A study of the interbacterial transfer of drug resistance (R-factor) in the human intestine

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A study of the interbacterial transfer of drug resistance (R-factor) in the human intestine
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x, 87 leaves : ill. ; 29 cm.
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Washington, Louis
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Drug Resistance, Microbial   ( mesh )
Intestines   ( mesh )
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bibliography   ( marcgt )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1972.
Bibliography:
Includes bibliographical references (leaves 79-86).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Louis Washington.

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University of Florida
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
        Page vii
    List of Figures
        Page viii
    Abstract
        Page ix
        Page x
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
    Materials and methods
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
    Results
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
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        Page 71
    Discussion
        Page 72
        Page 73
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        Page 76
        Page 77
        Page 78
    References
        Page 79
        Page 80
        Page 81
        Page 82
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        Page 84
        Page 85
        Page 86
    Biographical sketch
        Page 87
        Page 88
        Page 89
Full Text











A STUDY OF THE INTERBACTERIAL TRANSFER OF DRUG
RESISTANCE (R-FACTOR) IN THE HUMAN INTESTINE







By







LOUIS WASHINGTON






















A DISSERTATIONI PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA 1972















DEDICATION



This dissertation is dedicated to the memory of my parents, Mr. Mose Washington, Jr., and Ms. Isadora Washington; and of-my sister, Ms. Geraldine Washington. They had long since known that the grail was, indeed, the sparrow on which their eyes were cast.




Know the truth, and The truth will make you free Know it well and no one Will be able to deceive or make you afraid.


Charles C. Siefert, 1938













ACKNOWLEDGEMENTS



With humility, I acknowledge the help and counsel of my teacher, Dr. Herman Baer. I thank Dr. Ira Rosen for his many suggestions and support. For reviewing this manuscript, and generously offering valuable criticisms, I thank Dr. Charles Novotny. Indeed, I am appreciative for the suggestions and criticisms given by Drs. Parker A. Small and Peter Cerutti.

During the course of my tenure here, I have formed lasting bonds with some dear individuals -- too numerous to list here. From these dear sisters and brothers, especially Mr. and Ms. Emett McCaskill, I have drawn much comfort; and to them, I extend my thanks for the bond we share.

For the so many ways in.which my family -- past and present -have extended themselves in my behalf, I am thankful. I cherish my daughter, Tracie Leigh, for being my wellspring of enthusiasm.

I am most thankful for my wife, Ms. Geraldine T. Washington, for helping me in an infinite number of very personal and private ways.














TABLE OF CONTENTS

Page


ACKNOWLEDGEMENTS ...............................................ii

LIST OF TABLES ................................................. vi

LIST OF FIGURES .............................................. viii

ABSTRACT...................................................... ix

INTRODUCTION ................................................ I

History and Significance of R-Factors .......... ..........

Genetics and Molecular Biology of R-Factors.................. 3

Epidemiology of R-Factor Dissemination .......................7

Clinical Significance of R-Factor-Mediated Resistance .... 9

Present Study .............................................. 10

MATERIALS AND METHODS.......................................... 13

Clinical Setting.......................................... 13

Study Group............................................... 14

Bacteriological Techniques ......................... ........ 15

Antibiotic Sensitivity Determination........................ 18

Serological Procedures ..................................... 19

Bacteriophages .................................. .......... 21





IVN











TABLE OF CONTENTS (continued)

Page

R-Factor Elimination ................................... 23

Preparation of Nalidixic Acid-Resistant Mutants ........ 26 Invitro Transfer of R-Factor-Mediated Resistance ....... 27 RESULTS .................................................... 31

Invivo Transfer of R-Factor-Mediated Resistance ........ 31

Invitro Analyses of Bacterial Strains Involved
in R-Factor Transfer ................................... 54

DISCUSSION ................................................72

REFERENCES ....................................................... 79

B IOGRAPHICAL SKETCH ......................................... 87



























V













LIST OF TABLES

Table Page

I Gram-Negative Flora of 101 Patients Hospitalized
in Pediatric Intensive Care Unit and Newborn Intensive Care Unit ..................................... 33

2 Distribution of Gram-Negative Flora Among 101
Patients Included in Study of Invivo Transfer
of R-Factors ........................................42

3 Gram-Negative Flora of Patients in Whom Transfer or Segregation of R-Factors Possibly Occurred ................................................ 43

4 Gram-Negative Flora of Patients in Whom Probable Invivo Transfer of R-Factors Was Observed ........ 48

5 Gram-Negative Flora Fron 5 Patients, Each in
Whom the Antibiotic Sensitivity Patterns of
the Different Strains Were Identical .................. 53

6 Invitro Analyses of Strains, From 5 Patients, Who Exhibited Identical Antibiotic Sensitivity Patterns: Matings, R-Factor-Curing, and MaleSpecific Bacteriophage Propagation Experiments ........ 55

7 Resistant and Sensitive Gram-Negative Flora of
39 Patients. No Apparent Transfer of R-FactorMediated Resistance After Continuous Invivo CoIncubation ............................................ 56

8 Invitro Transfer of R-Factors. I. Matings of Donors (Who Were Suspected of Having Acquired Their Resistances Invivo) with Standard Laboratory Recipients. II. Using the Exconjugants Obtained
Frn (1) as Donors, the Original Sensitive Strains
(From Each Patient in Whom Probable Transfer of
R-Factors Occurred) Were Tested to Determine
Their Suitability as Recipients ....................... 61



vi










LIST OF TABLES (continued)

Table Page

9 Invitro Transfer of Antibiotic Resistances From Strains, Which Were Suspected of Having
Donated Their Resistances Invivo, to Standard
Laboratory Recipients .................................. 63

10 Results of R-Factor-Curing Experiments of 9 Strains Which Were Suspected of Having Acquired Their Resistance by Invivo Conjugation .......... 65

11 Adsorption and Propagation of Bacteriophage R17 by Strains Suspected of Having Acquired
Their Resistance by Invivo Transfer of R-Factors ....... 67

12 Propagation of Bacteriophage Ift by Strains Suspected of Having Acquired R-Factors Via
Invivo Conjugation ..................................... 69

13 Determination of the Frequency of Transfer of R-Factors by 8 Bacterial Strains Suspected
of Harboring Derepressed R-Factors ..................... 70

























vii














LIST OF FIGURES


Figure Page

1 Method For the Isolation of Exconjugants of
R-Factor Transfer Experiment When the Chromosomal Marker (Nalidixic Acid Resistance)
Is Carried by the Donor Strain ...................... 30






































viii












Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of Doctor of Philosophy


A STUDY OF THE INTERBACTERIAL TRANSFER OF DRUG
RESISTANCE (R-FACTOR) IN THE HUMAN INTESTINE By

Louis Washington

August, 1972


Chairman: Dr. Herman Baer
Major Department: Immunology and Medical Microbiology, College of Medicine


A study was undertaken to determine the frequency of invivo interbacterial transfer of R-factors among a population of hospitalized patients, who were undergoing a high rate of colonization by multiplyresistant, R-factor-containing bacteria.

The study group consisted of 101 patients confined to the Newborn Intensive Care Unit and the Pediatric Intensive Care Unit, Shands Teaching Hospital and Clinics, during the period 7 January, 1971 to 26 October, 1971.

In 57 patients, resistant and sensitive bacteria were simultaneously isolated on 2 or more successive days. The probable interbacterial transfer of R-factors was observed in 8 of these cases (14 percent). In 6 of the 8 cases, the apparent donors were Pseudomonas (75 percent), and



ix










Klebsiella accounted for 7 of the 8 suspected recipient strains (87.5 percent).

The statistically significant relationship between the resistant Pseudomonas and sensitive Klebsiella strains was examined by invitro mating experiments. No special qualities of the donor or recipient strains could be ascertained.







































X















INTRODUCTION



History and Significance of R-Factors


Emergence of Multiply-Resistant Bacteria

The emergence of bacteria simultaneously resistant to several antibacterial drugs was first noted in Japan, where these drugs were used extensively in the control of bacillary dysentery. At the end of the Second World War, various derivatives of sulphonamide were introduced for the treatment of dysentery, and proved effective. However, in 1949, an Increase in the incidence of dysentery was observed -- caused mosly by sulphonamideresistant mutants of Shigella. The peak incidence of dysentery occurred in 1952 (Watanabe, 1963).

The introduction of the newer antibiotics chloramphenicol, streptomycin, and tetracycline for the treatment of sulphonamide-resistant Shigcella was met by initial success, closely followed by the discovery of multiply-resistant strains of Shigella. Since 1956, the incidence of isolation of multiplyresistant strains in Japan has sharply increased. Though first observed in Japan, the recovery of multiply-resistant enteropathogenic bacteria is universal. Most recent surveys indicate that about 70 percent of the isolates of Salmonella, Shigella, and enteropathogenic Escherichia coli are resistant




2





to one or more of the antibacterial drugs (Lebek, 1967; Anderson, 1968; Davies, Farrant, and Tomlinson, 1968a, b; Watanabe, 1966). Transfer of Resistance by Coniugation

Ochiai et al. (1959) and Akiba et al. (1960) discovered that the multiple drug resistance of Shigella could be transferred to drug-sensitive strains of Escherichia coli and Shigella simply by growing them together. They further noted that cell-free filtrates of the donor culture could not transfer resistance, indicating the need for cell-to-cell contact. The subsequent experiments of Campbell (1962) and Jacob and Wollman (1958) showed that the multiple resistance was carried on, and transferred by, a sex factor. Watanabe (1963) introduced the term R-factor for this sex factor. Watanabe named that component of the R-factor which mediates its transfer, Resistance Transfer Factor (RTF), and to that part which contains the genetic determinants for drug resistance, he gave the name R determinants. Antibiotic Resistances Mediated by R-Factors

The first multiply-resistant organisms isolated carried an R-factor

which specified resistances to suiphonamides, chloramphenicol, streptomycin, and tetracycline. R-factors conferring the resistance to kanamycin and neomycin were later isolated (Lebek, 1963; Watanabe, Ogata, and Sato, 1964). Other R-factors have been isolated which mediate resistance to aminobenzyl penicillin, and nitrofuran derivatives (Anderson and Datta, 1965; Datta and Kontomichalou, 1965; Smith and Halls, 1966). D. H. Smith (1967a) isolated





3





many R-factors which conferred resistance to the aminoglycoside antibiotics gentamycin, spectinomycin, and viomycin. Smith (1967b) and Novick (1967) have shown that some R-factors confer resistance to the heavy metals, mercury, nickel, and cobalt.


Genetics and Molecular Bioloqy of R-Factors Compos i ti on

R-factors are closed circular extrachromosomal genetic elements, consisting of deoxyribonucleic acid (DNA). They are capable of autonomous replication, and possess the ability to effect their transfer from one

bacterium to another by conjugation. The R-factor is composed of a Resistance Transfer Factor and a Resistance determinant, which, in some host cells, may exist as two discrete molecules (Anderson and Lewis, 1965 a, b; Anderson, 1968). Rownd and Mickel (1971) proposed that the dissociation and reassociation of RTF and R determinants of the R-factor NRI

In Proteus mirabilis has a regulatory effect on the number of R determinants per host cell.

fi Types. Bacteria which transfer genetic material, via conjugation,

to other bacteria possess conjugation tubes (pill) which extend from the surface of the cell. Two types of pill have been described on the basis of their morphology, antigenic properties, and bacteriophage adsorption and propagation characteristics (Crawford and Gesteland, 1964; Brinton,




4






Gemski, and Carnahan, 1964; Orskov and Orskov, 1960; Ishibashi, 1967; Meynell and Datta, 1966a; Meynell and Lawn, 1967a, b). Two types of R-factors have been defined on the basis of the pili produced by the host cells. One group of R-factors contain genes for a pilus which is morphologically identical to the pilus produced by cells harboring the sex factor F, whereas the other group of R-factors code for a pilus resembling the I pilus first Identified in HFT cultures of Col + bacteria (Meynell and Lawn, 1967a).

R-factors which specify F pili produce a repressor which represses the production of its own pill in infected strains. This repressor suppresses the production of F pili in cells co-infected with F-like R- and F-factors. Resultingly, such cells are unable to transfer their sex factors. Thus, the F-like R-factors are said to be fertility-inhibiting (fi ). The second group of R-factors (I-like) does not suppress the sex factor F, and are designated fi Meynell and Datta (1967) isolated derepressed mutants of fi+ R-factors which no longer repressed production of their own pili, nor suppressed F pill. The fi+ pill are antigenically identical to F pili, and are sensitive to F-specific phage. These pill have been shown to be the structure to which the phage adsorbs (Meynell and Datta, 1966a; Datta, Lawn, and Meynell, 1966). By contrast, cells with fi R-factors do not propagate F-phages (Meynell and Datta, 1966a).

