Identification and Classification of Coryneform Bacteria Isolated from Bovine Mammary Glands

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Jeffrey L. Watts Western Michigan University

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This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. IDENTIFICATION AND CLASSIFICATION OF CORYNEFORM BACTERIA ISOLATED FROM BOVINE MAMMARY GLANDS

Jeffrey L. Watts

A Dissertation Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Department of Biological Sciences

Western Michigan University Kalamazoo, Michigan April 2000

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IDENTIFICATION AND CLASSIFICATION OF CORYNEFORM BACTERIA ISOLATED FROM BOVINE MAMMARY GLANDS

Jeffrey L. Watts, Ph. D.

Western Michigan University, 2000

Coryneform bacteria are frequently isolated from bovine mastitis and these

infections are associated with economic losses. Corynehacterium bovis, a lipid-

requiring species, has been the most frequently isolated coryneform from the milk of

infected bovine mammary glands. However, the taxonomic status of this organism is

uncertain. In the current study, a polyphasic approach was used to identify

coryneform bacteria isolated from bovine mastitis and determine the phylogenetic

relationships among the identified species. A total of 212 coryneform bacteria

isolated from bovine mastitis was obtained from mastitis reference laboratories in the

United States and Canada. Presumptive identification based upon Gram-stain,

oxidase, catalase, and Tween 80 stimulated growth classified 183 isolates as

Corynehacterium species. Eighty-seven strains were selected for species level

identification by 16S rRNA gene sequencing, the Biolog system and the API Coryne

system. Fifty strains were identified as Corynebacterium bovis by 16S rRNA gene

similarity studies: the Biolog and API Coryne systems identified 54.0 and 88.0% of

these strains, respectively. Antimicrobial susceptibility testing of 46 C. bovis and 14

C. amylocolatum strains determined these organisms were susceptible to ampicillin,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. oxacillin, cephalothin, ceftiofur, a combination of penicillin and novobiocin,

erythromycin, clindamycin, pirlimycin, tetracycline, florfenicol, enrofloxacin,

sarafloxacin, danofloxacin, and premafloxacin but not tilmicosin. Finally,

phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA

and phylogenetic analyses performed. All strains identified as C. bovis exam ined

clustered with the reference strains indicating that C. bovis is a well defined species

within the genus Corynehacterium. Rep-PCR of these strains indicated that only

minor genetic variation exists within strains of C.bovis. Corynehacterium bovis

ATCC 13722 was determined to be most closely related to Brevibacterium helvolum.

Based on phylogenetic analyses, this organism was placed in the genus

Brevibacterium as Brevibacterium neaveae sp. nov. Results of this study confirm

that the coryneforms isolated from bovine mammary glands are a heterogeneous

group of organisms. Furthermore, direct sequencing of the 16S rRNA gene appears

to be the most accurate method for identification ofCorynehacterium species isolated

from bovine mastitis.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright by Jeffrey L. Watts 2000

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS

The ability of a person to successfully complete a doctoral program at a later

stage in their career depends greatly on support from a number of persons. First. I

would like to thank and am greatly appreciative of the time and effort given to me by

my advisor. Dr. Silvia Rossbach. Through her willingness to mentor a student

working in an area different from her own. she has demonstrated the professional

agility to address a broad range of research areas. I would also like to thank the

members of my committee, Dr. Lenard Ginsberg, Dr. Robert Eisenberg, and Dr.

Chuck Ford. In particular, Dr. Ford has served as a source of encouragement and

support during my program.

I would also like to thank Pharmacia & Upjohn Animal Health for supporting

my program. I would also like to thank my many colleagues at P&U for their support

over the years. In particular, I want to thank Dr. David Lowery and Ms. Janet Teel

for their help in training me in the molecular techniques needed for this work. I also

want to thank Ms. Cathy Ditto for performing the Rep-PCR experiments in the study.

I also want to thank Dr. Robert J. Yancey, Jr. for encouraging me to complete my

doctorate and Dr. Richard C. Wardley for providing the time and resources to

complete this program.

Last and most important, I would like to thank my family. In particular, I

would like to thank my son, Eric, and daughter, Danielle, for sacrificing their time to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgments - Continued

allow me to complete this work. This is truly time that cannot be returned. To my

wife, Vickie, I offer my greatest thanks. Her sacrifice, dedication, and understanding

through this program have been my strength. At a time in my life that I had resigned

myself to never completing a doctorate, she kept the dream alive.

Jeffrey L. Watts

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGMENTS...... ii

LIST OF TABLES ...... vi

LIST OF FIGURES...... vii

CHAPTER

I. INTRODUCTION ...... I

Bovine Mastitis and the Corynebacteria ...... 1

Overview of Bovine M astitis...... 1

Mastitis Pathogens...... 2

The Role of Corynebacteria in Bovine Mastitis...... 5

Classification of the Corynebacteria...... 8

Historical Perspective...... 8

Taxonom y of C.bovis ...... 14

Study Hypothesis and Objectives ...... 17

II. M ATERIALS AND M ETH O D S...... 19

Methodology...... 19

Bacteria...... 19

Presumptive Identification ...... 21

Biolog System...... 22

API Coryne System...... 24

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents - Continued

CHAPTER

Minimum Inhibitory Concentration (MIC) Determinations 25

Ribosomal RNA Sequencing...... 27

Analysis of Sequence Data...... 28

Phylogenetic Studies...... 29

Rep-PCR...... 30

m. RESULTS AND DISCUSSION ...... 33

Identification of Corynehacterium bovis and Other Coryneforms Isolated From Bovine Mastitis...... 33

Antimicrobial Susceptibility of C. bovis and C. amylocolatum Isolated From Bovine Mammary Glands...... 41

Phylogenetic Studies on Coryneform Bacteria Isolated From Bovine Mammary Glands ...... 55

Transfer of C. bovis ATCC 13722 to the Genus Brevibacterium as Brevibacterium neaveae, sp. nov...... 62

Description of Brevibacterium neaveae sp.nov...... 63

IV. SUMMARY AND CONCLUSIONS...... 66

BIBLIOGRAPHY ...... 70

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

1. Comparison of Genera of Non-sporeforming Gram-positive Bacilli Described in Bergey’s Manual From 1957 to 1986 ...... 9

2. Chemical Characteristics of the Genus Corynehacterium and Related Genera ...... 11

3. Corynehacterium Species Described in Bergey’s Manual of Systematic Bacteriology in 1986...... 14

4. Corynehacterium Species and Related Taxa Described Since 1986 ...... 15

5. Source and Number of Isolates Used in the Study ...... 20

6. Reference Strains Used in the Study ...... 21

7. Test Substrates in the Biolog Gram-Positive Microbial Identification Panel...... 23

8. Primers Used for rRNA Gene Sequencing...... 28

9. Sequences From References Strains Used for Phylogenetic Analyses...... 31

10. Identification of Coryneform Bacteria Using rRNA Gene Similarity, the Biolog System, and the API Coryne System...... 36

11. Summary of Minimum Inhibitory Concentrations (MIC) for 46 Strains of C. hovis Isolated From Bovine Mammary Glands...... 43

12. Summary of Minimum Inhibitory Concentrations (MIC) for 13 Strains of C. amylocolatum Isolated From Bovine Mammary Glands ...... 53

