SS
RS
SS
S
S
S
S
R
S
S
RR
RR
RR
RR
S
SS
SS
SS
SS
S
S
RR
R
S
SS
SS
RS
RR
SS
RR
S
S
R
S
RR
RR
RR
RR
RR
RR
RR
SS
SS
SS
SS
SS
S
S
S
S
S
S
S
S
S
S
RR
R
S
SS
S
S
S
S
S
S
S
S
S
S
SS
SS
S
S
SS
SS
SS
SS
SS
SS
SS
S
S
SS
RR
RR
R
S
S
S
SS
SS
RR
RR
SS
SS
S
S
S
S
SS RR
SS RR
SS
Cb
SS
SS
SS
SS
SS RR
SS
SS
SS
SS
50 300
Amp
Stm
S
S
SS RR
* Group 2 includes isolates obtained during the second sampling period.
42
Table 13. Characterization of R Factor Types
R+
Isolates
Tc
5
D14421T1
D14421T2
D14421T15
D14422T6
D14422T7
D14424T2
D14424T4
D14424T6
D14437T12
D14437T14
D14437S4
D14437S13
D14443T1
D14443T13
D14443S9
D14452T1
D14452T13
D14452T17
D14452S2
D14458T1
D14458T5
D14458T16
D14459S1
D144511T13
D144511S6
D144511S12
D14462S5
D14462S13
D14465T4
D14465T17
D14466S5
D14467T11
D14474S3
D14474S7
D14474T11
D14475S11
30
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
SS
RR
RR
RS
RS
RR
RR
RR
RR
RS
Group 3
Antibiotic Concentration in p.g
Amp
Stm
C
Fd
K
2 10
50 300 2 10
5 30 5 30
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
RR
RR
SS
RR
SS
SS
SS
SS
SS
SS
SS
SS
SS
RR
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
RS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
RS
SS
SS
SS
SS
SS
SS
SS
SS
SS
RS
SS
RR
SS
RR
RR
RR
RR
RR
RS
RR
RS
RR
SS
RR
SS
RS
RR
RR
RS
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RR
RS
RR
RS
RS
RR
RR
SS
RR
RR
RR
RS
S
S
S
S
S
S
RR
RR
RR
RR
RR
RR
RR
RR
S
S
RR
RR
RR
SS
SS
SS
Cb
Gt
50
10
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
S
S
S
R
S
S
R
R
R
S
R
R
S
S
R
R
SS
S
S
S
S
S
S
S
S
S
S
S
S
S
RR
RR
SS
R
R
S
* Group 3 includes isolates obtained during the third sampling period.
43
Table 14. Characterization of R Factor Types - Group 4*
R+
Antibiotic Concentration in p.g
Amp
Fd
Stm
K
Tc
C
5 30
5 30
5
30
50 300
2
E14421T4
RR
SS
S
S
SS
E14421S10
RR
SS
S
S
E14423T6
RR
SS
S
E14423T9
RR
SS
E14437T13
RR
E14437S12
Cb
Gt
10
50
10
SS
RR
R
S
SS
RR
RR
R
S
S
SS
RR
SS
S
S
S
S
SS
SS
RR
R
S
SS
S
S
SS
RS
SS
S
S
RR
SS
S
S
SS
RR
RR
R
S
E14443T5
RR
SS
SS
SS
RR
RR
R
S
E14443T14
RR
SS
RR
SS
RR
SS
E14443S8
RR
SS RR
SS
RR
RR
E14459T12
RR
SS
S
S
SS
RR
SS
S
S
E14474T13
RR
SS
SS
SS
RS
RR
S
E14475S14
RR
SS RR
SS
RR
RR
Isolates
10
2
* Group 4 includes isolates obtained during the fourth sampling
period.