Colicinogenic factors (Col factors) are plasmids specifying the pro-





5




duction of proteins called colicins, which are elaborated by enteric bacilli and lyse other enteric bacilli (Gratia, 1932; Fredericq, 1957). The Col factor is not usually transferred by conjugation -- linkage with a sex factor is not a general feature. However, conjugal transmission has been demonstrated for several Col factors, including Col Ib (Ozeki and Howarth, 1961; Clowes, 1961; Meynell, 1961), Col IV, Col V and Col VI (Fredericq, 1963, 1965; Kahn and Helinski, 1964; Macfarren and Clowes, 1967; Nagel de Zwalg and Anton, 1964), Col Ela (Meynell and Lawn, 1967b), and Col B (Watanabe and Okada, 1965). The pili specified by fi R-factors resembles, both morphologically and antigenically, the Col pili. The tip of the fi R and Col pill is the receptor for the filamentous I phages (Lawn et al., 1967; Meynell and Lawn, 1968).

Both fi and fi R-factors are immune to superinfection by R-factors

of the same type, but not to R-factorsof a different type. Superinfection immunity is due, probably, to the modification of, or elimination of, surface receptor sites on the host cell (Watanabe, 1969). Association With Other Sex Factors

Sex factors (including R) are promiscuous with respect to their host range, their interactions with each other and their interactions with the DNA of the host cell. These sex factors, carrying a wide variety of genetic determinants, are transmitted between many species of enteric organisms (Meynell, Meynell, and Datta, 1968; Falkow, Johnson, and Baron, 1967).





6





It is the accessory genetic determinants, on a sex factor, which are usually responsible for its discovery, and which decide the category into which it is placed (Meynell and Datta, 1969). Approximately one-third of the fi Rfactors specify the production of Colicin I; however, since it was their resistance to antibiotics which led to their detection, they are designated R-factors rather than Col factors (Meynell and Datta, 1969).

Some of the characteristic interrelations between transmissible extrachromosomal elements follows. Resistance determinants of nontransferable Rfactors may be mobilized by the sex factors F, Col I, etc. (Anderson and Lewis, 1965a, b; Anderson 1965a, b, 1966a, b; Fredericq, Krvmiry, and Kettner, 1971). Fredericq (1966) demonstrated the recombination of Col B with R-factors and the conjugal co-transfer of the sex factors F (lac), R(T), Col B, Try.

The sex factor F, and the Col factor V must be closely related because they are the only wild type sex factors which continuously produce sex pili by all cells (Meynell and Datta, 1969), and F is the only factor with which Col V demonstrates superinfection immunity (Kahn and Helinski, 1964; Macfarren and Clowes, 1967). The similarity of F to the R-factor FI00-1 (a derepressed mutant of RIOO) was noted by the demonstration that mutations in 8 of the 10 cistrons, specific for transfer in F, could be complemented by RlO0-1 (Willets, 1971). R and F-infected cells differ in respect to their susceptibility to bacteriophage. F-factors contain the information for the restriction of phages iI and T 7, whereas R-factor-infected cells are





7





sensitive to these phages. The superinfection of F-infected cells with R

does not fully repress the immunity of the cells toward these phages (Morrison and Malamy, 1970).


Epidemiologly of R-Factor Dissemination


The early epidemiological significance of transferable drug resistance

was noted when sensitive strains of Shigella were isolated from some patients, whereas strains that were isolated from other patients in the same epidemic, and of the same serological group, were multiply-resistant (Akiba and Kimura, 1959; Matsuyama, Harada, Suzuki, Kameda and Komiyama, 1959; Ochiai, 1959). After treatment with a single antibiotic, patients harboring sensitive Shiqella and multiply-resistant Escherichia coli strains, began secreting multiply-resistant Shigella. This phenomenon was explained by the experiments of Akiba (1959), and Ochiai (1959), which demonstrated the invitro transfer of multiple resistance via conjugation, and suggested that such transfer occurred in the human intestinal tract. Lebek (1963) isolated a multiply-resistant Salmonella typhimurium from a patient in Munich; Escherichia coil with the identical resistances were isolated from the patient at an earlier time when he was excreting fully sensitive Salmonella.

The possibility of invivo transfer, as the vehicle for the dissemination of R-factors, has led to many experimental studies of this phenomenon.

Using human volunteers, Akiba et a]. (1960) and Kagiwada et al. (1960)




8





demonstrated the invivo transfer of multiple drug resistance in the intestinal tract. Harada, Suzuki, Kameda and Mitsuhashi (1960) and Nakaya et al. (1960) showed that the resistance factor could be transferred to the other members of the family Enterobacteriaceae, and R-factors have been transferred to Pseudomonas and Vibrio (Baron and Falkow, 1961).

Smith (1966) was successful in producing invivo transfer, in a single human volunteer, after unusually high orally administered doses (10 9). However, the results were transitory, the resistant strain was eliminated within several weeks. Roe et al. (1971) observed the changes in the antibiotic patterns of Pseudomonas strains among severely burned hospitalized patients, who were co-infected with multiply-resistant Escherichia coli strains, and demonstrated the invivo transfer of resistance from Escherichia coli to Pseudomonas in experimentally burned mice. Jones and Curtiss (1971) observed transfer of R-factors among Escherichia coli strains in the mouse intestine. Using human volunteers and laboratory animals, others have observed invivo transfer of R-factors (Kasuga, 1964; Jarolmen and Kemp, 1969; Walton, 1966) and uncovered epidemiological evidence for the transfer of R-factors in the human bowel (Watanabe, 1963; Davies et al., 1968a, b). Although these studies have lent credence to the possibility of the transfer of R-factors, via conjugation, within the human body, no conclusive data have been presented to show that invivo conjugation and R-factor transfer occurs under natural conditions. Moreover, it is presumed that Invivo




9





R-factor transfer is limited by the inhibitory effects of bile salts, fatty acids, anaerobic conditions and acid pH (Watanabe, 1963). In a prospective study of R-factor transfer in the human, Gardner and Smith (1969) found no evidence for transfer of R-factors, and concluded that selective factors, rather than invivo conjugation, may be the principal mode of R-factor dissemination.


Clinical Significance of R-Factor-Mediated Resistance


The clinical significance of bacterial resistance to chemotherapeutic drugs varies according to the nature of the mechanisms which control that resistance. Two mechanisms for antibiotic resistance are recognized -extrachromosonally determined resistance, and those resistances which are chromosomal ly control led.

Chrmosamal-mediated drug resistances are characterized by low mutation rates (10-8 to 10-10 ), so the selection of mutants will occur only in infections where the bacterial population is very large. The level of resistance of such first-step mutants is generally low enough to be overcome by increased dosage of antibiotics.- Treatment with a combination of unrelated antibiotics avoids the selection of mutants resistant to one type of antibiotic. In some cases, the mutant is metabolically defective, thus its pathogenicity may be reduced.

Resistance controlled by transferable extrachromosal elements is quite




10





different. Sometimes, resistances to all clinically available antibiotics are transferred en bloc between pathogenic and non-pathogenic bacteria. This transfer occurs between different taxonomic groups of gram-negative rods, such as Pasteurella, Vibrio, the Enterobacteriaceae and Pseudcmonas.

Since resistance of this type is usually mediated by enzymes which inactivate the antibiotic, the level of resistance is variable, and increases with the bacterial population. A single antibiotic may select for several resistances present on one R-factor, thus rendering combination antibiotic therapy useless. Bacteria harboring extrachromoscmal resistance factors are usually normal in all other respects.

Therefore, from a medical aspect, the threat posed by extrachromosomal transferable drug resistances to the efficiency of antibiotics is a very serious problem.


Present Study


General Conditions

This study was undertaken to determine the significance of invivo conjugation and transfer in the dissemination of R-factors among a population of patients who are undergoing a high rate of colonization by multiply-resistant, R-factor-containing bacteria. The study required daily cultures from the population of patients, and controlled environmental conditions.

In addition to a controlled population, and a predisposition by that











population toward acquisition of superinfecting strains, there must be present (1) a reservoir of R-factor-carrying bacteria to act as donors of the R-factors; (2) the presence of sensitive strains of bacteria among the patients, similarly-to serve as recipients of the R-factors; (3) a high incidence of antibiotic usage which acts as a selective force for the persistence of R-factor-containing bacteria; and (4) an effective means of monitoring the acquisition of, and antibiotic resistance changes in R-factor-carrying gram-negative bacteria. Experimental Approach

To effectively demonstrate invivo transfer, under the conditions of these experiments, the following conditions must be met:

1. A suitable recipient must be present. The recipient (that

organism which gains, by invivo transfer, additional antibiotic resistances) should be present upon admittance of the

patient, or acquired thereafter, and be characterizable biochemically, serologically, and by antibiotic sensitivity. Because extensive serological procedures are commercially available for Klebsiella and Escherichia strains only, the demonstration of invivo transfer was limited to organisms of these two

genera.

2. A resistant strain (donor) must be introduced or already be

present in the intestinal flora. This strain should be character-











izable in the same manner as the recipient; however, this is a

necessary condition only when the recipient strain and the prospective donor are of the same genus. (The order of acquisition

of recipient and donor is commutable.)

3. A change in the antibiotic resistance of the recipient must occur.

Proof of the occurrence of invivo transfer of R-factor-mediated resistance includes:

I. The recipient is biochemically and serologically identical both

before and after acquisition of resistance.

2. The resistances involved are R-factor-mediated, as evidenced by

(a) male-specific phage adsorption and propagation tests, (b) Rfactor-curing experiments, and (c) invitro transfer of antibiotic

resistances.

3. Evidence that the recipient, with the R-factor-mediated resistances observed, is not otherwise present in the population under

study.














MATERIALS AND METHODS


Clinical Setting


Newborn Intensive Care and Pediatric Intensive Care Units

The patients included in this study were confined to the Newborn

Intensive Care Unit (NICU), or the Pediatric Intensive Care Unit (PICU), Shands Teaching Hospital and Clinics, University of Florida, College of Medicine, Gainesville. The NICU consists of 2 large semi-independent rooms, each with a capacity of 8 bassinets, and connecting with a central nursing station. Entrance to either of the rooms is by way of the nursing station. PICU is a I-room facility, with a capacity of 10 beds, and the nursing station is centrally located along the forward wall. NICU and PICU Procedures

Upon entering these units, all personnel don clean gowns; caps and masks are not worn. A scrubbing area is situated immediately after the door, in both units, and personnel scrub their hands, fingernails and arms, to the elbows, with water and germicidal soap, prior to entering these units. The hands and arms are dried with clean paper towels, and hands are washed with germicidal soap and water between successive handling of patients.




13




14





Study Group

Patients

During the period of January to October, 1971, 101 successive patients admitted to NICU and PICU were included in this study. Most patients were confined to these units in excess of 5 days, thus minimizing the level of transiency. The confinement of this study to Pediatrics and Newborn ICU's was Ideal, because in these units a high rate of incidence of colonization with R-factor-containing gram-negative organisms has been demonstrated (Eisenach et al., 1972).

Patients confined to NICU were not included in this study after March, 1971, because of the introduction of the antibiotic gentamycin in the drug regimen of that unit. Although organisms with R-factor-mediated gentamycin resistance have been isolated (Smith, 1967c), none has been observed at this hospital. Thus, the acquisition of hospital strains of R-factor-containing organisms was greatly diminished in NICU. Cultural Techniques

The intestinal tract was the selected site for monitoring and observation of R-factor transfer, because of its importance as a reservoir in gramnegative nosocmial Infections (Gardner and Smith, 1969). The gram-negative intestinal flora of the patient was determined upon admission, and followed daily thereafter for the next 5 days, after which the patient was cultured every other day through day-9. It was our opinion that, if changes





15





in antibiotic resistance patterns had not become evident by day 9, then the likelihood of such a change occurring thereafter was not great enough to warrant continued monitoring.

For the purpose of these studies, the gram-negative bacteria which grew on MacConkey agar under aerobic conditions, were considered to constitute the patient's intestinal flora. Each strain of the patient's initial flora was identified, and its antibiotic sensitivity was determined. Subsequent specimens were identified and compared with the initial strains. Strains which possessed the same biochemical and physiological characteristics as the initial strain, but different in antibiotic sensitivities, were subjected to serological procedures to establish their identity with the initial strains. If serologic identity was confirmed, the recipients (as these subsequent cultures were called) were subjected to mating experiments, R-factor-curing procedures, and phage adsorption and propagation tests -- to confirm that the newly acquired resistance was R-factor-mediated.