13. Similarity of rRNA Gene Sequences for C.bovis Strains to C. bovis ATCC 7715 and C. bovis NCTC 3224...... 59

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

1. Schematic Representation of Sequencing Primers and rRNA...... 29

2. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Ampicillin...... 44

3. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Oxacillin...... 44

4. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Cephalothin...... 45

5. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Ceftiofur...... 45

6. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against a Combination of Penicillin and Novobiocin...... 46

7. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Erythromycin...... 46

8. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Tilmicosin...... 47

9. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Clindamycin ...... 47

10. Distribution o f MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Pirlimycin...... 48

11. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Tetracycline...... 48

12. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Florfenicol...... 49

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures - Continued

13. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Enrofloxacin...... 49

14. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Sarafloxacin...... 50

14. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Danofloxacin...... 50

15. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Strains Against Premafloxacin...... 51

16. Unrooted Tree Depicting the Phylogenetic Relationships among Strains of C. bovis and Other Members of the Corynehacterium urealyticum G roup...... 57

17. BOX-PCR Based Fingerprinting of 16 C. bovis Strains Isolated From Bovine Mammary Glands ...... 61

19. BOX-PCR Based Fingerprinting of 15 C. bovis Strains Isolated From Bovine Mammary Glands and Two Reference C.bovis S trains...... 62

20. Unrooted Tree Depicting the Phylogenetic Relationship of C.bovis ATCC 13722 and Members of the Genus Brevibacterium...... 64

viii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

INTRODUCTION

Bovine Mastitis and the Corynebacteria

Overview of Bovine Mastitis

Bovine mastitis or intramammary infections (IMI) are defined as an

inflammation of the mammary gland resulting from a bacterial infection (Bramley et

al., 1996; Eberhart et al. 1987; International Dairy Federation, 1987; Philpot &

Nickerson, 1991; Philpot. 1984). Bovine mastitis remains the most costly disease to

the dairy industry worldwide with losses estimated at 2 billion dollars per year in the

United States alone (Bramley et al., 1996; Eberhart et al. 1987). On a per cow basis,

losses are estimated at approximately SI85 per cow per year (Bramley et al.. 1996;

Eberhart et al. 1987). Most of these losses are due to reduced milk production but

other costs include fees for professional veterinary services, antibiotic therapy,

discarded milk, and early replacement of affected animals (Bramley et al., 1996;

Eberhart et al. 1987; International Dairy Federation, 1987; Philpot & Nickerson,

1991; Philpot, 1984). Bovine mastitis may be manifested as acute, clinical, or

subclinical forms of the disease (Bramley et al., 1996; Eberhart et al. 1987;

International Dairy Federation, 1987). Acute mastitis is characterized by sudden

onset, redness, swelling, hardness, pain, grossly abnormal milk, and reduced milk

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. yield. It is often accompanied by such systemic signs as fever, loss of appetite,

dehydration and depression. The secretion from infected udder quarters with clinical

mastitis is visibly abnormal and contains clots, flakes, or is watery in appearance.

Subclinical mastitis is the most prevalent form of the disease. Subclinical

mastitis is characterized by no observable changes in the milk appearance. However,

organisms can be cultured from the secretion and inflammatory changes such as

increased somatic cell counts (SCC) can be measured (Bramley et al., 1996; Eberhart

et al. 1987; International Dairy Federation, 1987). Chronic mastitis may be a subtype

of either clinical or subclinical mastitis and is characterized by a persistent infection

of the udder (Bramley et al., 1996; Eberhart et al. 1987; International Dairy

Federation, 1987).

Mastitis Pathogens

Over 135 organisms are known to cause mastitis (Watts, 1988). However,

the majority of IMI are caused by Gram-positive organisms such as staphylococci,

streptococci,Corynehacterium spp. and Gram-negative organisms including

Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, and Enterohacter spp.

(Bramley & Dodd, 1984; Bramley et al., 1996; Eberhart et al. 1987; International

Dairy Federation, 1987; Philpot & Nickerson, 1991; Watts, 1988). Historically,

mastitis pathogens have been categorized based on route of transmission or on

pathogenicity.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 Mastitis pathogens may be acquired by the cow by two different routes of

transmission. Those spread during the milking process are termed contagious

pathogens while those contracted from environmental sources between milking

intervals are termed environmentalpathogens (Bramley et al., 1996; Eberhart et al.

1987; International Dairy Federation, 1987). The contagious pathogens include

Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and

Corynehacterium hovis (Bramley & Dodd, 1984; Bramley et al.. 1996; Eberhart et al.

1987; Philpot & Nickerson, 1991; Watts. 1988). The environmental pathogens

include Streptococcus uheris, Enterococcus spp., Corynehacterium pyogenes, and

enteric bacilli (Bramley & Dodd, 1984; Bramley et al.. 1996; Eberhart et al. 1987;

Philpot & Nickerson, 1991; Watts, 1988). Development of mastitis control programs

have been primarily focused on controlling spread of the contagious pathogens in

dairy herds. A successful mastitis control program must control the rate of new

infections while reducing the persistence of existing infections (Bramley et al., 1996;

Eberhart et al. 1987; Philpot, 1984; Philpot & Nickerson, 1991). Components of

these programs have included post-milking teat antisepsis, use of properly

functioning milking machines, antimicrobial therapy during lactation and between

lactations (termed the “dry” period), and culling of chronically infected animals

(Bramley et al., 1996; Eberhart et al. 1987; Philpot, 1984; Philpot & Nickerson,

1991).

Mastitis pathogens have also been categorized based upon their pathogenicity.

The pathogenicity of mastitis pathogens has historically been measured using the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 magnitude of the inflammatory response to udder infections as indicated by an

increase in SCC (Bramley et al., 1996; Eberhart et al. 1987; International Dairy

Federation, 1987; Philpot, 1984; Philpot & Nickerson, 1991). Somatic cells in

normal milk are comprised of approximately equal populations of macrophages and

polymorphonuclear leukocytes (PMNs) (Bramley et al., 1996; Eberhart et al. 1987;

Philpot, 1984; Philpot & Nickerson, 1991). In response to a bacterial infection, SCC

rapidly rise with a shift toward PMNs as the predominate cell type. In an active case

of clinical mastitis, PMNs account for over 99% of cells and total SCC may exceed

106 cells per ml. Increased SCC are inversely correlated with reduced milk

production resulting in losses of 9 to 18% for counts between 4 x 105 cells per ml and

12 x 105 cells per ml and 19 to 25% above the latter figure.

Prior to 1980, SCC for normal mammary glands were considered to be 3-4 x

105 cells per ml; an SCC over 1 x 106 cells per ml was considered abnormal. Based

on this definition of normal and abnormal SCC, mastitis pathogens were categorized

as either major or minor pathogens. The major pathogens includedS. aureus, S.

agalactiae, S. dysgalactiae, S. uberis, C. pyogenes, and the enteric bacilli (Bramley et

al., 1996; Eberhart et al. 1987; International Dairy Federation, 1987). IMI with these

organisms usually resulted in SCC values over 10 x 105 cells per ml (Bramley et al.,

1996; Eberhart et al. 1987; International Dairy Federation, 1987). Minor pathogens

included the coagulase-negative staphylococci (often termed micrococci) and C. bovis

and IMI with the minor pathogens resulted generally in SCC considered to be normal

(i. e, 3-4 x 10s cells per ml) or only slightly elevated (Bramley et al., 1996; Eberhart

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al. 1987; International Dairy Federation, 1987). As a result of these definitions, an

individual mammary gland could be infected with minor pathogens and still be

considered normal. Indeed, early studies (Little & Plastridge, 1946; Counter, 1981)

on the prevalence of mastitis in dairy herds included mammary gland quarters

infected with minor pathogens in the uninfected category.