Table 15. Biochemical Characteristics of R+ E. coli Strains Isolated from Swine
Code Cit
312
380
316
344
376
300
Ind Urea Mot LD Glu Lac
+
-
+
Suc
H
2S
PD
OD VP Mal Arab Mann Ad
+
92
56
368
328
276
Abbreviations used are as follows:
citrate utilization
Cit
indole production
Ind
Urea hydrolysis of urea
motility
Mot
lysine decarboxylase
LD
Glu - fermentation of glucose
fermentation of lactose
Lac
fermentation of sucrose
Suc
production of hydrogen sulfide
H
2S
Iwo
PD
OD
VP
Mal
Arab
Mann
Ad
In
- phenylalanine deaminase
- ornithine decarboxylase
- Voge s-Prauskaue r
- malonate utilization
- fermentation of arabinose
- fermentation of mannitol
- fermentation of adonitol
- fermentation of inositol
In
45
Table 16. Identification of R4. Isolates - Group 1
R+
Isolate
Biochemical
Resistance
Determinants
Identification
Code
Present
A14443S5
356
Tc, K, Strn, Amp
E. coli
A14443S16
356
Tc, K, Strn
E. coli
A14443T18
316
Tc, K, Strn
E. coli
A144511S6
380
K, Strn
E. coli
A144511S11
316
Tc, K, Strn, Amp, Cb
E. coli
A14465S1
316
Strn , Amp, Cb
E. coli
A14465S18
316
Tc, K, Strn, Amp, Cb
E. coli
A14466S1
380
Tc, K, Strn, Amp
E. coli
A14466S3
572
K, Strn, Tc
Klebsiella
A14466S6
316
Tc, K, Strn, Amp, Cb
E. coli
A14467S1
328
Tc, Stm
E. coli
A14474S10
380
Tc, K, Strn, Amp
E. coli
46
Table 17. Identification of R+ Isolates - Group 2
Isolate
Biochemical
Code
Present
C14422S6
380
C14422S13
380
C14423S2
380
Tc, K, St=
Tc, K, Stm, Amp
Tc, K, Stm
C1442356
380
K, Stm
C14432S2
344
Stm, Tc
CI 4432S3
344
Stm
C14432S5
344
Stm
C14432S8
344
K, Stm, Amp, Cb
C14432S11
344
Stm
C14432517
380
Tc, K, Stm, Amp, Cb
C14432S18
344
Stm
C14432S19
344
Tc, Strn , Amp, Cb
C14437S6
344
Stm
C14437S10
344
Stm
014443513
C14443514
380
Tc, K, Stm
380
Tc, K, Stn
C14459S3
344
C1446552
380
C14465S5
92
C14475S1
380
Tc, Stm
Tc, K, Stm, Amp, Cb
Tc, Stm
Tc, Stm, Amp, Cb
C14475S12
376
C14475T16
312
R+
Resistance
Determinants
Tc, Stm
Tc, K, Stm
Identification
E. coli
E. coli
E. coli
E. coli
E. coli
E.
coli
......
E. coli
E. coil
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
47
Table 18. Identification of R+ Isolates
R+
Isolate
D14421T1
D14421T2
D14421T15
D14422T6
D14422T7
D14424T2
D14424T4
D14424T6
D14437T12
D14437T14
D14437S4
D14437S13
D14443T1
D14443T13
D14443S9
D14452T 1
D14452T13
D14452T17
D14452S2
D14458T1
D14458T5
D14458T16
D14459S1
D144511T13
D144511S6
D144511S12
D1446235
D14462S13
D14465T4
D14465T17
D14466S5
D14467T11
D14474S3
D14474S7
D14474T11
D14475S11
Biochemical
Code
380
380
380
380
316
380
380
380
316
312
316
300
300
312
300
312
316
376
376
380
312
376
380
380
312
312
344
312
312
376
723
380
312
312
312
368
Group 3
Resistance
Determinants
Identification
Present
Tc,Amp
Tc, Amp
Tc, Amp
Tc,Amp
Tc, Stm , Amp
Tc, Amp
Tc, Stm
Tc, Amp
Tc, Strn , Amp, Cb
Tc, Stm , Arnp , Cb
Tc, Strn , Amp
Tc, K, Stm
Tc, K, Strn
Tc, Stm, Amp, Cb
Tc, K, Stm
Tc, Strn , Amp, Cb
Tc, Stm, Amp, Cb
Tc, Amp., Cb
Tc, Strn , Amp, Cb
Tc,Amp
Tc, Stm, Amp, Cb
Tc, Stm , Amp, Cb
Tc, Stm
Tc, Strn , Amp
Tc, K, Stm, Amp, Cb
Tc, Strn, Amp, Cb
Stm
Tc, Stm
Tc, Stm
T c, Stm
Tc, Fd, Stm
Tc, Stm
Tc, Stm
Tc, Stm, Amp, Cb
Tc, Stm , Amp, Cb
Tc, Strn
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
T. coli
T. coli
E. coli
E. coli
T. coli
T_ .