Bacteriological Techniques


Stock Cultures

A number of strains of bacteria were kindly provided by Dr. George J. Hermann, Enteriobacteriology Unit, National Commnunicable Disease Control Center, Atlanta; Dr. Paul Chun, Depa-rtment of Biochemistry, College of liedicine, University of Florida; and Mrs. Kathleen D. Eisenach, Hospital Epi-











demniologist, Shands Teaching Hospital and Clinics, Gainesville. These
-1
strains included cultures of Escherichia coli 01:NM, R ; Ola:K:H7, R ;
+ + +
04:NM, R (S,T,Am,K); 04:K3:HS, R'; 06:NM, R (S,T); 06:NM, R (S,C,Am,K); 06:K2:Hl, R ; 075:NM, R (S,G,T,Am,K);2 075:KI:H5, R ; and Klebsiella +
pneumnoniae types 28, R (S,T,Am,K); 28, R-; 31, R'; 33, R'(Am,K); 33, R ; 39, R (T); 39, R ; 63, R (S,T,C,Am,K); and 63, R. Media

The media employed in the growth, propagation, and identification of the strains under study, were as follows:

I. MacConkey's Agar (Difco), and MacConkey's agar with antibiotics,

were used for initial cultivation and screening of rectal specimens.

The antibiotics incorporated into the media, and the drug concentrations were (a) tetracycline (Tetracyn, J. B. Roerig Company),

30pgm/ml; and (b) kanamycin (Kantrex, Bristol Laboratories),

30Agm/ml.

2. Mueller-Hinton Medium (Dlfco) and Mueller-Hinton Medium, to which

antibiotics were added, were used in all replica-plate experiments, and in determining antibiotic sensitivity by the KirbyBauer method. The antibiotics added, and their final concentration



NM Nonmotile; R = Organism does not harbor R-factor

2 +
R = Organism contains R-factor; S = streptomycin, C = chloramphenicol,
T = tetracycline, An = kanamycin, K = kanamycin




17





in the medium, were (a) tetracycline, 30 jAgm/m l; (b) kanamycin,

30 ,gm/ml; (c) streptomycin sulphate (Eli Lilly), lO10gm/ml;

(d) chloramphenicol (Chioromycetin, Parke-Davis Company), 30 .gm/ml;

(e) ampicillin (Penbritin S, Ayerst Laboratories), 10,ugm/ml; and

(f) nalidixic acid (Neggram, Winthrop), 30igm/ml.

3. L Broth and Agar (Lennox, 1955) was used for bacteriophage R 17

adsorption and propagation.

4. Worfel-Ferguson Agar (Difco) was used for growth and enhancement

of capsular antigen by Klebsiella strains.

5. Tryptic Soy Agar (Difco) and Tryptic Soy Broth were used for

growing Escherichia coli strains preparatory to serological

procedures.

6. Pennassay Broth (Difco) was used in all curing experiments.

7. Heart Infusion Broth (Difco) and Heart Infusion Broth with 0.35

percent agar were used for bacteriophage Ift adsorption and propagation.

8. Bacto-Blood Agar Base (Difco) was used for bacteriophage Ifj

adsorption and propagation.

9. Davis Minimal Broth (Difco) with I percent glucose was used for

Escherichia coli curing and selective enrichment procedures. Isolation and Identification of Bacterial Specimens

Isolation.- Initial rectal swabs were obtained from the patient upon











admission to the unit, and planted onto Mc, McT, and McK plates. The plates were streaked with a straight wire immediately thereafter to insure that invitro transfer of R-factors did not occur. Subsequent cultures were identically handled.

Biochemical and physiological procedures. Incubation of all cultures was at 370 C, unless otherwise indicated. After overnight incubation, representative colonies, of each morphological type, were picked with a straight wire and subjected to a standardized set of bacteriological tests (Baer and Washington, 1972). These strains were also inoculated into TSA deeps (for stock cultures) and TSB (for antibiotic sensitivity tests).


Antibiotic Sensitivity Determination


Antibiotic sensitivities were determined by using antibiotic discs in a single high concentration (Hyland) as reconmended by Bauer et a]. (1966). Enough bacterial growth from a pure culture of the strain to be tested was spread on Mueller-Hinton Meditmi plates (Difco) by means of moist cotton swabs to just produce confluent growth. After application of the sensitivity discs by means of an automatic dispenser, the plates were incubated overnight at 370 C. Zone sizes then were determined and the results read quailitatively as "sensitive," "resistant," or "Intermediately sensitive."


Mc MacConkey agar
McT MacConkey agar with tetracycline, 30jsgm/ml
McK MacConkey agar with kanamycin, 30jAgm/ml





19





The accuracy and reproducibility of the results was checked periodically by comparing the results obtained with the disc sensitivities with those obtained by performing dilution sensitivities in agar plates containing doubling dilutions of the antibiotic.


Serological Procedures


Of the members of the family Enterobacteriaceae. comprehensive serological procedures are commercially available only for Escherichia coli and Klebsiella pneumoniae. Hence antigenic identification of strains from other genera could not be determined. Therefore, no attempt was made to demonstrate invivo transfer of R-factors among genera other than Escherichia and Klebsiella.

The methods employed in serotyping these strains were those currently used by the Communicable Disease Center, Atlanta. Serotyping of Klebsiella

Inasmuch as the somatic (0) antigen is not assessable in many cases, the heat-stable capsular (K) antigen is used in serological procedures. Visual inspection of the colonial morphology was made to determine, grossly, the presence of a capsule (dry, wrinkled colonies indicated that the strain was not typable). The cultures were inoculated into Worfel-Ferguson agar, which contains 1.0 percent sucrose to enhance capsule production. A loopful of an overnight culture was inoculated into 0.5 ml of a physiological





20





saline solution, to which formalin (0.5 percent) had been added. From this tube, one loopful of the heavy suspension was taken and inoculated into 1.0 ml of formalinized saline. The presence of a capsule was determined by microscopic observation of "India Ink" slide preparations of the Kiebsiella strain. To determine the serological type, a light suspension of the encapsulated strain was placed onto a slide along with the specific type antisera (Klebsiella antisera, Difco), and microscopically looking for capsular swelling. Escherichia coli serotyping

Serological procedures were limited to the somatic (0) antigen. Escherichia coli antisera (Difco) were used in these procedures. The test strain was inoculated onto Tryptic Soy Agar (Difco) and incubated overnight. Subsequently, it was subcultured into duplicate 10 ml tubes of Tryptic Soy Broth (Difco) and incubated, with shaking, overnight. The strains were then steamed for one hour to destroy the heat labile antigens, and allowed to cool; then checked for "roughness". Those strains which "settled out" after cooling were discarded due to the lack of a typable

0 antigen. The cultures were diluted one-half with phenolized physiological saline (0.5 percent), and to 1.0 mi of the prepared antigen, 0.1 ml of the antisera under consideration, was added. The antigen-antisera mixture was incubated overnight at 500 C and checked for agglutination.











Bacteriophages

Propagation of R17 and Ifi

Greater than 95 percent of the bacterial strains harboring R-factors produce pill of either the f + or fi types (Lawn et al., 1967). The ability of the bacterial strains, which were suspected of containing Rfactors, to propagate the pili-specific bacteriophages R17 (f + ) and Ifl (fi') was tested.

R17. Escherichia coli K38 R F was used as a positive control in the propagation of R17. Suspected strains were inoculated into L broth and grown overnight at 370 C. The strain-was subcultured into fresh L broth (0.1 ml into 10 ml) and grown to a cell density of 107/ml, with shaking, in a 37 C water bath. To this tube, 10 phage particles were added to yield a final proportion of I phage particle per 100 bacterial cells. Onetenth mililiter of 1.0 mular calcium chloride was added to the phagebacteria mixture. Since the probability of a given bacterial cell (harboring a repressed R-factor) possessing a pilus is .01, the ratio of phage particles to bacterial cells (with pili) approached I : I (Meynell and Datta, 1965, 1966a; Arai and Watanabe, 1966; Datta, Lawn, and Meynell, 1966).

To the overnight phage-bacteria mixture, several drops of chloroform was added. The broth was filtered through .045 mm millipore filters, tenfold serial dilutions of the filtrate prepared, and the titre was determined by the soft agar overlay method, using Escherichla coli K38F+




22





as the seed layer, and scoring plaques.

Ifi. Escherichia coli Kl2 J53, carrying the derepressed fi R-factor 64-11 (Meynell and Lawn, 1968) was used for propagation of Ift, and as a positive control in tests for fi R-factors. Escherichia coli K12, Anber's strain 2395 (strain 803 of Wood, 1966) was used as the indicator strain. Test strains were inoculated into Heart Infusion Broth (Difco) and grown overnight at 370 C. Each strain was subcultured into fresh HIB and grown to a cell density of 107/ml, in a 370 C waterbath. Bacteriophage Ift were added, as above, to yield a concentration of 10 bacteria to 1 phage particle. The phage-infected cultures were incubated overnight at 370 C, then centrifuged and filtered. Tenfold serial dilutions were prepared, and the bacteriophage titres were determined, using a soft heart infusion agar overlay technique, with strain 2395 serving as the indicator. Phaqe Adsorption

R17. An alternate method employed for affirming the presence of fi Rfactors was the decrease in free phage titre in a phage-bacteria mixture, after a short incubation period of 30 minutes at 40 C. As in the phage propagation experiments, Escherichia coli K38R F was used as a positive control and as the indicator strain; Escherichia coli 01:NMR F served as a negative control. The cultures were grown overnight in L broth at 370 C. Each strain was reinoculated into fresh L broth (with CaCl2, 0.1 M) and grown to a concentration of 107 cells/mi. Phage R17 was added to a concentration of 105 part-





23





icles/ml and the mixture was incubated for 30 minutes at 40 C. Filtration was accomplished with 0.045 mm millipore filters fitted onto 5.0 ml syringes. Tenfold serial dilutions of the filtrate were prepared, and 0.1 ml of the filtrate was added to 5.0 ml of soft L agar (containing 0.1 M CaCl 2) and

0.1 ml of an overnight culture of the indicator strain. The soft agar mixture was poured onto L agar plates and incubated overnight at 370 C. Scoring of phage titre was accomplished by plaque count.

Ifi. Similarly, the adsorption of phage Ifi by suspected strains

was tested. Escherichia oli K12 J53 was used as a positive control, and 2395 as the indicator. The conditions of the experiments were identical to the phage R17 adsorption tests. However, the media used were HIB, HIA, and soft HIA (0.35 percent agar).


R-Factor El imination

Ethidium Bromide. Acridine Orange Treatment

Strains were inoculated into Pennassay Broth (Difco) and grown overnight at 370 C. One-hundredth milliliter of a one-hundredfold dilution of the cultures were subcultured into 100 ml of fresh Pennassay broth and incubated for 2 hours. Experimental titratlons had revealed that, at this point, the cell density of these subcultures had reached "105 cells/mi. ethidlum bromide (450 ,gm/ml) or acridine orange (35OjAgmhnl) was added to the cultures, which were then incubated with shaking for 18 hours. Tenfold




24.





serial dilutions of the cultures were made, and 0.1 ml of the treated strains were inoculated on Mueller-Hinton plates (4 plates per dilution), uniformly spread over the surface of the plates with a glass rod, and incubated overnight.

Those plates which yielded approximately 25 colonies were replicaplated onto Mueller-Hinton agar plates, containing the selected antibiotics (see section on Media). After overnight incubation; the antibiotic plates were compared with the master plates. The cured colonies (those colonies which grew on the master plate, but failed to grow on I or more of the antibiotic-containing plates) were picked from the master plates and purified by streaking onto a fresh Mueller-Hinton agar plate and incubating overnight at 37 0 C. Five representative colonies .from each plate were selected, inoculated into TS broth, and their antibiotic sensitivities were determined by the Kirby-Sauer method. Ultraviolet Irradiation

Those resistant strains which did not respond to the curing actions of ethidium bromide or acridine orange were subjected to ultraviolet irradiation prior to treatment with, the curing agents (Watanabe and Fukasawa, 1960; Iyer and Iyer, 1969). A Hanovia lamp, model No. 605000, was used as the source of UV light. The light was fixed at a distance of 26 cm above the surface of the culture to be irradiated. At this distance, an exposure time of 2 minutes yielded a survivor rate of approximately





25





I percent. Cells were grown and subcultured in Pennassay broth as in the preceding section. The subcultured strains, after incubation at 370 C for 2 hours, were poured into a sterile petri dish and irradiated. The strains were then transferred into tubes and left in the dark for I hour at 370 C. The strains were then treated with ethidium bromide or acridine orange. These cultures were then treated in the same manner as stated above.