The implementation of effective mastitis control programs over the last twenty

years has resulted in dramatic reductions in the incidence of the contagious pathogens

in dairy herds. This has resulted in an increase in the prevalence of the minor

pathogens such as coagulase-negative staphylococci and C.bovis (Bramley et al.,

1996; Hogan, 1999). During this same period, the definition of a normal SCC was

lowered from 3-4 x 10s cells per ml to 2.5-5 x 104 cells per ml (Bramley et al., 1996;

Eberhart et al. 1987; International Dairy Federation, 1987; Philpot & Nickerson,

1991; Philpot, 1984). This has resulted in a blurring of the distinction between the

major and minor pathogens as the increased SCC caused by the minor pathogens such

as C. bovis are now recognized to be associated with significant losses in milk

production (Bramley et al., 1996; Eberhart et al. 1987; International Dairy Federation.

1987; Philpot & Nickerson, 1991; Philpot, 1984).

The Role of Corynebacteria in Bovine Mastitis

A variety of coryneform bacteria have been isolated from bovine mastitis

(Watts, 1988; Hogan et al., 1999; Hommez et al., 1999). The two most commonly

isolated species are Arcanobacterium ("Corynehacterium") pyogenes, the causative

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 agent of summer mastitis, and C. bovis, a lipophilic species. Other corynebacteria

isolated from bovine IMI include Corynebacteria ulcerans, Corynehacterium

amylocolatum, Corynehacterium pseudotuberculosis, and Corynehacterium

minutissimum (Watts, 1988; Hommezetal., 1999).

As stated above, C. pyogenes is the causative agent of summer mastitis.

Summer mastitis is an acute, purulent form of mastitis that is often associated with

humid weather or low-lying areas (Bramley & Dodd, 1984; Bames-Pallesen et al.,

1987; Hogan et al., 1999). Flies, particularly Hydrotea irritans, are a known vector

of C. pyogenes and fly control is an important tool for IMI caused by this organism

(Barnes-Pallesen et al., 1987; Bramley & Dodd, 1984; Hogan et al., 1999). Summer

mastitis is much more prevalent in Europe than in the United States and may account

for up to 5% of total infections in dairy herds (Bramley & Dodd, 1984). C. pyogenes

infections are often associated with a strict anaerobe,Peptococcus indolicus, which

produces the characteristic pungent garlic-like odor associated with this disease

(Barnes-Pallesen et al., 1987; Bramley & Dodd, 1984; Hogan et al., 1999; Watts,

1988). C. pyogenes produces very small, B-hemolytic colonies on5% bovine blood

agar after 48 h incubation at 35-37° C. C. pyogenes was reclassified as Actinomyces

pyogenes in the early 1980s and, more recently, has been reclassified as

Arcanobacterium pyogenes (Hommez, 1999).

C. bovis is the most frequently isolated corynebacterium species from bovine

IMI (Brooks & Bamum, 1984a; Pankey et al., 1985; Linde et al., 1980; Woodward et

al., 1988). C. bovis readily colonies the teat canal of dairy cows and has been used as

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 an indicator of milking hygiene. In herds that do not practice post-milking teat

antisepsis (“teat-dipping"), it is not unusual for C. bovis to be isolated from more than

60% of quarter milk samples (Brooks & Barnum, 1984a; Pankey et al., 1985; Linde et

al., 1980; Woodward et al., 1988). Indeed, Pankey et al. (1985) demonstrated that

under experimental challenge conditions, the rate of new C. bovis IMI was nearly 30

times higher than that of S. agalactiae. However, this high infection rate was

considered to be due to teat canal colonization and subsequent contamination of milk

samples rather than true IMI (Pankey, 1985). Other studies (Brooks & Barnum,

1984a; Brooks & Barnum, 1984b; Pankey et al., 1985; Linde et al., 1980; Woodward

et al., 1988) have suggested that C. bovis is capable of infecting bovine mammary

glands and that these infections may be protective against infections caused by other

mammary gland pathogens. In all of these studies, mammary glands infected with C.

bovis have a decreased rate of additional infection with either S. aureus or S.

agalactiae.

Identification of corynebacteria isolated from bovine mammary glands has

been largely based on colony morphology, hemolysis, and growth requirements

(Barnes-Pallesen et al., 1984; Brown et al., 1969; Brown et al., 1981; Harmon et al.,

1990; Hogan et al., 1999; International Dairy Federation, 1981). For example, the

presence of small, B-hemolytic colonies after 48 h incubation is considered adequate

for presumptive identification ofA. pyogenes (Barnes-Pallesen et al., 1984; Brown et

al., 1969; Brown et al., 1981; Harmon et al., 1990; Hogan et al., 1999; International

Dairy Federation, 1981). Identification ofC. bovis is based largely on the presence of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 small, white, non-hemolytic colonies on5% bovine blood agar after 48 h incubation

at 35-37° C. Moreover, C. bovis tends to grow well only in areas of visible milkfat

due to a requirement for oleic acid (Barnes-Pallesen et al., 1984; Brown et al., 1969;

Brown et al., 1981; Harmon et al., 1990; Harrigan, 1966; Hogan et al., 1999;

International Dairy Federation, 1981). This high reliance on presumptive

identification has limited the ability of most mastitis microbiology laboratories to

recognize and delineate corynebacteria species. Indeed, other corynebacteria species

have only been reported in those studies where more extensive characterizations were

conducted (Cobb, 1962; Femandez-Garayzabal, 1997; Hommez, 1999).

Classification of the Corynebacteria

Historical Perspective

The genus Corynebacterium was first described in 1896 to accommodate the

causative agent of diphtheria, Corynebacterium diphtheriae (Breed et al., 1957; Jones

& Collins, 1986). This organism, originally described by Kruse in 1886, was

originally placed in the genus Bacillus as Bacillus diphtheriae (Breed et al., 1957;

Jones & Collins, 1986). The creation of the genusCorynehacterium allowed the non-

sporeforming, irregular, Gram-positive bacilli to be separated from the spore-forming,

Gram-positive bacilli such as Bacillus (Breed et al., 1957; Jones & Collins, 1986). In

the interceding years, a number of new species were added to the genus

Corynebacterium until it became a collection of organisms generally characterized as

non-sporeforming, Gram-positive bacilli (Breed et al., 1957; Jones & Collins, 1986;

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 Pascual et al., 1995). In the 7lh edition of Bergey’s Manual of Determinative

Bacteriology (Jones & Collins, 1986), the familyCorynebacteriaceae contained 6

genera with the genus Corynehacterium containing 33 species (Table 1). Many of

these species were ill-defined and their relationships to other species within the genus

were not well characterized.