coli
E. coli
E. con
E. coli
E. coli
E
T. coli
Proteus mirabilis
E. coli
F. coli
T. coli
T. coli
T. coli
48
Table 19. Identification of Rt Isolates - Group 4
R+
Isolate
Biochemical
Code
Resistance
Determinants
Identification
Present
E14421T4
276
Tc, Amp, Cb
E. coli
E14421S10
312
Tc, Stm, Amp, Cb
E. coli
E14423T6
376
T c, Stm
E. coli
E14423T9
56
Tc, Amp, Cb
E. coli
E14437T13
312
Tc, Stm
E. coli
E14437S12
312
Tc, Stm, Amp, Cb
E. coli
E14443T5
312
Tc, Stm, Amp, Cb
E. coli
E14443T14
300
Tc, K, Stm
E. coli
E14443S8
312
Tc, K, Stm, Amp
E . coli
E14459T12
376
Tc, Stm
E.
E14474T13
312
Tc, Strn , Arnp, Cb
E. coli
E14475S14
312
Tc, K, Stm, Amp
E. coli
coli
49
Table 20. Distribution of R Factors Among Major E. coli Biotypes
R Factor
E. coli Biotype
312
380
316
Tc, K, Stm
5%
16%
10%
Tc, K, Stm, Amp, Cb
5%
8%
30%
11%
12%
Tc, K, Stm, Amp
K, Stm
11%
100%
8%
20%
68%
68%
K, Stm, Amp, Cb
8%
Tc, Amp
28%
Tc, Stm, Amp
Tc, Amp, Cb
300
10%
Stm
Tc, Stm, Amp, Cb
376
8%
Stm, Amp, Cb
Tc, Stm
344
63%
4%
20%
4%
10%
16%
16%
10%
5%
16%
10%
Total Identified
18
25
10
12
6
4
50
MS-2 Bacteriophage Sensitivity Studies
MS-2 bacteriophage sensitivity tests were done on all of the R+
isolates whose antibiotic sensitivity patterns and identification data
are indicated in previous tables. While the positive control, E. coli
CSH 63, showed numerous plaques, indicating the production of F-like
pili, no test R+ organisms showed any evidence of phage activity. It
can be concluded from these data that the test organisms did not har-
bor R factors which coded for the synthesis of F-like pili. Seed controls of the test organisms were also negative for phage activity.
Generic Range of Transfer Among R+ Isolates
A number of R+ isolates exhibiting various combinations of
resistance determinants were further tested to determine the generic
range and frequency of transfer of their R factors. Transfer data
from these donors into E. coli W3110 are shown in Table 21. As
noted in this table, some determinants present in the donors were not
expressed in the recipients.
Data obtained from transfers into Salmonella enteriditis are
shown in Table 22. Transfer from citrate positive donors could not
be tested, since citrate utilization was used as the selective technique
to recover drug resistant recipients.
Transfer into two different Klebsiella strains was tested.
51
Table 21. Frequency of Transfer of R Factors from R+ Isolates into
E. coli W3110
Donor
Resistance
Determinants
Present
Frequency
of Transfer
in Donor
A14443S5
Tc, K, Stm, Amp
A14443T18
Tc, K, Stm
A144511S6
K, Stm
A144511S1 1
Tc, K, Stm, Amp, Cb
A14465S1
Stm, Amp, Cb
A14465S18
Tc, K, Stm, Amp, Cb
A14466S1
Tc, K, Stm, Amp
A14466S3
Tc, K, Stm
A14466S6
Tc, K, Stm, Arnp, Cb
A14467S1
Tc, Stm
A14474S10
Tc, K, Stm, Amp
C14432S2
Stm, Tc
Tc, Stm
C14465S5
C14475S12
Tc, Stm
C14475T16
Tc, K, Stm
D14421T1
Tc, Amp
D14422T7
Tc, Stm, Amp
D14424T6
Tc, Amp
D14437T12
Tc, Strn , Amp, Cb
D14437S13
Tc, K, Stm
D14452T13
Tc, Stm, Amp, Cb
E14423T6
Tc, Stm
E14443T5
Tc, Stm, Amp, Cb
E14474T13
Tc, Stm, Amp
2.0 x 10 -1
-1
1.2 x 10
> 10
-1
Resistance
Determinants
Expressed in
Recipient
K, Stm
Tc, Strn
K, Stm
1.7 x 10-2 K, Stm
4.3 x 10 6Stm, Amp, Cb
3.1 x 10-7 Tc, Stm, Amp, Cb
4.6 x 10 -1 K, Stm
5.7 x 10-5 K, Stm
7.6 x 10-5 K, Stm, Amp, Cb
3.5 x 10-3 Tc, Stm
6
K, Stm
5.8 x 10
5.6 x 10-2 Stm
> 10
-1
-1
1.4 x 10
8.7 x 10-2
4.4 x 10-3
1.9 x 10-3
3.1 x 10-3
4.5 x 10-2
4.0 x 10 -2.