Isolation of Cured Cells by Selective Killing

Since the proportion of cured Escherichia co.i cells, obtained after

combined treatment with UV and acridine orange, was very small (41 percent), a modification of the penicillin screening method of Watanabe and Fukasawa (1960) was used to increase the frequency of recovery of these sensitive cells. R-factor-containing strains were inoculated into Davis Minimal Broth (Difco) with 1 percent glucose, and grown with shaking, at 370 C to a density of I05 10 6 The cells were then exposed to UV light, incubated for I hour in the dark, and then acridine orange was added to the cultures. The cultures were incubated for 20 hours with shaking, then centrifuged, washed with 5.0 ml of Pennassay broth, recentrifuged and washed twice, and resuspended in Pennassay broth. After incubation for 40 minutes at 370 C with shaking, either chloramphenicol or tetracycline was added (at a concentration of 30j)gm/mi).

Those cells which had lost their resistance to these bacteriostatic





26





antibiotics were inhibited by the drugs, whereas the cells which retained the R-factors continued to grow.

The cultures were incubated for another hour, and then subjected to a massive inoculation of penicillin (1004gm/ml). The cultures were then incubated for an additional 3 hours. During this period, the actively metabolizing cells were subject to the bactericidal action of penicillin, but the chloramphenicol or tetracycline-sensitive cells were not affected since they were not growing.

The culture was then centrifuged, washed in Pennassay broth, and resuspended in fresh Pennassay broth. The washing procedure was repeated twice. Then the cultures were resuspended in 10 ml of Pennassay broth and incubated, with shaking, for 4 hours. Tenfold serial dilutions of the cultures were prepared, and 0.1 ml of each dilution was spread onto tueller-Hinton plates and incubated overnight. Velvet replication plates were prepared as in the other curing procedures, and examinated after overnight incubation.


Preparation of Nalidixic Acid-Resistant Mutants


Nalldixic acid-resistant ( >lOOjugm/ml), multistep chromosomal mutants of Escherichia coli K12 F R and Klebsiella pneumoniae 33 R F were isolated by the Szybalski Gradient Plate Technique, as modified by Webb and Washington (1966). Square style, 100 x 15 nun petri dishes (Integrid, Falcon





27





Plastics) were inclined such that when 20 ml of melted Mueller-Hinton agar was poured into each dish, the thickness of agar at the lower side of the dish was 8 mm, whereas the "agar-front" just reached the side of inclination. After allowing the agar to harden, the plates were placed onto a level surface, and overlayed with 20 ml of Mueller-Hinton agar to which nalidixic acid (100jgm/ml) had been added. Calcium alginate swabs of TS broth cultures were streaked onto the prepared plates. Diffusion of the drug fran the upper layer to the lower layer provided a solid-surface concentration gradient fram which spontaneous nalidixic acid-resistant mutants could be isolated. These mutants were observed as isolated clones, growing in a forward area of higher nalidixic acid concentration, ahead of the streak of confluent bacterial growth.

The foremost clone from each plate was picked, inoculated into TS broth, and restreaked onto similarly prepared'plates, with the exception that the level of nalidixic acid in the upper layer was 200jAgn/ml. This second step was repeated until colonies, which grew to a point beyond the medial of the plate, were isolated. The level of drug resistance was confirmed, for each strain, by serial tube dilution.


Invitro Transfer of R-Factor-Medlated Resistance


The donors for these experiments were those strains which were suspected of having acquired their resistance by an invivo conjugation event. The re-





28





cipients were the nalidixic acid-resistant mutants of Escherichia coli K12 and Klebsiella pneumoniae type 33 which were prepared in our laboratory, and which harbored no sex factors. The exconjugants (those cells which acquired antibiotic resistances as a result of this cross) were then used as donor cells, and the original sensitive strains, obtained from the same patients, were the recipients in experiments to test the ability of the latter to function as recipients.

Both donor and recipient cells were incubated overnight in Pennassay broth or R broth. The overnight cultures were then diluted fiftyfold into fresh Pennassay broth or L broth, and incubated with shaking in a 37 C water bath for 2 hours. The donors were mixed with the recipients in a ratio of I : 1, and incubated, without shaking for an additional 2 hours. At the end of this period, the mating was interrupted by agitation for 30 seconds, and the mixed culture was diluted hundredfold.

Tenfold serial dilutions of the mixture were plated onto a set of 6 Mueller-Minton agar plates, each one of which contained either nalidixic acid at a concentration of lOOjxgm/ml, or one of the antibiotics (see Media, p. 16). Colonies from the antibiotic-containing plates were picked and streaked onto Mueller-Hinton plates and incubated overnight. Representative colonies from these plates were grown in TS broth, and tested for antibiotic resistance by the Kirby-Bauer method.

Mating mixtures of exconjugants (paragraph 1) and test strains were





29





serially diluted as above, and plated onto Mueller-Hinton medium (Figure I). After overnight incubation, these plates were velvet-replicated onto NuellerHlinton plates containing the selective antibiotics or nalidixic acid. After overnight incubation, the master plate was compared with the nalidixic-acidcontaining replicate, and those which failed to grow on the latter were circled on the master plate, and then the circled colonies were compared with the other antibiotic-containing replicates. Nalidixic acid sensitive, antibiotic-resistant exconjugants were purified by single colony transfers, and tested for drug sensitivity as above.





A. Mating mixture of donor EXAMPLE
and recipient
Donor: E. coil R Na (obtained
from mating E. coil R+Na x
E. coll R-NaR)
B. Inoculate onto master plates, E. coil RNaR )
Incubate overnight. S
incubate overnight, tenfold serial dilution Recipient: Klebsiella R Na


C. Replicate onto antibiotic Colonies on master plate:
O 000Colonies oi matr+plte
plates and plate contain- 1. E. coil R+Na
ing nalidixic acid. 2. Klebsiella R'Na
3. Kiebsiella R+NaS

iD. Master plates (B) are corn- Colonies on nalidixic acid plate:
pared with anlidixic repli -+ R
cates. Those colonies on S T C .Am E. coil RNaa
master plates which do not /\ /
master plates which do not Colonies on antibiotic plates:
grow on nalidixic replicates
are circled. 1. E. coil R+NaR
2. Klebsiella R+Na

E. Master plates now compared with Colonies circled on master plates:
antibiotic replicates. Colonies 1. Klebsiella R Nas
on'replicates, which correspond 2. Klebsiella R+NaS
to circled colonies on master
plates, are recipients. Circled colonies on master plates,
also growing on antibiotic replicates:
1. Klebsiella R+NaS


Figure 1. Method For the Isolation of Exconjugants of R-Factor Transfer Experiment When the Chromosomal Marker (Nalidixic Acid Resistance) Is Carried by the Donor Strain













RESULTS



Invivo Transfer of R-Factor-Mediated Resistance


Gram-negative bacteria were isolated from 91 of the 101 patients included in this study. A synopsis of these 101 patients and their respective gram-negative flora is presented in Table I. Those patients from whom no bacteria were recovered were hospital born and did not .acquire a gramnegative intestinal flora during the period which they were cultured. An additional 9 newborn patients, who were admitted to INICU without gram-negative flora, acquired a single gram-negative strain during their hospital

tenure. The flora of 25 additional patients was inconsequential and did not contribute to the study.

Anong 18 of the remaining patients, it was reasonably suspected that a transfer of R-factor-mediated resistance occurred. In the remaining 39 cases sensitive and resistant organisms were simultaneously isolated from the patients on 2 or more consecutive days, without any apparent transfer of R-factors. The distribution of patients within these groups is shown in Table 2.


Cases of Possible Invivo Transfer, or Segregation, of R-Factors

There were some indications of the possible invivo transfer, or loss,

of R-factors among the gram-negative flora in 5 cases (Table 3). This




31













Key to Symbols and Abbreviations used in Table I







Antibiotics

STR streptomycin

C chloramphenicol

T tetracycline

An ampicillin

K kanamycin



R resistant S sensitive I intermediately sensitive, number following the letter denotes
zone size in millimeter ND not done





33


Table I

Gram-Negative Flora of 101 Patients Hospitalized in Pediatric
Intensive Care Unit and Newborn Intensive Care Unit



PATIENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T Am K

IKiebsiella 7 R R R R R

2 Pseudomonas sp. 2-5 R R R R R
E. cloacae 2-5 R S S R S

3 E. Cloacae 3 5 5 5 S S
Pseudomonas sp. 3 R S R R R

4 E. coli 1-2 S S S S S
E. coli 2-3 5 R R R R
Proteus sp. 1-3 5 R R R R

5 E. coli 1-4 5 5 R S S
Kiebslella j-4 5 5 5 R S

6 Klebsiella 2 R R R R R

7 No growth

8 E. coli 1-4 5 5 R S S

9 E. cloacae 1-2 5 S S R S
Kebslella 2 R R R R R

10 E. coli 1-3 5 S S S S
E. coli 3 R 5 117 R S
E. cloacae 1-4 R R R R R
Klebsiella 2-4 R S I R S
Klebsiella 3 R S I R R
Klebsiella 4 5 5 I R S
Kiebsiella 4 R R R R R
Proteus sp. 4 R R R R R

11 Pseudomonas sp. R S R R R
E. coli 1 R S S R S
E. coli 2-4 5 5 S S S





34



Table I (continued)



PATIENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER' ORGANISMS ISOLATED STR C T Am K

12 Klebsiell]a I R S R R S
Klebsiella 2 S S 115 R S
E. coli 1-2 R S S S R
E. coli 2 R S R S S
E. col 2 5 S R S S
Proteus sp. 1-2 S S; R S S
Proteus sp. 2 5 s S R

13 Proteus sp. 2 R R R R R
E. coil 2-3 5 S; S S S;
E. coil 2-3 5 S R S S

14 No growth

15 E. coil 1-9 5 S S S S

16 Proteus sp. 1-2 R S R S S
E. coil 2-4 R S R S S

17 E. cloacae 1-5 S S S S S
E. cloacae 2-5 R R R R R
E. coil 2-5. 5 S S S S
Protus sp. 5-7 S R R R R

18. E. coil I S S; S S S
Proteus sp. I 5 Rt R. R R

19 Klebsiel la 4-5 R R R R R

20 Proteus sp. 1-2 5 S R S, S
Proteus sp. 2-4 5 R R R R
Klebsiell]a 2-3 5 S R S S
E. coil 2-3 5 S; R S S

21 No growth

22 No growth




35




Table I (continued)



PATIENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T An K

23 Proteus sp. 1-3 S S ft S S
E. coli 1 S 5 116 R S
E. coil 2-3 R S S R S

24 Kiebsiella 1-7 S S J15 R S
Klebsiella 9 R 5 It R S
Pseudamonas sp. 2-9 R R R R R
Proteus sp. 1-2 5 S Rt S S
Kiebsiella 9 R R Rt R R

25 Pseudomonas sp. 1-4 R S R R R
E. coil 3-4 5 S 115 R S
E. coli 4 5 5 S S S
Klebsiella 4 R R R R R

26 E. cloacae 4 R S S R S
Pseudamonas sp. 4 R R R R R

27- E. coli 1 5 S 116 R S

28 No growth

29 E. coli 1-4 5 5 R S S
E. cloacae 1-2 R S S S R

30 E. coli 1-2 5 5 S S S
E. coil 2 R 5 11.6 R S
E. cloacae 1-2 5 5 S R S
E. cloacae 1-2 R R R R R

31 Pseudomnonas sp. 1-2 R R- R R R
Kiebsiella 1-2 5 5 S R S
Klebsiella 2 .R R It R R

32 Pseudomonas sp. 1-7 R R R R R

33 E. coil 1-2 5 5 117 5. 5
E. cloacae 1-3 R R It R R





36


Table I continuede)



PATIENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T Ain K

34 Pseudomona aeruginosa 1-3 R S R R R
E. coli 1-2 S S S S S
E. coli 3 5 S R S S

35 Proteus sp. 1-7 5 S R S S
E. coli 1-7 5 S S R S
E. cloacae 1-7 ft S, S R S

36 Kebsiella 1-3 ft S S S S
Proteus 2-4 R R R R R
Kebsiella 4 R R R R R

37 E. cloacae 1-3 R S R ft ft
Proteus sp. 1-3 5 R R R R
E. coli 3 5 R S ft R

38 E. coli 1-4 R S I17 R S
Klebsiella 1-4 5 S, S ft S
Pseudanonas sp. 2-4 R R R R R

39 No growth

40 E. cloacae 1-2 R R R ft R
Pseudomnonas sp. 1-2 R Rt R ft R

41 E. coli 1-4 S S S. S S
E. coli 3-4 5 S S ft S

42 Pseudamonas sp. 1-7 R S ft ft R
Pseudcxnonas sp. 1-7 R R R ft R

43 Pseudomonas sp. 1-5 ND ND ND ND ND
Kiebsiel Ia 1-4 S S 116 R S
Klebsiella 4 5 S R R S
Kebsiella 5 R R ft ft ft

44 Klebsiella 1-4 S S S R S
E. coli 1 5 S ft S S
E. col 2 R Rt R R R
Pseudomonas sp. 2 Rt R Rt Rt R





37


Table 1 (continued)