Table 1

Comparison of Genera of Non-sporeforming Gram-positive Bacilli Described in Bergey’s Manual From 1957 to 1986

Genera 1957 1986 Corynehacterium Caseohacter Listeria Corynehacterium Erysipelothrix Aureobacterium Microbacterium Curtobacterium Cellulomonas Microbacterium Arthrohacter Arthrohacter Brevibacterium Renibacterium Agromyces Cellulomonas Oerskovia Rothia Actinomyces Arcanobacterium Propionibacterium Arachnia Gardnerella Bifidobacterium Eubacterium Acetobacterium Butyrivibrio Thermoanaerobacter Lacnospira

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 During the time span from the 1960s to the 1980s, chemical cellular studies

had begun to elucidate the relationships between the various organisms within this

group (Cummins, 1971: as reviewed in Jones and Collins, 1986). These studies

identified specific characteristics for classification of the coryneform bacteria and, for

the first time, allowed a rational approach to the taxonomy of this group of organisms

(Jones & Collins, 1986; Schliefer & Kandler, 1972). Based on these data,

characteristics useful in differentiating the various genera of the non-sporeforming,

Gram-positive bacilli were as follows (Jones & Collins, 1986; Coyle and Lipsky,

1990; Funke et al., 1997; Clarridge & Spiegel, 1995: Funke & Bernard, 1998): (a) cell

wall type, (b) menaquinone type, (c) presence or absence of mycolic acid, (d) fatty

acid type, and (e) % G+C (27). The genus Corynebacterium was determined to

belong to the Actinomyces group of organisms and to be closely related to the

mycobacteria and rhodococci (collectively termed the CMR group) (Jones & Collins,

1986). Jones and Collins (1986) included the genus Corynebacterium with the non-

sporeforming. Gram-positive bacilli along with 22 other genera (Table 1). An

overview of the chemical characteristics separating the genus Corynehacterium from

other genera containing the Gram-positive, irregular, bacilli is presented in Table 2.

In general, the genus Corynebacterium was limited to those organisms with the

following characteristics: (a) Gram-positive, non-sporeforming, club-shaped bacilli

with a palisade or “Chinese-character” arrangement; (b) catalase-positive; (c)

peptidoglycan type A; (d) m-diaminopimelic acid type; (e) short-chain mycolic acids;

and (0 Mol% G+C content ranging from 51 to 63% (Jones & Collins, 1986; Coyle

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. G-t-C4 71-75 71-76 53 47-53 60-67 68-75 51-68 65-67 ______Type 11, 11, 12 10-12 69-75 7 ,8 8 ,9 59-70 9 71-76 ______S, A, 1 7 S, A, I 9 A 9, 10 S, A, I S, A, IS, A, I 11, 12 9 67-70 S, A, I S, A, I Characteristic Table 2 No No No S, A, 1 No and Related Genera (Modified from Coyle & Lipsky, 1990; Funke et al., No S, A, 1 No No No No Type2 Acids Present Type3 Lys Lys ///-Dap No S, A, 1 9, 10 Corynebacterium 1997; Jones & Collins, 1986; Schleifer & Kandler, 1972) ______G roup1 Peptidoglycan Diamino Acid Mycolic Fatty Acid Menaquinone Mol% A Lys No A AA L-Orn Lys B Dab BB D-Orn D-Orn A Lys A ///-Dap B A A, B3 ///-Dap, Dab6 Yes S, U,T 8,9 ______Genus ______Chemical Characteristics ofthe Genus Curtobacterium Oerskovia Corynebacterium Caseobacter Cellulomonas Rothia Brevibacterium Renibacterium Microbacterium Aureobacterium Arthrobacter Agromyces

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IO 63-65 Mol% 53-68 G+C4 48-52 Menaquinone 9 - 42-44 Type 9 A, 1A 9 S, U Fatty Acid S, U 10 57-69 Type3 Characteristic No No No Mycolic Acids Present Lys Lys, Orn Diamino Acid //j-Dap Type2 A L-Dap No A A A L-Dap, A Lys No S, U G roup1 , r . . „ . « 1. Gardnerella 2//i-Dap, //H'.v«-diaminopimelic acid; 3S, Dab,straight diaminobutyric chain; U, monounsaturated; acid; D-Orn, D-ornithine;A, anteiso-methyl-branched; Lys, lysine; L-DAP; iso-methyl-branched; 1, L-diaminopimelic T,acid. tuberculostearic acid. Genus Peptidoglycan Propionibacterium 5 Animal 5 pathogenic species possess type A peptidoglycan while plant pathogenic species possess type B. Table 2 - Continued Arachnid Arcanobacterium Actinomyces 4The Mol% G+C for the animal pathogen corynebacteria^ h e diaminois -65 51 and acid67-68 type for for the the plantplant pathogenicpathogenic species. species is Dab.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 and Lipsky, 1990; Funke & Bernard, 1998; Funke et al., 1997; Clarridge & Spiegel,

1995).

The type species for the genus is Corynebacterium diphtheriae (Jones &

Collins, 1986). At this time, the genus Corynebacterium contained 18 species

pathogenic to humans and animals and 7 pathogenic to plants (Table 3; Jones &

Collins, 1986).

The advent of genetic techniques such as DNA hybridization and DNA-rRNA

hybridization during the 1970s and 1980s allowed for greater insight into the

phylogeny of the genusCorynebacterium (Pascual et al., 1995; Ruimy et al., 1995).

Precise phylogenetic studies needed an accurate molecular tool to elucidate the

relationships of organisms within the genusCory nebacterium (Woese. 1987). In the

1980s, direct sequencing of the 16S RNA of the small ribosomal subunit and

comparison of the sequence data to those of type strains of known species provided

such a tool (Woese, 1987). Use of these data has generally established the accuracy

of the earlier chemical studies as well as defined a number of new genera (Femandez-

Garayzabal et al., 1995; Funke et al., 1997; Jones & Collins. 1988; Pascual et al.,

1995; Pascual et al., 1996; Pascual et al., 1998; Rainey et al., 1995; Reigel et al.,

1997a; Reigel et al., 1997b; Ruimy et al., 1995; Sjoden et al. 1998; Woese, 1987;

Zimmerman et al., 1998). A summary of the new genera and species described since

1986 is presented in Table 4.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3

Corynebacterium Species Described in Bergey’s Manual of Systematic Bacteriology in 1986

Host Range (Pathogenecity) Humans and Animals Plants C. diphtheriae C. michiganense C. pseudotuberculosis C. insidiosum C. xerosis C. iranicum C. pseudodipthericum C. nebraskense C. kutscheri C. sepedonicum C. minutissimum C. tritici C. striatum C. rathayi C. renale C. cystitidus C. pilosum C. mycetoides C. matruchotii C. flavescens C. vitarumen C. ylutamicum C. callunae C. bovis1 C. paurometabolum1 Species incertae sedis

Taxonomy ofC. bovis

Until recently, the taxonomic relationship ofC. bovis to the other

corynebacteria is best characterized as tenuous (Jones & Collins, 1986). C. bovis was

originally placed in the genus based on phenotypic characterisitics. That is, C. bovis

was a pleomorphic, Gram-positive, catalase-positive, non-sporeforming bacillus.C.

bovis exhibited a distinct lipophilism but other corynebacteria also displayed this

characteristic to varying degrees (Clarridge & Spiegel, 1995; Coyle and Lipsky,

1990; Funke & Bernard, 1998; Funke et al., 1997; Jones & Collins, 1986).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 Table 4

Corynebacterium Species and Related Taxa Described Since 1986 (Modified from Clarridge & Spiegel, 1995; Coyle & Lipsky, 1990; Funke & Bernard, 1998; Funke et al., 1997)