1.3 x 10-4
1.4 x 10-2
1.6 x 10 -1
5.0 x 10-2
Stm
Tc, Stm
Tc
Tc,Amp
Tc, Stm, Amp
Tc, Amp
Tc, Stm, Arnp, Cb
Tc, Stm
Tc, Strn,Arnp, Cb
Tc, Stm
Tc, Stm, Amp, Cb
Tc, Stm, Amp
52
Table 22. Frequency of Transfer of R Factors from R+ Isolates into
Salmonella enteriditis
Donor
Resistance
Determinants
Present
Frequency
of Transfer
in Donor
A14443S5
Tc, K, Stm, Amp
A14443T18
Tc, K, Stm
A144511S6
K, Stm
A144511S11
Tc, K, Stm, Amp, Cb
A1446551
Stm, Amp, Cb.
A14465S18
Tc, K, Stm, Amp, Cb
A14466S1
Tc, K, Stm, Amp
A14466S3
K, Stm, Tc
A14466S6
Al 4467S1
Tc, K, Stm, Amp, Cb
Tc, Stm
A14474S10
Tc, K, Stm, Amp
C14432S2
Stm, Tc
Tc, Stm
Tc, Strn
Tc, K, Stm
Tc, Amp
Tc, Stm, Amp
Tc, Arnp
Tc, Strn , Amp, Cb
Tc, K, Stm
Tc, Stm, Amp, Cb
Tc, Strn
Tc, Strn , Amp, Cb
Tc, Stm, Amp
C14465S5
C14475S12
C14475T16
D14421T1
D14422T7
D14424T6
D14437T12
D14437S13
D14452T13
E14423T6
E14443T5
E14474T13
Resistance
Determinants
Expressed in
Recipient
2.6 x 10-6
5.0 x 10-7
K, Stm (Tc)
Tc, K, Stm
5.9 x104.8 x 10-6
K, Stm
1.5 x 10-6
6.8 x 10-5
Stm, Amp, Cb
>
10-4
Stm (K, Amp, Cb)
Tc, K, Strn, Amp, Cb
K, Stm
NT
NT
5.2 x 10 -5
3.3 x
4.4 x
1.4 x
9.2 x
5.8 x
8.7 x
10-7
K, Stm, Arnp, Cb (Tc)
Tc (Stm)
Stm (Tc, K, Amp)
10-4
Stm
10-5
Tc, Stm
Tc, Stm
Tc (K, Stm)
10-7
10-4
10-7
6.4 x 10 -8
7.5 x 10-5
8.9 x 10-5
1.1 x 10-4
1.2 x 10 -4
Tc, Amp
Tc, Strn , Amp, Cb
Tc, K, Stm
Tc, Stm, Amp, Cb
Tc, Stm, Amp, Cb
53
Klebsiella 190-3, an isolate from river water, has been shown to
have 100% DNA homology with the ATCC Type 3 Klebsiella pneumo-
niae, while Klebsiella 104-A, a vegetable isolate, has only 4% DNA
homology with the ATCC Type 3 strain (41). Frequency data from
transfers into Klebsiella 190-3 are given in Table 23, and those into
Klebsiella 104-A appear in Table 24.
The R+ isolates were also tested for transfer into Proteus vulgaris, and frequency data from these transfers are shown in Table 25.
The frequency data into the above recipients showed consider-
able variation, and generally reflected the genetic relatedness between donors and recipients. Where Aeromonas liquefaciens was
used as a recipient, no evidence of transfer of R factors from the
above R+ isolates was indicated. Since this organism, isolated from
salmon, is classified in an order (Pseudomonadales) distinct from
that in which the Enterobacteriaceae (Eubacteriales) are classified,
negative results here are again consistent with the lack of genetic
relatedness to the donors tested.