PATIENT DAY (S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T Am K

E. coli 3 5 5 5 5 S
E. coli 4 5 5 R R R
Proteus sp. 4 R R R R R

45 E. cloacae I R S S R S
Pseudomnonas sp. 1-3 R S R R R
E. coli 2-3 S S R S R

46 E. coli I S S S S
E. cloacae I 5 5 5 R S

47 E. coli 1-2 5 5 R S S
E. coli 1-3 S S R R R
Proteus sp. 1-3 R R R R R

48 Pseudomonas sp. I S R R R R

49 E. cloacae 1-3 5 S S R S
Pseudanorias sp. 1-3 R R R R R

50 E. coli 1-4 5 5 S S S
Pseudomnonas sp. 1-4 R S R R R
Klebsiella 1-4 5 5 5 R S

51. E. coli I S S S S

52 Kebsiella 1-2 S S S R S
E. coli 1-3 5 s S R S
Pseudanonas sp. 2-3 R S R R R

53 Kiebsiella 1-4 5 S S R S

54 E. coli 1-7 5 5 5 R S

55 Pseudanonas sp. 1-2 R R R R R

56 E. coli 1-5 S S S S S

57 Pseudcnonas sp. I -R S R R R





38


TablIe 1 (continued)



PATIENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T Am K

58 E. cloacae 1-3 R S S R S
E. cloacae 4 R S S S R
Pseudonionas sp. 1j-4 R S R R R
Kiebsiella 1-2 5 s 116 R S
Klebsiel ]a 3 R 5 117 R S
Klebsiella 4 R S R R R

59 No growth

60 Proteus sp. 1-2 5 R R R R
Pseudomonas sp. 2 R R R R R

61 E. coil 1-7 5 5 5 5 S

62 Kiebsiella 1-3 5 5 5 R S
E. coli 1-2 5 S S S S

63 E. coil 1-9 S S S S S
Pseudanonas sp. 4-9 R R R R R

64 E. cloacae 2-4 5 5 S R S
E. coil 2-4. 5 5 5 S S

65 Pseudonionas sp. 1-4 R S R R R

66 E. coil I S S S. S S
E. coil 2 R S 116 S 5
E. coil 2 5 S R S S
E. coil 3 R 5 117 R S
E. coli 3 R S R R S
Kebsiella 1-2 5 S S R S
Pseudanonas sp. 1-3 R R. R R R

.67 E. coil 1-3 5 S S S S
Proteus sp. 1-3 5 S 5 R

68 No growth

69 E. coil j-4 R S R S R
E. coil 2-4 5 5 5 5 5
Pseudaxnonas sp. -2-4 R S S, R S





39,


Table I (continued)



PAT IENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T Am K

70 E. cloacae 1-3 R R R S R

71 Kiebsiella 1-7 S S I R S
E. coil 1-7 R S S R S

72 E. cloacae j-4 S, 5 5 R S

73 E. coil 1-3 R S R R S
Pseudomonas sp. 2-3 R R S R S

74 E. coli 1-5 S R R S R

75 E. coil 1-9 S S S R R
Pseudomonas sp. 5-9 R R R R R

76 E. coil 1-7 R S R S R
Klebsiella 1-4 5 5 S R
Kiebsiella 5-7 R R R R R
Pseudamonas sp. 3-7 R R R R R

77 No growth

78 E. cloacae 1-7 5 5 5 5 S

79 E. coil 1-3 R S R R S

80 E. coil 1-7 R S S S S

81 E. coil 1-4 5 5 5 R S
E. coil 1-4 R S R R S

82 E cloacae 1-9 s 5 R R S
E. coil 1-9 5 S S R S

83 Kiebslella 1-4 2 R R R R
E. coil 1-4 R R A2 R R

84 No growth

85 E. coil 1-7 5 S S S S
Kiebsiella 4-7 5 S, R- R S




40


Table 1 (continued)



PAT IENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T Am K


86 Proteus sp. 1-4 R R R R R
Pseudomonas sp. 2 R S R R R*

87 E. coli 1-3 S S S S S
E. coli 1-3 S S R S R

88 Pseudomonas sp. 1-5 R S R R R
Kiebsiella 1-5 S S S R S

89 Proteus sp. 1-4 R R R R R

90 Kiebsiel ]a 1-2 R S S R S
Klebsiel ]a 3-7 R 5 115 R R
E. coil 2-7 R S R S R
Kiebsiella 4-7 R S R R R

91 E.1cloacae 1-4+ R S S R S

92 Pseudoinonas sp. 1-9 R R R R R

93 Proteus sp. 1-5 5 5 R S S

94 E. coil 1-5 S S S R S
E. cloacae 1-5 R S S R S

95 E. coli 1-6 5 S S R S
Pseudomonas sp. 1-6 R S R R R

96 Klebsiella 1-5 S S 117 R S
Proteus sp. 3-5 5 S S S S

97 E. coli 1-4 5 S S R S
Kebsiel la 1-4 5 S, S R S

98 E. coil 1-9 5 5 R R S
Proteus sp. 1-9 5 S R S, S




*.Pseudomonas was possibly a contaminant




41


Table 1 (continued)



PATIENT DAY(S) ANTIBIOTIC RESISTANCES
NUMBER ORGANISMS ISOLATED STR C T An K


99 E. cloacae 1-3 R S R R R
Klebsiella 1-3 R R R R R
E. coli 1-3 S S S S S

100 E. coli 1-4 R S S R R
Proteus sp. 1-4 S S S S S

101 E. coli 1-3 R S S R S
Klebsiella 1-3 R S S R S
E. cloacae 1-3 R S S R S











Table 2

Distribution of Gram-Negative Flora Among 101 Patients
Included in Study of Invivo Transfer of R-Factors



PERCENT OF
GROUP, ACCORDING TO GRAM-NEGATIVE FLORA PATIENTS


Newborn, acquired no flora 10

Newborn, acquired one strain only 9*

I strain on admission, acquired no additional strains 18

I or more strains on admission, cultured 1 day only 7

2 or more different strains on admission with identical
antibiotic pattern, acquired no additional strains 5

2 or more different strains of varying antibiotic
patterns, no transfer of R-factors 39*

2 or more different strains of varying antibiotic
patterns, probable transfer of R-factors 8*

2 or more different strains of varying antibiotic
patterns, possible transfer, or segregation, of
R-f actors 5*




*34 of the 101 patients acquired at least I gram-negative organism during their hospital confinement. Nine were newborn, 13 comprised
the latter 2 groups, and the other 12 were included in the large
group of 39 patients.




*43






Table 3

Gramn-Negative Flora of Patients in Whom Transfer or Segregation of R-Factors Possibly Occurred



PAT IENT NUMBER OF DAYS RESISTANCE PATTERNS
NUMBER ORGANISMS ISOLATED TOGETHER STR C T Am K

2 Pseudcxnonas aeruginosa 3, R. R R R R
Enterobacter cloacae 1R S S R S
Enterobacter cloacae )R S S S R

10 Enterobacter cloacae + R R R R R
Escherichia coi (Indol )S S S S S
Kiebsiella 63 R R R R R
Klebsiella NT 1-4 R 5 118 R S
Kiebsiella, NT R 5 118 R R
Kiebsiella, NT S S 117 R S
Escherichia coli (Indol ) R S 117 R S

12 Kiebsiella 20 R S R R S
Escherichia coli R S S S R
Escherichia col 1-2 R S R S S
Kiebsiella 20 S S R R S
Escherichia coli S S R S S

23. Escher Ich ia coil iS 5 116 R S
Proteus sp. 2S S R S S
Escherichia coli R S S R S

49 Enterobacter cloacae 2 S S 5 R S
Pseudaxnonas aerug inosa '~~)R R R R R
Enterobacter cloacae )R. S S R S





44





could not be confirmed by serological procedures -- either because the s.trains were nontypable, or because there are no commercial serotyping procedures for these organisms. Each of these cases is discussed below.

I. (Patient # 2). The first-day cultures from this patient yielded

no growth. His cultures, from day 2, grew out a multiply-resistant Pseudomonas and an Enterobacter cloacae, resistant to

streptomycin and ampicillin. The flora remained unchanged until

day 5, when, in addition to these organisms, an Enterobacter

cloacae sensitive to ampicillin but resistant to streptomycin

and kanamycin, was isolated. Biochemically, the second Enterobacter was identical to the first, however, no serological procedures were available to establish identity of the two strains.

2. (Patient # 10). Initially, this patient's flora consisted of a

nonmotile Escherichia coli, and a multiply-resistant Enterobacter
R R
cloacae (SCTAmK)R. He acquired a Klebsiella (SAM)R on day 2;

on the third day a Klebsiella NT(SAmK)R was isolated, along with
R
a type 63 Klebsiella (SCTAmK) Again on day 4, 2 nontypable Klebsiellae were isolated, one of which was resistant only to ampicillin, and the other was both ampicillin and streptomycin

resistant. Whether this was a demonstration of the acquisition and segregation of an R-factor, or the result of superinfection

with several different serotypes of Klebsiellae, could not be




45





ascertained because of the nontypable nature of the Klebsiella

isolates.

3. (Patient # 12). On day 1, this patient's flora consisted of
R R
Klebsiella (STAn)R, Escherichia coli (SK)R, and 2 strains of
R
Proteus. On the second day, 3 Escherichia coli strains (ST) ,
R R .R
(SK) (T) were isolated together with a Klebsiella (TAm)

The 2 Klebsiella isolates were of the same serotype (type 20),

indicative of a possible loss of resistance by segregation of the

resistance determinant; however, each of the Escherichia coli

strains was rough, and therefore nontypable.

4. (Patient # 23). An Escherichia coli, sensitive to streptomycin,

was isolated from this patient on day 1. All of the other bacteria

of his flora were also sensitive to streptomycin. On the second

day, a streptomycin resistant Escherichia coli of the same biochemical and physiological characteristics was isolated from the

patient. There was no obvious donor of this resistance, and

attempts to serotype the Escherichia coli strains were not met

with success. This could either have been a superinfection of a different strain, or the acquisition of streptomycin resistance,

by the first isolate, from an unidentified source. Too, the

possibility exists that both the streptomycin sensitive and the streptomycin resistant strains were present on both day I and 2.





46





The inability to detect this possibility was a limitation which

could not be eliminated by design. Indeed, if 2 different strains

(serotypes) of the same organism, with identical resistance patterns (or with resistances identical for tetracycline and

kanamycin) were present, it would not have been observed unless these strains differed in their biochemical and/or physiological

characteristics, or if a chance isolation of the 2 strains occurred on successive days (ie., strain "A" is isolated on day 1,

and strain "8" on day 2).
R R
5. (Patient # 49). Enterobacter (Am) and Pseudomonas (SCTkiK) were

isolated from this patient's flora on day I and 2. On day 3, an

Enterobacter cloacae resistant to streptcnycin was isolated. The

biochemical and physiological characteristics were identical.

Invitro studies demonstrated the transferability of the streptomycin determinant, but not ampicillin. Exconjugants of the PseudoR
monas-Enterobacter (An) mating received all resistances. Identity

of the 2 Enterobacter strains, by sarological procedures, could

not be demonstrated, as previously explained. Cases of Probable Invivo Transfer of R-Factors

In 8 cases, strains of Escherichia coli or Klebsiellae of varying antibiotic sensitivities were isolated. In each case, the original isolate was sensitive to one or more antibiotics, to which it demonstrated marked re-





47





sistance on subsequent isolation. These cases are discussed below and shown in Table 4.

1. (Patient # 24). Strains of Klebsiella (A)n)R, Pseudomonas (SCTAmK)R,

and Proteus (T)R were isolated franom this patient through the seventh

day, with no apparent change in resistance patterns. But on day 9,

in addition to the Proteus and Pseudomonas, 2 Klebsiellae strains were isolated. Upon examination, their resistances were (STAm)R
R
and (SCTAmK)R. Serotypes of the 3 strains were all type 21.