Year Corynebacterium spp. Other genera 1987 C. diphtheriae (redefined) Jonesia denitrificans C. ammonia genes 1988 C. pseudotuberculosis Dermabacter hominis Tsukamurella paurometabolum 1991 C. xerosis 1992 C. pseudodiphthericum 1993 C. kutscheri C. minutissimum 1994 C. jeikeium A ureobacterium 1995 C. pilosum Microbacterium spp. C. mycetoides Cellulomonas spp. C. matruchotii Sanguibacter suarezii C. flavescens Sanguibacter keddieii C. vitarumen Dietzia maris C. glutamicum C. callunae 1996 Brevibacterium otitidis Arthrobacter cumminsii Sanguibacter inulinus 1997 C. coyleae A rcanobacterium pyogenes C. lipophiloflavum Arcanobacterium bemardiae C. imitans C. mucifaciens C. singulare C. durum C. mastitidis 1998 C. falsenii A ureobacterium resistens C. riegelii Rothia dentrocariosa C. thomssenii Curtohacterium spp. C. kroppenstedtii C. confusum C. sundvallense C. sanguis C. phocae C. camporealis

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 The availability of data from chemical studies questioned whether C. bovis

belonged in the genus (Jones & Collins, 1986). These data indicated that C. bovis

differed from other corynebacteria by possessing very short chain (22-36 carbon)

mycolic acid and high levels of tuberculostearic acid. Additionally, C. bovis displays

a Mol% G+C of 67-74 compared to 51-65 for the “true" corynebacteria. As a result

of these data, Jones and Collins (1986) indicated that C. bovis should be excluded

from the genus.

In 1995, Pascual et al. (31) conducted a phylogenetic analysis of the genus

Corynebacterium based upon 16S rRNA gene sequences which included the type

strain of C. bovis (ATCC 7715). The results of this study indicated that C. bovis

clustered within the confines of the genus and was most closely related to other

lipophilic corynebacteria such as C. jeikeium. Thus, C. bovis should be considered a

true member of the genus (Pascual et al., 1995). These data expanded the Mol% G+C

for the genus Corynebacterium sensu stricto from 5 1 -65 to 51 -74.

In a similar study, Ruimy et al. (1995) examined the phylogeny of the

corynebacteria also based on the small subunit ribosomal RNA gene sequences.

Again, C. bovis ATCC 7715, the type strain, was included in this study. Results of

this study were similar to those of Pascual et al. (1995). That is, C.bovis clustered

closely within the genus toC. jeikeium. However, the robustness of this association

was not high in either study with bootstrap values ranging from 55 to 75% (Pascual et

al., 1995).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 More recently, another lipophilic corynebacteria,Corynebacterium mastitidis,

isolated from mastitis in sheep was described (Femandez-Garayzabal et al., 1997).

This organism contains short chain mycolic acids but no tuberculosteric acid. This

organism does not appear to be closely related to C.bovis based upon 16S RNA gene

sequence analysis (6-10% divergence).

Study Hypothesis and Objectives

In summary, the importance of the corynebacteria will increase as the dairy

industry focuses on reducing SCC in milk below 2 x 105 per ml. Recent taxonomic

studies have dramatically restructured the genus Corynebacterium as several

organisms previously included in the genus are now placed in other genera.

Moreover, recent taxonomic studies have described several new species of

Corynebacterium. Nevertheless, the primary objective of the taxonomic studies have

been to define the relationship between a new species and currently defined species.

Thus, these studies have included only a single reference strain for comparative

purposes.

The impact of the taxonomic changes in the genusCotynebacterium on the

epidemiology and economics of bovine mastitis has not been defined. Moreover, the

current methods employed in mastitis bacteriology laboratories are inadequate for

identification of the Gram-positive, non-sporeforming bacilli. This has resulted in

many of these organisms being placed in C. bovis. At present, no studies have been

conducted that have examined a large number of strains of C. bovis isolated from

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 bovine mammary glands using both phenotypic and current genetic taxonomic

methods.

The primary hypothesis of this study is that C. bovis, as presently identified,

represents a heterogenous group of organisms belonging to other species within the

genus Corynebacterium as well as other genera. Moreover, the antimicrobial

susceptibility patterns of corynebacteria isolated from bovine mastitis is unknown and

may differ significantly from those of humans. The final hypothesis is that strains

defined as C. bovis based upon the current species description may represent several

new species as well.

In order to test these hypotheses, a polyphasic approach utilizing phenotypic

characteristics and 16S rRNA gene sequencing was used to fulfill the following

objectives:

1. Identification of a large collection of coryneform bacteria isolated

from bovine mastitis based on the current taxonomy of the genus utilizing both

phenotypic and rRNA gene sequence data.

2. Determination of the antimicrobial susceptibility ofCorynebacterium

species to a variety of antimicrobial agents.

3. Determination of the phylogenetic status of organisms classified as C.

bovis and other coryneform bacteria.

4. Determination of the genetic diversity of strains identified as C. bovis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CH APTER II

MATERIALS AND METHODS

Methodology

Bacteria

Isolates used in the study were requested from mastitis research laboratories

throughout the United States and Canada. All organisms were isolated from bovine

mammary glands. All isolates were identified using standard criteria used in mastitis

bacteriology techniques for identification of C.bovis (Bames-Pallesen et al., 1987;

Hogan et al., 1999). A total of 237 isolates were received from seven locations. The

source and number of isolates received are summarized in Table 5. Isolates from Dr.

K. Leslie, University of Guelph, were originally obtained by Dr. Don Barnum and

described in a previous study (Brooks & Barnum, 1984a). Isolates from Dr. Larry

Fox, University of Washington, included strains originally obtained by Dr. John

McDonald, National Animal Disease Center, Ames, IA. Thus, the strain obtained

represented historical isolates as well as recent field isolates. In addition, nine

reference strains of Corynebacterium species and related genera were obtained from

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 Table 5

Source and Number of Isolates Used in the Study

Source No. Isolates Culture Collection No. Received Dr. Allen Britten 8 21-28 Udder Health Systems Bellingham, Washington

Dr. Jim Cullor 8 32-39 University of California School of Veterinary Medicine Davis, CA

Dr. Ken Leslie 10 230-240 School of Veterinary Medicine University of Guelph Guelph. Ontario, Canada

Dr. Larry Fox 156 68-97 School of Veterinary Medicine 100-225 Washington State University Pulllman, WA

Dr. Steve Nickerson 27 40-47 Mastitis Research Laboratory 49-67 Louisiana State University Homer, LA

Melrose Veterinary Clinics 18 1. 3 ,4 New Ulm, MN 6-20

Pharmacia & Upjohn Animal Health 7 2 Kalamazoo, MI 29-31 226-227

Dr. Al Harper 3 5 Veterinary Outlets 228-229 New Bolton, TX

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 Table 6

Reference Strains Used in the Study

Organism ATCC No. Arcanobacterium pyogenes ATCC 19411 Corynebacterium bovis ATCC 13722 Corynebacterium bovis ATCC 7715 Cellulomonas cellulans ATCC 27402 Corynebacterium diphtheriae ATCC 11050 Corynebacterium jeikeium ATCC 43734 Dermabacter horn in is ATCC 49369 Rhodococcus equi ATCC 6939 Sanguibacter keddieii ATCC 51767

the American Type Culture Collection (Rockville. MD) for comparative purposes

(Table 6). Upon receipt, all isolates were maintained in the Pharmacia & Upjohn

Animal Health culture collection in 1.0 ml of Trypticase Soy Broth (TSB; B-D

Microbiology Systems, Cockeysville, MD) containing 10% glycerin at -70° C.