Acridine Elimination of R Factors from R+ Isolates
Eight R+ isolates were treated with acridine orange in an
attempt to eliminate the R factors. As seen in Table 26, elimination
was effective in two strains, A14443T18 and C14475S12. The latter
strain retained tetracycline resistance, but when tested for transfer
54
Table 23. Frequency of Transfer of R Factors from R+ Isolates into
Klebsiella 190-3
Donor
Resistance
Determinants
Frequency
of Transfer
Present
in Donor
A14443S5
Tc, K, Stun , Amp
A14443T 18
Tc, K, Stm
A144511S6
K, Stm
A144511511
Tc, K, Stm, Amp, Cb
A14465S1
Stm, Amp, Cb
A14465S18
Tc, K, Stm, Amp, Cb
A1446651
Tc, K, Stm, Amp
A14466S3
K, Stm, Tc
A14466S6
Tc, K, Stm, Amp, Cb
A1446751
A14474S10
Tc, Stm
Tc, K, Strn, Amp
C14432S2
Stm, Tc
C14465S5
Tc, Stm
C14475S12
Tc, Stm
C14475T16
Tc, K, Strn
D14421T1
Tc, Amp
D14422T7
Tc, Stm, Amp
D14424T6
D14437T12
Tc, Amp
Tc, Stm, Amp, Cb
D14437S13
Tc, K, Stm
D14452T13
E14423T6
Tc, Stm, Amp, Cb
E14443T5
Tc, Stm, Amp, Cb
E14474T13
Tc, Stm, Amp
Tc, Stm
* Recipient resistant to Amp, C
.
7.7 x 10-7
4.3 x 10 -8
2.5 x 10-8
3.9 x 10 -7
3. 6 x 10-7
7.4 x 10-7
-8
Resistance
Determinants
Expressed in
Recipient*
K, Stm
Tc, Strn (K)
K, Strn
K, Stm
Stm
Tc, Stm
7.3 x 10
4.1 x 10 -4
1.2 x 105
-5
3.0 x 10
K, Stm
6.0 x 10-6
4
1.6 x 10
-4
3.5 x 10
Tc, Stm
9. 6 x 10-6
-6
Tc
Tc, K, Stm
Stm
Tc, Strn
Stm
Tc, Stm
2.1 x 10
1.0 x 10-7
1.4 x 10-5
3
2.4 x 10
Tc
Tc, Stm
2.5 x 10-4
-7
2.9 x 10
1.7 x 10-2
Tc, Stm
Tc, Strn
Tc
Tc, Stm
Tc, Stm
55
Table 24. Frequency of Transfer of R Factors from R+ Isolates into
Klebsiella 104A
Donor
Resistance
Determinants
Present
Frequency
of Transfer
in Donor
A14443S5
Tc, K, Stm, Amp
A14443T18
Tc, K, Stm
A144511S6
K, Stm
A144511S11
Tc, K, Stm, Amp, Cb
A14465S1
Stm, Amp, Cb
A14465S18
A14466S1
Tc, K, Stm, Amp, Cb
Tc, K, Stm, Amp
A14466S3
K, Stm, Tc
A14466S6
Tc, K, Stm, Amp, Cb
A14467S1
Tc, Stm
A14474S10
Tc, K, Stm, Amp
C14432S2
C14465S5
Strn, Tc
Tc, Stm
C14475S12
1.8 x 10-8
2.7 x 10-8
Resistance
Determinants
Expressed in
Recipient*
K, Stm
K, Strn, Cb
-
3.0 x 10 -7
8.3 x 10-9
9.1 x 10-4
3.4 x 106
1.8 x 10-6
K, Stm, Cb
Stm
Tc, Strn
9.3 x 10-8
4.5 x 10-7
6.5 x 10-4
C14475T16
Tc, K, St
1.
x 10 -6
Tc
D14421T1
Tc, Amp
5.9 x 10-8
Tc
D14422T7
Tc, Stm, Amp
D14424T 6
Tc, Amp
1.3 x 10-5
D14437T12
Tc, Stm, Amp, Cb
Tc
Tc, Stan, Cb
D14437S13
Tc, K, Stm
D14452T13
Tc, Stm, Amp, Cb
Tc, Stm
E14423T6
E14443T5
E14474T13
Tc, Stm, Amp, Cb
Tc, Stm, Amp
* Recipient resistant to Amp.