2. (Patient # 31). On the first day, this patient's flora consisted
R
of a single Klebsiella strain (Am) Cultivation of the second

day's culture revealed 2 Klebslellae strains (SCTAmK)R, (Am)R
R
and Pseudomonas (SCTAmK)R. It is improbable that the multiplyresistant Klebsiella was present on the first day; had it been

present, it would have been noted on the MacConkey agar plates which contained either tetracycline or kanamycin. Serotyping confirmed all of the Klebsiellae strains to be of the capsular

type 63.

3. (Patient # 36). The initial isolates from this patient were R R
Klebsiella (SAm) and Proteus (SCTAmK)R. On day 2, an Escherichia
R
coll (TAm) was isolated, in addition to the original isolates.

The flora remained unchanged until day 4, when Klebsiella (SCTAnK)R

was isolated. All Klebsiella strains were shown to belong to




48


Table 4

Gram-Negative Flora of Patients in Whomi Probable Invivo Transfer of R-Factors Was Observed



PAT IENT DAY(S) RESISTANCE PATTERNS
NUMBER ORGAN ISMS-SEROTYPES ISOLATED STR C T Ain K


24 Kiebsielia 21 1 S, S 116 R S
Pseudanonas sp. 3 R. R R R R
Proteus sp. 1 S S R S S
Klebsiella 21 9 R S R R S
Klebsiella 9 R R R R R

31 Klebsiella 63 1 5 5 S R S
Klebsiella 63 2 R R R R R
Pseudoionas sp. 2 R R R R R

36 Klebsiella 63 1 R S S R S
Proteus sp. I R R R R R
Escherichia coli 2 5 5 116 R S
K1.bsiella 63 4 R R R R R
Proteus sp. 4 R R R R R

43 Kiebsiella 63 1 S S I17 R S
Pseudomonas sp. 1 ND ND ND ND ND
Klebsiella 63 -4 S S R R S
Klebsiella 63 5 R R R R R
Pseudomonas sp. 5 ND ND ND ND ND

58 Klebsiella 39 1 S S I R S
Enterobacter cloacae 1 R S S R S
Klebstella 39 3 R S, I R S
Enterobacter cloacae 3 R S S R S
Pseudcmnonas sp. 3 R S R R R
Klebsiella 39 4 R S R R R
Pseudamonas sp. 4 R S, R R R
Enterobacter cloacae 4 R S S S R

66 Escherichia coi NT 1 S S S S S
Kiebsiella I S S S, R S
Pseudomonas, sp. 1 R R R R R
Escherichia coli NT 2 R S; I S S
Escherichia coli NT 2 S S R S S




49


Table continued)



PATIENT DAY(S) RESISTANCE PATTERNS
NUMBER ORGANISMS-SEROTYPES I ISOLATED STR C T Am K


Klebsiella 2 S S S R S
Pseudomonas sp. 2 R R R R R
Escherichia coli NT 3-4 R S R R S
Pseudomonas sp. 3-4 R R R R R

76 Klebsiella 39 1 S S S R S
Escherichia coli I R S R S R
Klebsiella 39 3 S S S R S
Escherichla coli 3 R S R S R
Pseudomonas sp. 3 R R R R R
Klebsiella 39 5-7 R R R R R
Escherichia coli 5-7 R S R S R
Pseudornonas sp. 5-7 R R R R R

90 Klebsiella 9 1 S S S R S
Klebsiella 9 1 R S S R S
Escherichia coli 2 R S R S R
Klebsiella 9 2 S S S R S
Escherichia coli 3-7 R S R S R
Klebsiella 9 3-7 R S 115 R R




50






serotype 63.

4. (Patient # 43). From this patient, on day 1, a Kiebsiella (An)R

and a Pseudomonas were isolated. (The resistance pattern of the

Pseudomonas was not recorded; subsequent attempts to revive the organisms from stock culture have failed.) On the fifth day of

culturing, multiply-resistant Psuedomonas and Klebsiella (SCTAmK)R

were isolated. The serotype of the Klebsiella isolate and its

4 predecessors was type 63.

5. (Patient # 58). This patient's flora consisted of Enterobacter

cloacae (SAm)R and Klebsiella (Am)R when first cultured. The resistance patterns remained unchanged through the second day.

On day 3, a streptomycin-resistant Klebsiella was isolated along with Enterobacter cloacae (SK)R and Pseudomonas (STAmK)R. On the

fourth day, a multiply-resistant Klebsiella was isolated, which

had the same antibiotic resistance pattern as the Pseudomonas

strain (STAmK)R, along with the Pseudomonas and an Enterobacter
R
cloacae (SK)R. It is possible that the patient's flora contained ampicillin-resistant, kanamycin-sensitive Enterobacter cloacae on the third and fourth day, and that they were not observed due to the selective media employed. Biochemically and physiologically, the Enterobacter strains of days 1 to 4 are identical. From the

serological data (all Klebsiellae strains were type 39) and the





51





antibiotic resistance patterns (Pseudomonas = Klebsiella), an apparent transfer of resistance had occurred from the Pseudomonas to the Kilebsiella.

6. (Patient # 66). A fully sensitive Escherichia coli, an ampicillinresistant Klebsiella and a Pseudomonas (SCTAmK)R were cultured on the first day, and on day 2, Escherichia coli of 2 different
R R
antibiotic patterns were isolated (ST) and (T)R. Escherichia

coli, cultures from the third day were resistant to streptomycin,

tetracycline and ampicillin. The Klebsiella were not isolated

past day 2, and the patterns of resistance of the Escherichia coli and Pseudomonas strains remained stable through day 4. Biochemical and physiological results indicated identity among the Escherichia strains. Equivocal results were obtained from serological experiments and the strains were forwarded to the Enteric Bacteriology Unit of the Communicable Disease Center, Atlanta, for serologic identification. The strains were determined to be nontypable by

this Unit.

7. (Patient # 76). Escherichia coli (STK) and Klebsiella (An)R were

cultivated from this patient's flora for 4 consecutive days, without change. On the fourth day, a multiply-resistant Pseudomonas (SCTAmK)R was isolated. Cultures on the fifth and seventh days

revealed Escherichia coli (STK)R, and multiply resistant (SCTAmK)R




52






Pseudomonas and Klebsiella. The serotypes of all strains of

Klebsiella were type 39.

8. (Patient # 90). This patient acquired an Escherichia coi (STK)R
R
on the second day, after having only a Klebsiella (Am) on day 1.

The Kiebsiella isolated on the third day was resistant to streptomycin and kanamycin and demonstrated a borderline resistance to

tetracycline. The Klebsiella was isolated through day 7, and

all isolates were of the capsular type 9.


Cases of Invivo Transfer, Suspected on the Basis of Retrospective Analyses

The possibility existed that a transfer event had occurred, within a given patient's intestinal tract, prior to the advent of his inclusion in these studies. Among individuals who have acquired R-factor-containing organisms, it has been observed that the entire gut flora has acquired the resistance pattern of the original infecting strains (Akiba et al., 1961; Lebek, 1967; Mitsuhashi et al., 1961). Consistent with this, in 5 of the patients included in this study, the flora consisted of 2 or more different strains, and in each case, the antibiotic resistance patterns of the different strains were identical. These strains and their resistances are shown in-Table 5.

If the antibiotic patterns, observed in these 5 cases, were the result of the invivo transfer of R-factors, then the antibiotic resistances should be transferable, and the R-factors should be of the same fi type. According-





53






Table 5

Gram-Negative Flora From 5 Patients, Each in Whom the Antibiotic Sensitivity Patterns of the Different Strains Were Ident i cal


PATIENT NUMBER OF DAYS RESISTANCE PATTERNS
NUMBER ORGANISMS ISOLATED TOGETHER STR C T Am K


16 Escherichia coli 4 R S R S S
Proteus sp.


40 S. marcescens 2 R R
Pseudomonas aerugi nosa


83 Klebsiella 4 R R R R R
Escherichia coli


97 Escherichia coli 4 R S S R R
4 R S S R R
Kiebsiel la


101 Escherichia coli
Klebsiella 3 R S S R S
Enterobacter cloacae





54





ly, invitro mating experiments and bacteriophage propagation studies, as well as curing techniques, were performed on all strains isolated from the patients shown in Table 5 (Table 6).

In cases # 16, 82, 97 and 101, these results demonstrated that the antibiotic resistances were R-factor-mediated, and that the fi types of the R-factors were the same.

In patient # 40, no transfer of resistances was achieved. However, both of the patient's strains were readily cured of their resistances, and while the Pseudomonas strain propagated the male-specific phage R17, neither R17 nor Iff was propagated by the Serratia.

The results further indicated that, in each case, the R-factors carried by the different strains were possibly identical. Simultaneous Occurrences of Sensitive and Resistant Organisms. Without Transfer of R-Factor

In 39 cases, the flora consisted of 2 or more different strains, of differing antibiotic sensitivities, for periods up to 9 days, without apparent transfer of resistances (Table 7). Twelve of these patients were included in the group of 34 patients who, upon admittance, possessed a gram-negative intestinal flora, and in addition, acquired at least 1 gram-negative organism during their hospital tenure.

Invitro Analyses of Bacterial Strains Involved in R-Factor Transfer

The suspected recipient and donor strains, from the 8 cases in which






Table 6

Invitro Analyses of Strains, From 5 Patients, Who Exhibited Identical Antibiotic Sensitivity Patterns: Matings, R-Factor-Curing, and Male-Specific Bacteriophage Propagation Experiments


PATIENT RESISTANCES CURING EXPERIMENTS: BACTERIOPHAGE PROPAGATION
NUMBER ORGANISMS TRANSFERRED DETERMINANTS LOST R17 If


16 Escherlchia coil S, ST S, T, ST +
Proteus sp. S, ST T, ST +


40 S. marcescens none SK, STCAmK
Pseudomonas aeruginosa none T, ST, STCAnK + +


82 Klebslella ST, T, CKST CKST, SK +
Escherichia coil ST, AmCT, CKSTAm STK, Am, CKST +


97 Escherichia coli SK, SKAm, Am S, SK, Am +
Klebslella S, SKAm SK +


101 Escherichia coli SAm SAm +
Klebsiella S, Am S +
Enterobacter cloacae SAm SAm +
/~





56


Table 7

Resistant and Sensitive Gram-Negative Flora of. 39 Patients.
No Apparent Transfer of R-Factor-Mediated Resistance After Continuous Invivo Co-Incubation



PATI ENT NUMBER OF DAYS RESISTANCE PATTERNS
NUMBER ORGANISMS ISOLATED TOGETHER STR C T An K


5 Escherichia coli 4S S R S S
Kiebsiella S S S R S

ii Pseudomonas sp. 2 R S R R R
Escherichia coli R S S R S

13 Proteus sp. IR R R R R
Escherichia coli NM 2 S S S S S
Escherichia coli S S R S S

17 Enterobacter cloacae IS S S S S
Escherichia coli 4 S 5 5 5 5
Enterobacter cloacae 2R R R R R
Proteus sp. S R R R Ri

20 Kiebsiella 2 5 5 R S S
Escherichia coli S S R S S
Proteus S R R R R

25 Pseudomonas sp.2 R S R R R
Escherichia coi S S 1 16 R S
Kiebsiella JR R R R R

29 Escherichia coli 2 5 S R S S
Enterobacter cloacae S S S S R

30 Enterobacter cloacae S S S R
Escherichia coil (Indol ))S S S S S
Enterobacter cloacae + I R R R R R
Escherichia coil (Indol )R 5 116 R S

33 Escberichia coli1 S S 116 S S
Enterobacter cloacae R R R R R





57


Tab I e 7 (cont i nued)



PATIENT NUMBER OF DAYS RESISTANCE PATTERNS
NUMBER ORGANISMS ISOLATED TOGETHER STR C T Am K

34 Escherichia coli NM S S S S S
Pseudomonas aeruginosa 3 R R R R R
Escherichia coli S S R S s

35 Proteus sp. s S R S s
Escherichia coli 7 S S s R S
Enterobacter cloacae R S s R S

37 Enterobacter cloacae 3 R S R R R
Proteus sp. S R R R R

38 Escherichia coli R S 117 R S
Klebsiella 4 s S S R S
Pseudomonas sp. R R R R S

41 Escherichia coli NM 2 s S S s S
Escherichia coli s S s R S

44 Escherichia coli 06 2 S s S s S
Klebsiella S S S S S
Escherichia coli 04 1 S S R R R
Proteus sp. R R R R R
Escherichia coli NT s S R s s
Escherichia coli 075:motile I S s R s S
Klebsiella s s S R S
Escherichia coli 075:NM R R R R R
Pseudomonas R R R R R