All isolates were revived for presumptive identification and phenotypic

studies by streaking onto Trypticase Soy Agar (TSA; Becton-Dickinson

Microbiology Systems, Cockeysville, MD) supplemented with 5% sheep blood and

1% Tween 80 (Sigma Chemical Company, St. Louis, MO).

Presumptive Identification

All isolates were presumptively identified based on Gram-reaction.

morphology, catalase reaction, hemolysin production, and the effect of lipid on

growth (Lauderdale et al., 1999). The effect of lipid on growth was determined by

subculturing each isolate onto unsupplemented TSA, TSA supplemented with 5%

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sheep blood, and TSA supplemented with I % Tween 80. Organisms exhibiting lipid

dependency exhibited no growth on unsupplemented TSA, poor or no growth on TSA

supplemented with 5% sheep blood, and good to luxuriant growth on TSA

supplemented with 1% Tween 80. Catalase-positive, Gram-positive, non-

sporeforming bacilli were presumptively identified as Corynebacterium spp. with

isolates exhibiting lipid-dependency presumptively characterized as C. bovis. All

isolates were identified using the Biolog system (Biolog, Inc., Hayward. CA) and the

API Coryne System (Vitek Systems, St. Louis, MO).

Biolog System

The Biolog microbial identification system consists of a 96 well microtiter

plate containing 95 dehydrated substrates (Table 7). Test organisms were prepared

by subculturing on Biolog Universal Growth Medium supplemented with 5% sheep

blood (BUGM; Biolog, Hayward, CA). For organisms exhibiting lipid-dependency.

isolates were subcultured into 1 ml of TSB supplemented with 1% Tween 80 and

incubated for 4 h at 35° C under aerobic conditions. After incubation, isolates were

then subcultured onto the surface of a BUGM plate. All plates were incubated for 24

h at 35° C under aerobic conditions. A bacterial suspension was prepared by

removing colony material from the surface of a BUGM plate with a sterile cotton

swab and agitating in 5 ml of 0.85% saline. The bacterial suspension was vortexed

and then standardized to 35-42% transmittance using a turbimeter (Biolog, Inc.). A

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 Table 7

Test Substrates in the Biolog Gram-Positive Microbial Identification Panel

water L-arabinose a-D-lactose a-cyclodextrin D-arabitol Lactulose 13-cyclodextrin arbutin M altose dextrin cellobiose Maltotriose glycogen D-fructose D-mannitol inulin L-fucose D-m annose mannan D-galactose D-melezitose tween 40 D-galacturonic acid D-melibiose tween 80 gentiobiose a-methyl-D-galactoside JV-acetyl-D-glucosamine D-gluconic acid B-methyl-D-galactoside yV-acetyl-D-mannosamine a-D-glucose 3-methyl-glucose L-pyroglutamic acid glucose-6-phosphate D-L-a-glycerol phosphate D-tagatose 1 act amide 6-methyl-D-glucoside D-trehalose D-lactic acid methyl ester a-methyl-D-mannoside turanose L-lactic acid Palatinose xylitol D-malic acid D-psicose D-xylose L-malic acid D-raffinose acetic acid methyl-pyruvate L-rham nose a-hydroxybutyric acid methyl-succinate D-ribose B-hydroxybutyric acid propionic acid Salicin Y-hydroxybutyric acid pyruvic acid Sedoheptulosan p-hydroxyphenyl-acetic acid succinamic acid D-sorbitol a-ketoglutaric acid succinic acid Stachyose a-ketovaleric acid W-acetyl-L-glutamic acid Sucrose alaninamide adenosine L-serine D-alanine 2’-deoxy-adenosine Putrescine L-alanine inosine 2,3-butanediol L-alanyl-glycine thymidine Glycerol adenosine-5’-monophosphate thymidine-5’- U ridine-5’- monophosphate monophosphate fructose-6-phosphate glucose-1 -phosphate Glycyl-L-glutamic acid L-asparagine uridine L-glutamic acid amygdalin m-inositol a-methyl-D-glucoside

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 150 (j.1 aliquot of this suspension was dispensed into each well of a Biolog Gram-

positive identification panel. Panels were incubated for 24 h at 35° C and then read

using the Biolog microplate reader using the Biolog Microstation 3.5 software and the

Gram-positive database (version 3.7). The Biolog software compared the results

obtained with the test strain to the database and provided an identification based on

distance calculations. Data from each isolate were entered into a user-defined

database using the Microstation software and a dendrogram generated using the

MCLUST software (Biolog).

API Corvne System

The API Coryne System consists of a gallery of 20 microcupules containing

dehydrated substrates. The system allows for the determination of the following

biochemical tests: nitrate reduction, pyrazinamidase, pyrrolidonyl arylamidase,

alkaline phosphatase, 13-glucuronidase, 13-galactosidase, a-glucosidase, N-acetyl-13-

glucosaminidase, esculin, urease, gelatin hydrolysis, a fermentation (negative)

control, glucose, ribose. xylose, mannitol, maltose, lactose, sucrose, and glycogen.

All isolates were tested using the procedures recommended by the manufacturer

except for isolates exhibiting lipid dependency. Colony material from each isolate

was subcultured into 0.3 ml of sterile distilled water and this suspension swabbed

onto the surface of a TSA agar plate supplemented with5% sheep blood. For

organisms exhibiting lipid dependency, colony material from each isolate was

inoculated into 1 ml of TSB supplemented with 1% Tween 80 and incubated for 4 h

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 at 35° C under aerobic conditions. After incubation, this suspension was spread over

the entire surface of a TSA agar plate supplemented with 5% sheep blood. All plates

were incubated for 24 h at 35° C under aerobic conditions.

After incubation, plates were removed from the incubator and a cotton swab

used to remove colony material from the surface of the plate. The colony material

was suspended in 3.0 ml of sterile distilled water to a turbidity equivalent to a No. 6

McFarland standard. The first 11 microcupules of the test strip gallery were

inoculated with this suspension. A 0.5 ml aliquot of this suspension was then added

to 3.0 ml of Gram-Positive medium (GP medium; provided by API), mixed using a

vortex mixer, and used to inoculate the nine fermentation test microcupules. Sterile

oil was used to overlay the urease, negative control, and fermentation test

microcupules. The test strips were incubated aerobically at 35° C for 24 h.

After incubation, the appropriate reagents were added to each well and color

reactions read after 10 min at ambient temperature using the provided color chart.

The previously determined catalase test results were used as the final test. Reactions

were recorded on the provided test result sheets and a seven digit octal code

generated. This numerical profile was interpreted using the profile index provided by

the manufacturer for a final identification.

Minimum Inhibitory Concentrations (MIC) Determinations

Prior to the MIC determinations, all isolates were revived by subculture in

TSB supplemented with 5% sheep blood and 1.0% Tween 80 and incubated for 24 h

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 at 35 to 37°C. The MIC determinations were performed using a broth microdilution

method (Sensititre, Westlake, OH). This method adheres to the guidelines of the

National Committee for Clinical Laboratory Standards (NCCLS, 1999). The MIC

panels consisted of commercially prepared 96-well microtiter plates (Sensititre)

containing the following antimicrobial agents: ampicillin, oxacillin, cephalothin,

ceftiofur, penicillin + novobiocin, erythromycin, clindamycin, pirlimycin.

premafloxacin, enrofloxacin, sarafloxacin, danofloxacin, tetracycline, florfenicol, and

tilmicosin. The dilution ranges tested were 0.06 to 64.0 fig/ml for all the

antimicrobial agents except for penicillin + novobiocin (0.06 )lg/ml of penicillin: 0.13

(ig/ml of novobiocin to 64.0 pg/ml of penicillin: 128.0 pg/ml of novobiocin), and

premafloxacin (0.0078 to 8.0 |ig/ml). In addition to the strains tested, the following

NCCLS recommended American Type Culture Collection (ATCC) quality control

strains were included with each batch of organisms tested: S. aureus ATCC 29213,

Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and

Pseudomonas aeruginosa ATCC 27853.