>
10-3
K, Stm, Cb
Tc, K, Stm
Stm, Cb (K)
Tc, Stm
Stm
Tc, Stm
7.2 x 10-9
4.8 x 10-7
Stm
9.9 x 10-5
Tc, Stm, Cb
Tc, Stm, Cb
56
Table 25. Frequency of Transfer of R Factors from R+ Isolates into
Proteus vulgaris
Donor
Resistance
Determinants
Present
Frequency
of Transfer
in Donor
A14443S5
Tc, K, Stm, Amp
A14443T18
Tc, K, Stm
A144511S6
K, Stm
A144511S11
Tc, K, Strn, Amp, Cb
A14465S1
Stm, Amp, Cb
A14465S18
Tc, K, Stm, Amp, Cb
A14466S1
Tc, K, Strn, Amp
A14466S3
Tc, K, Stm
A14466S6
Tc, K, Stm, Amp, Cb
A14467S1
Tc, Stm
Tc, K, Stm, Amp
Al 4474S10
C14432S2
C14465S5
014475512
C14475T16
D14421T1
D14422T7
D14424T6
D14437T12
Stm, Tc
Tc, Stm
Tc, Stm
Tc, K, Stm
Tc, Amp
Tc, Stm , Amp
Tc, Amp
D14437513
Tc, Stm, Amp, Cb
Tc, K, Stm
D14452T13
Tc, Strn, Amp, Cb
E14423T6
Tc, Stm
Tc, Stm, Amp, Cb
E14443T5
E14474T13
Tc , Stm , Arnp
Resistance
Determinants
Expressed in
Recipient
1.2 x 10-1
Tc
4.0 x 10-2
6.9 x 10 -3
5.2 x 10-4
Tc
Tc
9.1 x 10-3
Tc
Tc
Table 26. Acridine Elimination of R Factors
R+
Biochemical
Isolate
Percent
Code
Present
Elimination
312
Tc, K, Stm, Amp, Cb
-*
380
T c , Amp
316
Tc, K, Stan
344
Tc, Stm
376
Tc, Stan
300
Tc, K, Strn
572
Tc, K, Strn
E. coli
D144511S6
R Determinants
Phenotype of Strain
Showing Elimination
E. coli
D14421T1
E. coli
A14443T18
1%
Tc S,
1%
TcR, StmS
Stm8
E. coli
C14432S2
E. coli
014475512
(Tc not transferable)
E. coli
D14437513
Kleb siella
A14466S3
E. coli
-ESH23
Positive
Control
* (-) indicates no elimination.
F 'lac
94%
Lactose Negative
58
into E. coli W3110, no transfer of the tetracycline determinant
occurred.
Compatibility Testing of Transfer Factors
Compatibility testing of R factors from R+ isolates was limited
to a few organisms due to overlapping of r determinants in most of
the test organisms. Table 27 is a summary table of experimental
results obtained with the R+ isolates tested. Data indicate that while
the frequencies of transfer varied significantly, recipients in each
case expressed r determinants from both the donor and recipient R
factors.
Table 27. Compatibility of Transfer Factors Among R+ Isolates
1
Frequency
of Transfer
Recipient
Donor
Experiment
Markers Expressed
in Recipient
E. coli
E. coli
E. coli
A14443S16
D14424T6
D14424T6
(TcR*, KR, StmR)
(Tc
(NaS)
(Na
Klebsiella
E. coli
Amp
)
10-3
(Tc R ,
R
R
R
K,
Stm,Amp)
E. coli
-514424T6
3.2 x 10-5
R
R)
1.2 x
12
R)
D14424T6
A14466S3
(TcR, KR, Stm
,
(Tc ,Amp
R
,KR,Stm R , AmpR)
NaR)
(NaR)
E. coli
E. coli
F. coli
D14424T6
.514424T6
C14432S8
3
(KR,Strn R
R
R
(NaS)
* R - indicates resistance to the antibiotic.
R
(NaR)
R
3.2 x 10-2
R
,StmR,
p
R,CbR)
60
DISCUSSION
The incidence of R+ bacteria among drug resistant enteric
isolates from swine which were fed Lactobacillus concentrate over a
three month period showed marked fluctuation, comparable to that
encountered in the negative control animals.
A significant increase in R+ isolates was observed in both
bottle fed and negative control animals during the third sampling
period, three days postweaning. Characterization of R factor types
obtained from these animals demonstrated a predominance of one
specific R factor (Tc, Amp). Subsequent identification of a limited
number of isolates harboring the (Tc, Amp) R factor implicated one
unique biotype of E. coli, 380.