45 Enterobacter aerogenes R S S R S
Pseudomonas aeruginosa R s R R R
Escherichia coli 2 s S R s R

47 Escherichia coli Lac- S S R S S
Proteus 3 R R R R R
Escherichia coli Lac s s R R R

50 Escherichia coli s s s S s
Pseudomonas aeruginosa 3 1 R S R R R
Klebsiel I a S S S R S
Proteus sp. R R R R R




58


Table 7 (continued)



PATIENT NUMBER OF DAYS RESISTANCE PATTERNS
NUMBER ORGANISMS ISOLATED TOGETHER STR C T Am K

52 Klebsiella 3 J. S S S R S
Escherichia coli 2 S S S R S
Pseudanonas sp. R S R R R
Escherichia coli NM R R R R S

62 Klebsiella 3 S S S R S
Escherichia coli I S S S S S
Pseudomonas sp. R S R R R

63 Enterobacter cloacae 4 S S S S S
Escherichia coli S S S R S

64 Escherichia coli 4 S S S S S
Pseudomonas sp. R R R R R

67 Escherichia coli 3 S S S S S
Proteus sp. S S S R S

69 Escherichia coli R S R S R
Escherichia coli NM 3 2 S S S S S
Pseudomonas sp. R S S R s

71 Klebsiella 6 S S 117 R S
Escherichia coli R S S R S

73 Escherichia coli 2 R S R R S
Pseudomonas sp. R R S R S

75 Escherichia coli 4 S S S R R
Pseudornonas R R R R R

81 Escherichia coli NM 4 s S S R S
Escherichia coli R S R R S

82 Enterobacter aerogenes 7 S S R R S
Escher I ch I a col I S S S R S

85 Escherichia col i 4 s S s S S
Klebsiella S S S R S





59


Table 7 (continued)



PATIENT NUMBER OF DAYS RESISTANCE PATTERNS
NUMBER ORGANISMS ISOLATED TOGETHER STR C T Am K


87 Escherichia coli NM Lac 3 s S S S S
Escherichia coli M Lac+ S S R S S

88 Pseudomonas sp. 5 R S R R R
Klebsiella s S S R S

94 Escherichia coli 5 S S S R S
Enterobacter cloacae R S S R S

95 Escherichia coli 6 S S S R S
Pseudanonas aeruginosa R S R R R

96 Klebsiella 3 S S 117 R S
Proteus sp. s S S S s

98 Escherichia coli 7 s S R R s
Proteus sp. S S R S S

99 Enterobacter aerogenes R S R R R
Klebslella 3 R R R R R
Escher I ch I a col i S S S R S

100, Escherichia coli 4 R S S R R
Proteus sp. S s S S S

42 Ps. aeruginosa 7 R S R R R
PS. aeruginosa (non-pigmented) R R R R R




60





invivo transfer of R-factors was thought to have probably occurred, were analyzed, invitro, to determine their ability (1) to transfer R-factors,

(2) be cured of their antibiotic resistance, and (3) to adsorb and propagate the male-specific bacteriophage R17 and Ifl. Invitro Transfer of Resistance

If, as suspected, an invivo transfer of resistances had occurred in each of these cases, then these events should have been reproducible invitro. Therefore, both the original sensitive isolates and the presumed

R
exconjugants (e.g. in Table 4, patient # 24: Klebsiella (Am) isolated
R
on day I = sensitive isolate; Klebsiella (SCTAmK) =.presumed exconjugant) from those cases in which transfer was suspected, were used as recipients and donors, respectively (Table 8). First, the presumed exconjugant was mated with one of the standard laboratory recipients. The progeny of this cross was then back-crossed with the original sensitive isolate (see Figure ).

When performing the latter experiment, some difficulty was encountered. It is apparent that this is due to the nature of the experimental design. Invitro transfer of R-factor-mediated resistance via conjugation is of low yield. The maximum frequency of such transfer, under ideal conditions is
-2
-i0 2 (Watanabe, 1969). One reason for such a low rate of transfer is because, in a given wild-type R+ culture, most cells contain a repressor, hence, I in 100 cells possesses a specific pilus at a given time (Meynell and Datta, 1965; Datta, Lawn, and Meynell, 1966). This is contrasted to




61

Table 8

Invitro Transfer of R-Factors. I. Matings of Donors (Who Were Suspected of Having Acquired Their Resistances Invivo) with Standard Laboratory Recipients. II. Using the Exconjugants Obtained From (1) as Donors, the Original Sensitive Strains (From Each Patient in Whom Probable Transfer of R-Factors Occurred)
Were Tested to Determine Their Suitability as Recipients

RESISTANCE PATTERNS
DONOR RECIPIENT
OF EXCONJUGANTS

24(SCTAmK)R 29(AmNa)R T, ST, CKST
29(CKST)R NaR 24(Am)R CT, KST

31(SCTAmK)R 29(AmNa)R ST, CT, KTS
29(KTS)R 31(Am)R KTS

36(SCTAmK)R 29(AnNa)R CKST
29(CKST)R 43(Am)R none

43(SCTAmK)R 29(AmNa)R T, KS, CKST, CK
29(CKST)R 43(Amn)R none

58(STAmK)R 29(AmnNa)R ST, K, KST
29(KST)R 58(Am)R none

76(SCTAmK)R 29(AmnNa)R STK, T, CT
29(CT)R 76 (Am) R none
29(SKT)R 76(Am)R STK

90(STAK)R 29(AmNa R K
29(K)R 90(SAmd none
29(K)R 90(Am) K

66(STAm)R LW(Na)R A, AS, AT, AST
LW(AST)R 66(Sens) none


Code of strains = first number is the patient's number; the letters in
parentheses = antibiotic resistances.
Strain # 29(AmNa)R is a multistep nalidixic acid resistant Kiebsiella
prepared in our laboratory.
Strain LW(Na)R is a multistep nalidixic acid resistant Escherichia coli
prepared in our laboratory.
Strain 90(Am)R derived from strain 90(STAK)R in curing experiment
(Table 9).




62





invitro transfer of the sex factor F, where the rate of transfer approaches 100 percent. In these experiments, in which the donor was resistant to the selective agent nalidixic acid, it was necessary to not include nalidixic acid in the medium on which the mating mixture was plated. Since the frequency of transfer of antibiotic resistances may have been as low as 106 (Watanabe, 1969), it was necessary to inoculate at least 40 plates for each dilution of the mating mixture and to allow the mating to proceed for 16 hours. Even with these modifications, it was not possible to detect exconjugants of a mating frequency of less than 10-3. However, invitro transfer was observed among 4 of the 10 mating pairs.

When the exconjugant 29(K)R was used as the donor and 90(SAn)R and

90(Am)R were the recipients, the kanamycin resistance was transferred only to the latter. Suspecting that this may have been due to the presence of
+ R
a fi factor mediating streptomycin resistance in 90(Sn) this strain was infected with the male-specific bacteriophage R17, and tested for propagation of the phage. Strain 90(SAm)R did propagate the phage, and treatment of the strain with ethidium bromide resulted in the loss of streptomycin resistance. From these results, it was concluded that the streptomycin resistance in 90(SAm)R was carried on a fi+ factor. Invitro Transfer of Resistances from Suspected Donor Strains

The suspected donor strain, from each case under consideration, was

tested for its ability to transfer its resistance by conjugation (Table 9).




Table 9

invitro Transfer of Antibiotic Resistances From Strains, Which Were Suspected of
Having Donated Their. Resistances Invivo, to Standard Laboratory Recipients


DONOR EXCONJUGANTS
PATIENT NUMBER RECIPIENTREITNEPTRS
RESISTANCE PATTERNREISTNEPTRS


R R
Pseudomonas 2'4(SCTAmK) 29(AmNa) C CI(S, STCK



Proteus 36(SCTAmK)R 29(AnNa) R STCK, TC


R R

Pseudonionas 66(SCTAmK) RLW(Na) RTC, CKS, STkiK Pseudomonas 76(SCATITK) R 29 (Ama) R STK, K, STCK


Pseudomonas 76(SCTAmK) RE. coil (STK) Rnone Escherichia coil 90(STK) R 29(AmNa)R ST K




64





If the suspected donor could not transfer its resistances invitro, then the possibility of invivo transfer was most unlikely. In each case, the recipient was a multi-step nalidixic acid resistant strain of that species which was the presumed recipient invivo. Since the Pseudomonas strain from case # 43 was not available, no determination could be made. In those experiments, in which Pseudomonas was the donor, transfer of resistances was effected with little or no difficulty. Transfer with Proteus was of a much lower frequency, 3x1O o The results of these experiments showed that the resistances of the suspected donors were extrachromosomal and transferable.

It is supposed that the failure to transfer any resistances from the Pseudomonas strain, from patient # 76 to the resistant Escherichia coli strain, obtained from the same patient, was due to superinfection immunity. Therefore, the Escherichia strain was incubated with phage R17. The phage titre of the supernatant medium, after incubation, was approximately a hundredfold greater than the titre of the inoculum. R-Factor-Curi ng Experiments

If the antibiotic resistances (carried by the strains isolated in the above 8 cases) arose from an invivo transfer event, then these resistances should be amenable to the curing action of ethidium bromide or acridine orange, or a combination of UV light and ethidium bromide or acridine orange. The results of these experiments, shown in Table 10,




Table 10

Results of R-Factor-Curing Experiments of 9 Strains Which Were Suspected of Having Acquired Their Resistance by Invivo Conjugation


PATIENT ORIGINAL PROGENY
NUMBER STRAIN-SEROTYPE CURING AGENT RESISTANCE PATTERN RESISTANCE PATTERN

24 Klebslella 21 ethidium bromide SCTAmK Am, SCAmK, CTA

31 Klebsiella 63 ethidium bromide SCTAmK SCAmK, SCAm, CTAm, Am

36 Klebsiella 63 ethidium bromide SCTAmK SCAmK, Am, (SCTAmK)S

43 Klebsiella 63 ethidium bromide SCTAmK Am

58 Klebslella 39 ethidium bromide SCTAmK Am, CTAm

66 Escherichia coli NT UV and AO STAm Am,(SCTAmK)S

76 Klebsiella 39 ethidium bromide SCTAmK CTAm, SCAmK, SKAm

90 Klebsiella ethidium bromide ST*AcnK Am



Tetracycline resistance zone size = 15 mm; after treatment with curing agent, zone was greater than
21.mm.


/




66




were definitive for all strains except # 76. Even though a loss of all of the resistant determinants was not detected in any one of the progeny clones of # 76, the spectrum of resistances lost by the population of cells for that strain indicated that all resistances were extrachrcmosomal.

Earlier experiments had shown that ethidium bromide (490J gm/ml)

gave better results with Klebsiella, but was ineffective with Escherichia coil, even after pretreatment with UV light. Curing of Escherichia coil R-factors was accomplished by a combination of ultraviolet irradiation followed by acridine orange treatment. Phage Adsorption and Propagation

R17. The presumed recipient, from each case, was tested for its ability to adsorb and propagate the male-specific bacteriophage R17 (Table ii). Positive scores are assigned those organisms which demonstrated approximately a hundredfold increase and decrease in the propagation and adsorption experiments, respectively. As shown in the table, these experiments were performed on the strains both before and after treatment with the curing agents, in an effort to correlate the loss of resistances with the concomitant loss of bacteriophage adsorption and propagation properties. The Klebsiella strains, from patients # 58 and # 76, were not of the fi+ type, as evidenced by their inability to adsorb and propagate the phage.




Table 11

Adsorption and Propagation of Bacteriophage R17 by Strains Suspected of Having Acquired Their Resistance by Invivo Transfer of R-Factors



BACTERIOPHAGE TITRE
PATIENT ORGANISMS-SEROTYPES PROPAGATION ADSORPTION
NUMBER BEFORE CURING AFTER CURING BEFORE CURING AFTER CURING


24 Klebsiella 21 1.38xl07 8.3x10 4.2xl02 9.1xlO4

31 Klebsiella 63 2.27x107 8.1xl04 1.3xlO0 8.6x1o4

36 Klebsiella 63 2.23x107 8.0xlO4 2.1xlO3 8.4xi04
ON
43 Klebsiella 63 2.5x107 7.9x104 1.76xi03 7.7x104

58 Klebsiella 39 7.7xl0 8.0xlO4 8.1xlO4 8.3x104

66 Escherichia coil NT 3.6x107 7.8x104 1.7xlO3 8.2x104 *

76 Kiebsiella 39 9.3x104 9.0xl04 7.6x10 7.7x104

90 Klebsiella 9 4.2x107 8.7x104 5.0xlO3 8.4x104
negative Klebsiella FR 7.9xi04 ND I.ixlO5 ND
control
positive Escherichia coli K38F R 1.96xi0o ND 1.4xlO0 ND
control

/
*
A loss of susceptibility to the bacteriophage R17 was concomitant with the loss of tetracycline resistance. However, those strains which did not lose tetracycline resistance during the curing procedures (see Tablelo) remained susceptible to the phage.
**Not done
Not done




68





IfI. Each of the presumed recipients was tested for their

ability to propagate the bacteriophage Ifl (Table 12). The Klebsiella strains from patients # 58 and # 76, which did not propagate R17, did propagate If2. Additionally, the Klebsiella strain from patient # 24 propagated the phage.