MIC determinations were performed as per the manufacturer’s instructions for

the commercially prepared panels or as per NCCLS guidelines for the manually

prepared panels (NCCLS, 1999). All panels were read after 18 h incubation at 35°C

under aerobic conditions. The first dilution with no visible growth was considered

the MIC for each strain. The MIC at which 50% (MIC5 0) and 90% (MIC 9 0 ) of the

isolates that were equal to or below, as well as the minimum and maximum MIC

values (range) were calculated.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 Ribosomal RNA Sequencing

Sequencing of the ribosomal RNA (rRNA) gene was used for final

identification and phylogeny studies. The polymerase chain reaction (PCR)

procedure used to amplify the rDNA sequence was performed as follows:

1. A 0.01 ml aliquot was removed from each frozen stock culture and

used to inoculate 60 JJ.1 of sterile distilled water.

2. A 1 pi aliquot of this suspension was added to the 49 fil of PCR

mixture (22.7 pi of distilled water, 15.2 fj.1 of 3.3X XL buffer II [PE Biosystems,

Foster City, CA], 4 pi 10 mM dNTP mix, 1.6 pi 25 mM magnesium acetate, 2.5 pi

of a 10 pM solution of 5’ primer [pA], 2.5 pi of a 10 pM solution of a 3’ primer [pH]

and 0.5 pi vTth DNA polymerase [PE Biosystems, Foster City, CA]).

3. The PCR was then performed in a Perkin-Elmer Model 9600 Thermal

Cycler (PE Biosystems, Foster City. CA) (94° C - 1. 5 min, 94° C - 20 s, 55° C - 45

s, 72° C - 4 min, 72° C - 7 min for 35 cycles).

4. The PCR mixture for each isolate was then purified using a QIAquick

PCR Purification Kit (Qiagen, Valencia, CA). Sequencing was performed by adding

5 pi of template to 5 pi of dye terminator ready reaction mix (Biodye, PE Biosystems,

Foster City, CA), and 3 pi of 1 pMol primer and amplification by PCR using the

previously described program. Excess dye terminators were removed from the

sequencing reaction mixture by passage through a sephadex column (Centri-Sep Spin

Columns, Princeton Separations, Adelphia, NJ).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 5. Sequencing was performed using a ABI Prism Model 377 Sequencer

(PE Biosystems, Foster City, CA).

The sequences of the primers used in the study are presented in Table 8. A

schematic representation of the rRNA genes sequenced are presented in Figure 1.

Approximately 100 bases were trimmed from the 5’ end of each sequence to

eliminate the hypervariable region as previously described (Pascual et al., 1995).

Sequences were assembled using Sequencher 3.0 for the Macintosh and manually

corrected.

Table 8

Primers Used for rRNA Gene Sequencing

Primer Sequence (PNU No.) (5’-> 3 ’) pA (1812) 5 ’ - AG AGTTTGATCCTGGCTC AG-3 ’

pH (1813) 5-AAGGAGGTGATCCAGCCGCA-3’

(1831) 5 ’ -G AGG AAC ACCG ATGGCG A AGGC-3 ’

(1832) 5 ’ -GCCCCCGTC A ATTCCTTTG AGTT-3 ’

Analysis of Sequence Data

Bacterial Identifications

The completed sequences for each isolate were exported in FASTA format

and compared to the rRNA gene sequence EMBL database using the BLAST search

engine of the NCBI taxonomy browser (NCBI, 1999). An isolate was considered

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 identified to the species level if similarity with a reference sequence was equal to or

greater than >98% (Stackebrandt and Goebel, 1994).

pA (1-749)

^ 1831 (67-795) *

1832 (618-1366) ► ______pH (732-1480)______

rRNA Gene

Figure 1. Schematic Representation of Sequencing Primers and the rRNA Gene.

Phylogenetic Studies

All phylogenetic analyses were performed using the PHYLIP group of

phylogeny analysis programs (Felsenstein, 1989). Distance matrices were produced

using the program DNADIST as previously described (Pascual et al., 1995). Trees

were constructed by the neighbor-joining method with the program NEIGHBOR of

PHYLIP. The stability of each group was assessed using the programs SEQBOOT,

DNADIST, NEIGHBOR, and CONSENSE (Pascual et al., 1995; Fernandez-

Garayzabal, 1997). A total of 100 bootstrap trees were generated. The phylogenetic

relationship of each isolate was compared to the reference strains listed in Table 9.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 Rep-PCR

Rep-PCR was performed on the isolates identified as C. bovis to determine the

genetic variation within the species. Rep-PCR was performed as previously

described (Versalovic et al., 1994). The 22-mer BOX AIR primer (5’-

CTACGGCAAGGCGACGCTGACG-3’) (Versalovic et al., 1994) was synthesized

by Sigma-Genosys (The Woodlands, TX). PCR amplifications were performed in 25

(il of a solution containing IX Gitschier Buffer, 10% DMSO (Sigma, St. Louis, MO):

1.25 mM of each dNTPs (Strategene, LaJoIla, CA), 55 pmol of BOX AIR primer and

2U of Taq DNA polymerase (Promega, Madison, Wl). Gitschier Buffer contained

16.6 mM (NH4)2S04; 67 mM Tris HC1, pH 8.8: 6.7 mM MgCL; 6.7 pM EDTA; 30

mM (3-mercaptoethanol (Sigma) and 170 pg/ml BSA (Strategene) (Versalovic et al.,

1994). A I pi loopful of C.bovis culture from a blood agar plate was added as

template. Amplifications were performed with a DNA thermocycler (Perkin Elmer

9600) with the following temperature profile:

Table 9

Sequences From References Strains Used for Phylogenetic Analyses

Species EMBL Accession No. Corynebacterium actoacidophilum X84240 Corynebacterium afermentans X81874 Coi-ynebacterium ammoniayenes X 84440 Corynebacterium amvlocolatum X84244 Corynebacterium bovis X84444 Corynebacterium bovis D38575 Corynebacterium callunae X 84251 Corynebacterium cystidis X84252

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 Table 9 - Continued

Species EMBL Accession No. “ Corynebacterium fastidiosum " X 84245 Corynebacterium flavescens X8444I “Corynebacterium genitalium ” X84253 Corynebacterium jeikeium X84250 Corynebacterium kutscheri X8187I Corynebacterium matruchotii X84443 Corynebacterium minutissimum X84678 Corynebacterium mycetoides X84241 Corynebacterium propinquiun X84438 Corynebacterium pilosum X84246 Corynebacterium pseudodiphthericum X84258 “Corynebacterium pseudogenitalium ” X81872 Corynebacterium pseudotuberculosis X84255 Corynebacterium renale X84249 “Corynebacterium segmentosum " X84437 Corynebacterium striatum X84442 “Corynebacterium tuberculostearicum X84247 “Corynebacterium ulcerans” X84256 Corynebacterium urealyticum X84439 Corynebacterium variabile [ X53185 Corynebacterium vitarumen X84680 Corynebacterium xerosis X84446 Unidentified bacterium gene ABO 12595 Coryneform bacterium (strain LMG 3820) AJ222817 Brevibacterium otitidis X93593 Brevibacterium mcbrellneri X93594 Brevibacterium linens X76566 Brevibacterium helvolum X77440 Brevibacterium iodinum X83813 Brevibacterium epidermidis X76565 Brevibacterium casei X76564 Clavibacter michiganensis subsp. nebraskensis U96182 Clavihacter michiganensis subsp. tessellarius U96181 Curtobacterium luteum X77437 Curtobacterium citreum X77436 Rcithayibacter rathayi D45062 Rathavibacter tritici X77438

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 cycle at 95°C for 7 min; 35 cycles at 90°C for 30s. at 53°C for 1 min, at 65°C for 8

min; and 1 cycle at 65°C for 16 min (Versalovic et al., 1994).