The emergence of one particular R factor type which had not
been encountered in earlier samples may have been due to the ability
of this biotype to predominate over other intestinal flora during the
physiological stress conditions associated with weaning. The epidemic
like spread of this R factor through animals in close proximity is suggested by the fact that the bottle fed and negative control animals were
kept in the same enclosure. The water fed animals, which were
housed in a separate enclosure, did not show a significant increase in
this particular R factor type at weaning.
The persistence of R+ organisms in the test animals in the
61
absence of antibiotic feeding would suggest that such organisms successfully competed in a population where the number of enteric bac-
teria decreased due to the feeding of lactobacilli (39). Such persistance might be due to saturation of the immediate environment with
R+ organisms as a result of previous widespread use of antibiotics;
therefore, further experiments would be indicated in the absence of
reservoirs of R+ organisms resulting from extensive antibiotic feeding to evaluate the use of lactobacilli as a feasible alternative.
The types of R factors present in test and control animals encompassed a wide spectrum of various combinations of r determinants. Such R factors could have conceivably arisen from recombi-
nation of single determinants or alternatively, from segregation of r
determinants from one parental R factor containing all of the r determinants detected. It is important to note that the R factors charac-
terized could contain additional r determinants which were not tested
for in this study.
In some cases, a resistant phenotype present in the donor was
not transferred, or was not expressed in the recipient. Such a phenomenon could be explained by segregation of individual r determi-
nants as noted by Anderson (2), or by restriction or repression in the
new host. Separation of r determinants as observed in these isolates
is supportive of the concept of R factors existing as plasmid aggregates. It must be pointed out, however, that nontransferable
62
resistance in these donors could be chromosomal in nature.
The utilization of the numerical coding system for the identifi-
cation of R+ isolates revealed a clustering of certain R factor types
with unique E. coli biotypes. Although this phenomenon would merit
further testing, it is suggestive of a permissiveness or nonpermissiveness on the part of the host in accepting unique R factor DNA,
and subsequently expressing resistance or repressing transcription
of the foreign DNA.
MS-2 bacteriophage sensitivity studies done on a representative
number of R+ isolates indicated that the R factors isolated from test
and control swine did not produce F-like pili, and therefore could not
be classified as F-like R factors. Confirmation of the R factor pili
as I-like, however, would necessitate further testing with I specific
bacteriophages, which were not available at the time this investigation was conducted. Since most fi+ R factors are F-like, the lack of
F-like pili would suggest that these R factors are fr, although further testing with F+ organisms should be done. The possibility exists
that the R factors could be classified as being neither F-like nor Ilike, as discussed by Lawn (25).
The host range of the R+ isolates tested encompassed several
genera of the family Enterobacteriaceae. A wide range of transfer
frequencies into E. coli W3110 indicated the presence of wild type
repressed R factors, as well as derepressed R factor mutants whose
63
transfer frequencies were as high as 10-1. The lack of expression of
some r determinants was noted in several transfers, again explainable by repression or restriction by the new host, or by segregation
of the r determinants.
Transfer frequencies into Salmonella enteriditis showed a significant decrease in comparison to the frequencies observed when the
same R factors were transferred into E. coli. Again, segregation of
some r determinants was evident, where populations harboring only
part of the total r determinant complement were observed in close
proximity to organisms expressing total resistance. Four of the
strains tested showed no transfer into Salmonella. Negative trans-
fer could possibly be due to restriction of the R factor DNA entering
the recipient.
Similar results were obtained in transfers into Klebsiella. Frequencies were intermediate, in general, between those observed in
E. coli and Salmonella. Data obtained showed correlation between
frequency of transfer and genetic relatedness between donor and recipient. It was interesting to note that atypical E. coli such as D1443-
7T12, which morphologically resembled Klebsiella, showed much
higher frequencies of transfer into Klebsiella than did those E. coli
strains which were biochemically typical.
Frequency of transfer into Klebsiella 190-3 and Klebsiella 104-A
differed significantly in some cases. Klebsiella 104-A, which had
64
been shown to be atypical, showed lower frequencies of transfer when
used as a recipient, than did Klebsiella 190-3, a typical strain. Negative transfer was observed in three strains using Klebsiella 190-3,
while seven strains showed no transfer into Klebsiella 104-A. Such
observations would again be consistent with host restriction of the
foreign R factor DNA.