Invitro Mating to Detect Derepressed Mutants

Since derepressed mutants of R-factor-containing mutants have been Isolated from human feces (Lee and Linton, 1969), one explanation for the invivo transfer of R-factors which probably occurred in these 8 cases, is that the R-factors of the donors were derepressed thus increasing the chances of an invivo conjugation event. Therefore, mating experiments wore performed with the suspected donors of each case (with the presumed exconjugant in case # 43) and the standard laboratory recipients to determine the frequency of transfer of R-factors. From the results, shown in Table 13, it was determined that none of the suspected donors carried derepressed R-factors. Evaluation of Antibiotic Usage

An evaluation of the hospital records of those 8 patients, in whom the probable invivo transfer of R-factors had occurred, was undertaken to determine the extent of antibiotic therapy they had received during their hospital tenure. Similarly, the records of the 39 patients, from




69




TablIe 12

Propagation of Bacteriophage Ifi by Strains Suspected of Having
Acquired R-Factors Via Invivo Conjugation



PATI ENT*
NUMBER ORGANISMS-SEROTYPES BACTERIOPHAGE TITRE


24 Klebsiella 21 1 .3xl0 8

31 Klebsiella 63 5. OxI o6

36 Klebsiella 63 2.0O 06

43 Kiebsiella 63 3. 5x1O

58 Klebsiel 'a 39 2. 7x1 o8

66 Escherichia coli NT 2.3X105

76 Kiebsiella 39 2.1 xl 08

90 Kiebsiella 9 1 .4x105

positive Escherichia ""'Ii 1-tanJ32xO9
control- K2sriJ5

cngtol Kiebsiella FR 8.0x105




Propagation mixture contained 10/lbacteria, an .xO6/ml phage
particles. Incubation was for 16 hours at 370 C.




Table 13

Determination of the Frequency of Transfer of R-Factors by 8 Bacterial Strains Suspected of Harboring Derepressed R-Factors


DONOR NUMBER OF FREQUENCY
PATIENT NUMBER COLONIES RESISTANCES OF
RESISTANCE PATTERN RECIPIENT TESTED TRANSFERRED TRANSFER

Escherichia coil K12J53(ST) LW(Na) 123 ST 2.3x10-1

Pseudomonas 24(SCTAmK)R 29(AmNa) R

Pseudomonas 31(SCTAmK)R 29(AmNa)R 237 None e10- 2

Proteus 36(SCTAmK) R 29(AmNa) R 183 None <10-2

Pseudomonas 58(STAK)R 29(AmNa) R 262 None <10-2

Pseudomonas 66(SCTAmK)R LW(Na)R 411 None <10-2

Pseudomonas 76(SCAmTK)R 29(AmNa)R 361 None <10-2

Escherichia coli 90(STK)R 29(AmNa) 335 None <10-2

Klebsiella 43(SCTAmK)R 29(AmNa)R 453 None <10-2



Since the Pseudomonas, which was the suspected donor in case 43, was lost, the presumed recipient
of the R-factor was used.




71







whomi sensitive and resistant organisms were simultaneously recovered, without apparant R-factor transfer, were evaluated.

Five of the 8 patients (71 percent) received antibiotic therapy,

whereas 22 of the 39 patients (56 percent) in the latter group received drugs.













DISCUSSION



Well documented observations of interbacterial transfer of R-factors invivo under natural conditions are scarce. Watanabe (1963) attributed this to the adverse conditions for bacterial conjugation (pH, anaerobiasis) that exist in the human intestinal tract. For the most part, experimental infections with R-factor-containing organisms, and the subsequent observation of invivo R-factor transfer, have been performed with gnotobiotic animals (Jones and Curtiss, 1970); newborn animals remiss of a normal bacterial flora (Walton, 1966); or in cases where antibiotics were first administered (Smith, 1969).

On the basis of a prospective study in which invivo transfer of Rfactors was not observed, Gardner and Smith (1969) concluded that this phenomenon was not of significance; rather, the dissemination of R-factors appeared to be due to the colonization of patients by R-factor-containing bacteria. The persistence of these organisms was due, according to these investigators, to the selective forces exerted by antibiotic usage. Eickhoff (1970) concurred with this view in his investigation, although, in 4 of his cases, the evidence for invivo transfer of R-factors among his Klebsiella strains seemed compelling.

The conditions necessary for the demonstration of invivo R-factor





72




73





transfer -- antibiotic resistant and sensitive gram-negative bacteria simultaneously colonizing the gut -- were present in 57 of the 101 patients included in this study (Table 1). In 8 of those 57 patients (14 percent), invivo transfer of R-factors probably occurred. Several factors, presented here, argue for the invivo transfer of antibiotic resistance to initially sensitive strains, rather than the superinfection by antibiotic resistant hospital strains, or the selection of preexisting resistant strains through the course of antibiotic therapy.

I. The serotypes of the nosocomial strains of Kiebsiella and

Escherichia coli isolated from patients confined to the

units included in this study are Klebsiella type 63, and Escherichla coli types 01, 04, 06, and 075. The hospital

strain of Klebsiella carries the resistances for streptomycin, chloramphenicol, tetracycline, ampicillin, and kanamycin, and

during the course of this study, no antibiotic-sensitive

hospital strains of type 63 Klebsiella were isolated. Strains

of Klebsiella type 63 were involved in 3 of the 8 cases with probable Invivo transfer (Table 4). In all 3 patients, serotype 63 was present in admission culture. In 1 case, this was

.followed by the recovery of a tetracycline-resistant strain

on day 4, and a completely resistant strain on the fifth day.

Since only multiply-resistant strains of this type have been





74





observed as hospital flora, these results are not likely accounted for by superinfection and subsequent selection.

Moreover, these 3 patients were not clustered within the

sequence of patients included in this study. The patients

were spread over a 4-week period; 2 were confined to the

NICU, the third to the PICU. Thus, if the type 63 Klebsiella

isolated from these 3 patients is assumed to have been of

nosocomial origin, then most of the other patients cultured

during this period should also have acquired this strain.

Antibiotic-sensitive Klebsiella strains and Klebsiella with

partial resistance patterns were isolated in 31 patients.

The serological typing of all these isolates yielded no

additional type 63.

Since Klebsiella type 9 has not been observed in the

hospital environment, and types 21 and 39 are not frequently observed, superinfection seems to be an unlikely explanation

In the cases involving these strains.

2. Several antibiotic resistance patterns were noted in the same

strain Isolated on several occasions from the same patient (cases # 24, 43, and 58). The superinfection of a patient

by a strain with identical serotype, but different antibiotic

resistance, must be an unlikely event.





75





3. If the patient harbored an antibiotic resistant strain of

the same serotype as the antibiotic sensitive dominant strain, this resistant strain would have been observed during the initial culturing procedures. The experimental design included planting the rectal swab onto tetracycline- and kanamycin-containing plates. Since greater than 99 percent of the R-factormediated resistance encountered in this study included either

I or both of these resistances, a low titre-resistant strain

would have been detected and identified.

Farrar et al. (1972) noted the influence of antibiotics (oral kanamycin) on invivo R-factor transfer, in their report of a case of invivo interbacterial transfer of kanamycin resistance. In our cases reported here, the administration of antibiotics did not appear to favor, or have an adverse effect on, invivo R-factor transfer. The incidence of antibiotic usage among the 8 cases of probable invivo transfer (Table 4) was 71 percent (5 of 8). In 39 cases, where both sensitive and resistant organisms were simultaneously isolated, without apparent transfer, the incidence of antibiotic administration was 56 percent (22 of 39).

Among the 39 cases, where the conditions for invivo transfer existed (coexistence of a resistant and a sensitive strain), but where no actual transfer was observed, the following potential recipient strains were found: Klebsiella, 12 (31 percent); Escherichia coli, 24 (61 percent).




76






Pseudomonas, Proteus, and Enterobacter cloacae were each found there. Of the 46 resistant strains, acting as potential donors, 19 (41 percent) were Pseudomonas, 6 (13 percent) Klebsiella, 9 (20 percent) Enterobacter cloacae 8 (17 percent) Escherichia coli, and 4 (9 percent) Proteus. Thus, Kiebsiella acted significantly more often as a recipient and Pseudomonas as a donor in the cases of probable invivo transfer than would be expected from their frequency of isolation (P < 0.01, and 0.02 > P >0.01, respectively).

This suggests that some factors may exist which favor the invivo transfer of R-factors between Pseudomonas and Klebsiella strains. One such factor, the existence of derepressed R-factor mutants among the Pseudomonas strains, was tested by invitro mating experiments (Lee and Linton, 1969). The frequency of transfer of R-factors by those strains was less than 10-2, indicating that derepression was not the cause. Invitro mating experiments of the recipient Klebsiella strains with other R-factor-containing gram-negative bacteria (Table 8) did not reveal any extraordinary characteristics of these strains as recipients. It may be that there exists an affinity between Pseudomonas and Klebsiella strains, which neither strain expresses for other gram-negative bacteria.

Although these results indicate a rate of invivo transfer of

14 percent among those patients who were initially colonized with a




77





sensitive strain and subsequently infected with resistant bacteria, it is quite possible that the actual frequency of transfer was higher. We were unable to assess transfer in any strains other than Klebsiella and Escherichia coli. In 2 cases involving Pseudomonas aeruginosa and Enterobacter cloacae (Table 3, # 2 and # 49), invivo transfer may have occurred, but could not be confirmed due to lack of typing procedures. Transfer may also have taken place in 2 other cases where untypable strains of Escherichia coli and Klebsiella were involved (Table 3, # 10 and # 12).

After oral administration of antibiotics, patients who had initially excreted sensitive bacteria were noted to excrete bacteria of the same serotype, with resistances identical to the resistance pattern of other R-factor-containing bacteria colonizing their intestinal tract (Akiba, 1959; Lebek, 1963). Similarly, the presence of 2 or more bacterial strains within a single patient, with the same R-factor, seems indicative of the possible invivo transfer of R-factors. Five patients were admitted to this study with 2 or more different strains, all possessing the same resistance patterns (Table 5). Mating experiments, R-factor elimination and bacteriophage propagation procedures confirmed that the R-factors, possessed by the different strains, in each patient, were identical.

Summarily, contrary to the conclusions of Gardner and Smith (1969)




78






and Eickhoff (1970), we feel on the basis of our prospective study, that invivo transfer of R-factors may occur with considerable frequency in a suitable population of patients. In addition to the spread of R-factor-carrying bacteria, invivo transfer of the R-factors may therefore be an important mode of the nosocomial dissemination of antibiotic resistance.












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Wood, W.B. (1966). Host Specificity of DNA Produced by Escherichia coli: Bacterial Mutations Affecting the Restriction and Modification of DNA. J. Molec. Biol., 16:118-133.















BIOGRAPHICAL SKETCH




Louis Washington was born in New Orleans, Louisiana, and graduated from the Walter L. Cohen Senior High School of that city. He was a member of the United States Army from September, 1954 to August, 1957. In June, 1967, he received the degree of Bachelor of Science with a major in Biology from Southern University in New Orleans. Since September, 1967, he has been a student in the Department of Immunology and Medical Microbiology, College of Medicine, University of Florida. During this period, he has been supported by NIH Training Grant 5TI-Al-0128.

Louis Washington is married to the former Geraldine Elizabeth Ann Thompson, and is the father of a daughter, Tracie Leigh.

















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I certify that Fhave read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Herman Baer, Chairman
Associate Professor of Immunology and Medical Microbiology


I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Peter Cerutti
Chairman and Professor of Biochemistry


I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.


gae .
Ira Rosen
Assistant Professor of Immunology and Medical Microbiology


I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Pa r ke r Sm a I I ,
Chairman and Professor of Immu"nology and Medical Microbiology











This dissertation was submitted to the Dean of the College of Medicine and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1972



Dean, College of Medicine





Dean, Graduate School