Five strains showed definite transfer into Proteus vulgaris,
although the only r determinants expressed were those selected for
after conjugation. One strain which showed transferable tetracycline
and streptomycin resistance, A14443T18, was transferred into Proteus, and an attempt was made to select Proteus strains containing or
expressing both determinants by plating on MacConkey agar supple-
mented with both tetracycline and streptomycin. No such strains
were obtained, indicating segregation of the r determinants had
occurred. These data were in agreement with previous observations
regarding the dissociation of R factors in Proteus (35).
No R factor transfer into Aeromonas was evident. Since this
organism is removed phylogenetically from the Enterobacteriaceae,
(a member of the family Pseudomonadaceae, order Pseudomonadales
negative transfer data is presumed to be due to lack of significant
homology of R factor DNA with the host DNA. The possibility must
be considered, however, that the recipient contained a plasmid which
was incompatible with the R factors isolated in this investigation.
65
Acridine elimination of R factors from eight test strains was
achieved in only two organisms, at a low frequency, approximating
1%.
Such low frequency of elimination was consistent with published
frequencies of elimination, and could indicate chromosomal integra-
tion of the R factors. One strain, C14475S12, retained tetracycline
resistance, but the resistance was no longer transferable, indicating
that the resistance transfer factor as well as the determinant for
streptomycin resistance, had been eliminated. It is possible that this
strain contained chromosomal resistance to tetracycline in addition to
transferable tetracycline resistance, or that the tetracycline determinant had recombined with the host chromosome.
A limited number of R+ isolates were tested for R factor com-
patibility, and in all cases, the recipient which was selected for,
expressed r determinants from both R factors, indicating that the
R factors were compatible. Such compatibility would suggest that if
the R factors are I-like, they would fall into different subclasses,
since R factors of the same subclass would, by definition, be incompatible. The low frequency of transfer of the R factor from Klebsiella A14466S3 into E. coli D14424T6 might, however, indicate
incompatibility where recombination has occurred to prevent exclusion.
The recipient recovered from the transfer of E. coli C14432S8
did not express kanamycin resistance, which could be explained by
66
segregation of this determinant.
As is evident from this investigation, R factors from the R+
isolates obtained from test and control swine are transferable in vitro
to a variety of bacteria from different environmental sources. Since
some of these organisms are considered opportunistic human patho-
gens, it would be of interest to conduct further investigations to as-
certain what role, if any, these resistant organisms might play in the
in vivo dispersion of R factors through animals and animal handlers
into areas where the presence of such R factors would constitute a
serious hazard to human health.
67
SUMMARY
The incidence of R+ organisms in swine which were fed con-
centrates of Lactobacillus showed marked fluctuation, but was com-
parable to that noted in negative control animals, indicating that such
organisms were able to persist in the absence of antibiotic feeding
where the number of coliforms decreased.
A wide spectrum of R factor types encompassing several of the
r determinants tested was observed among R+ isolates from test and
control animals. A predominance of one R factor (Tc, Amp) was evident in the post weaning samples in the bottle fed and negative control
animals, and appeared to be associated with one biotype of E. coli,
380.
This R factor was not present in significant numbers in the
water fed animals, which were housed in separate enclosures from
the bottle fed and negative control animals.
Clustering of certain R factor types with unique E. coli biotypes was observed with several different R factors.
The predominant species identified among the R
isolates was
E. coli, although Klebsiella, Salmonella, and Proteus were also
encountered.
A wide range of transfer among several different genera was
demonstrated with a representative number of R+ isolates. Recipients included E. coli W3110, Klebsiella 190-3, Klebsiella 104-A,
68
Salmonella enteriditis, and Proteus vulgaris. Frequencies of transfer showed correlation, in general, with the genetic relatedness of
donors with recipients. No transfer was evident when Aeromonas
liquefaciens was used as a recipient.
MS-2 bacteriophage sensitivity studies done on selected R+
isolates were negative, indicating that the test group did not produce
F-like pili.
Acridine elimination experiments were done on eight test
strains. Effective elimination was observed in two of the eight
strains, with a frequency of elimination approximating 1%. One
strain, C14475512, retained tetracycline resistance, but when this
strain was further tested for transfer of tetracycline resistance into
E. coli W3110, no transfer was evident, indicating that the resistance
transfer factor, as well as streptomycin resistance, had been eliminated.
Limited compatibility experiments were done on four different
R+ isolates. Data indicated that a degree of compatibility existed
among the organisms tested, although the low frequency of transfer
in one experiment could have been indicative of incompatibility, with
the subsequent occurrence of recombination to prevent exclusion of an
incompatible R factor.
69
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