Rep-PCR products (2 |il) were separated by electrophoresis on a 1.5% agarose

gel (SeaKem LE agarose, FMC Bioproducts,Rockland, ME) in 0.5X TAE buffer (20

mM Tris-acetate, 0.5 mM EDTA, pH8.0) at a contant voltage of 3 V/cm. After being

stained with ethidium bromide, the gel was photographed under UV transillumination

with Polaroid 665 film. DNA molecular weight markers TJHindHI and

O X 174 IH aelll (Gibco/BRL, Gaithersburg, MD) were used as size standards.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III

RESULTS AND DISCUSSION

Identification of Corynebacterium bovis and Other Coryneforms Isolated From Bovine Mammary Glands

The majority of mastitis bacteriology laboratories use phenotypic

characteristics to presumptively identify strains of C. bovis (Cummins, 1971; Coyle

&Lipsky, 1990; Linde et al., 1990; Funke et al., 1997; Hogan et al., 1999). Basically,

those organisms exhibiting a small colony type after 48 h incubation in the area where

butterfat was deposited on the agar surface are presumptively considered to be C.

bovis. Some laboratories, recognizing the inaccuracy of this system, simply report

out these organisms generically as “coryneforms”. Indeed, the primary reason for

identification of coryneform bacteria in mastitis laboratories is to differentiate the

organisms from the more pathogenicNocardia spp. (Brown et al., 1969; Brown et al.,

1951; Harmon et al., 1990; Hogan et al., 1999). Thus, the first objective of this study

determined the accuracy of currently accepted methods for identification of C. bovis.

Of the 237 putative isolates submitted as C. bovis, 25 obtained failed to grow

on subculture. Of those that grew, 183 of 212 (86.3%) organisms identified as C.

bovis by mastitis bacteriology laboratories were correctly identified presumptively as

coryneform bacteria. Of the strains misidentified, 1 was identified as a yeast, 2 as

Bacillus spp., 11 as Enterobacteriaceae, 18 as staphylococci, 1 asa Streptococcus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34 spp., and 1 as an Enterococcus spp. These results are similar to the identification

levels for corynebacteria from clinical laboratories that indicate that 30% of

corynebacteria are misidentified (Coyle & Lipsky, 1990).

The remaining 183 strains identified as coryneforms based upon Gram-stain,

catalase production, and nitrate reduction were further characterized based upon their

requirement for lipids. The effect of lipid supplementation on the growth of

coryneforms can be characterized as either an absolute requirement for lipids or

stimulation of growth by lipids (referred to as lipid stimulated growth or lipophilicity)

(Coyle & Lipsky, 1990: Funke et al., 1997). Since determination of the absolute lipid

requirement necessitates the use of lipid free medium, we chose to determine if

growth of the organism was stimulated by supplementation of the media with either

5% sheep blood or 1% Tween 80. Of the 183 strains tested, 51 strains grew on

unsupplemented TSA while the growth of 108 (59.3%) strains was stimulated by lipid

supplementation. Blood supplementation has been reported to meet the lipid

requirement of coryneforms such as C.jeikeium , a coryneform isolated from humans

(Funke et al., 1997). Of the 108 strains demonstrating lipid stimulated growth, all but

7 grew on blood supplemented TSA. However, growth on blood supplemented TSA

was much less luxuriant than growth on the Tween supplemented medium. Colony

sizes generally were 0.5 mm or less after 24 h on blood supplemented media

compared to 1 to 2 mm for the same strain on the Tween supplemented medium.

The isolates identified as coryneform bacteria were further characterized

based on rRNA gene similarity studies or using commercial biochemical based

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 systems. The results of these studies are presented in Table 10. Direct sequencing of

the ribosomal small subunit of the 16S ribosomal RNA gene and comparison of

sequences of unknown organisms to those of type strains has become the reference

method for taxonomic studies (Fernandez-Garayzabal, 1997; Fernandez-Garayzabal,

1998; Pascual et al., 1995; Ruimy et al., 1995). In the current study, the rDNA

similarity identification was considered the correct identification and used to

determine the accuracy of the Biolog and Coryne systems. All the reference strains

tested were correctly identified by direct rDNA sequencing except C. bovis ATCC

13722. In addition, both the Biolog and API Coryne systems failed to identify the

type strain of Dermabacter. However, this strain is not in the identification databases

for either system.

C. bovis ATCC 13722 was identified as a Microbacterium arhorescens by the

Biolog system and as a Corynebacterium group ANF by the API Coryne system.

Previous studies by Dopfer (as cited in Jones & Collins, 1986) and Cummins (1971)

determined that the cell wall type of this strain contained peptidoglycan based upon

diaminobutyric acid rather than the diaminopimelic acid common to the

corynebacteria. These and other investigators have concluded that this organism may

represent a new taxon. This organism grew well on unsupplemented TSA and 16S

rRNA gene similarity studies indicated a >99% similarity with an unidentified

bacterial 16S rRNA gene. These data concur with the conclusions of previous

investigators (Cummins, 1971; Jones & Collins, 1986) that this organism does not

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n U> O

/Rhodococcus group ANF) (1) (6) Group 1(1) group ANF group ANF (1) spp. (1) Identification (No.) (1) (1) (1) (44) (Corynebacterium (1)

(2) bovis Group I (3) C. C. C. C. diphtheriae Corynebacterium Corynebacterium Corxnebacterium. Low ID C. jeikeium C. C. minutissimum/jeikeium Corynebacterium No ID (2) jeikeium C. (I) No ID (1) C. jeikeiumC. No ID (1) C. C. bovis equi Table 10 spp.) (1) (1) (1) spp. (1) (1) (1) (1) (Corynebacterium (11) Biolog System API Coryne System Identification (No.) (1) (2) (1) (20) (4)

(27) bovis pseudodiphthericum C. C. afermentans C. C. afermentans C. C. amycolatum C. bovis C. C. accolens C. afermentans C. lilium/i’lutamicum C. Corynebacterium muteC. call jeikeium C. C. C. jeikeiumC. CDC Group G (6) (15) (2) (50) Identification ofCoryneform Bacteria Using rRNA Gene Similarity, the Biolog System, and the API Coryne System rRNA Similarity Identification (No.) C. C. amylocolatum C. C. bovis C. C. ammimiagenes C. C. confusion

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U> (1) (1) ) (I Identification (No.) API Coryne System (1) (3) bovis C. No ID (3) C. xerosis C. C. bovis Rhodococcus equi C. C. pseudotuberculosis/ulcerans No ID (1) Rhodococcus equi (I) spp.) (1) (1) (2) No ID (2) ) 1) 1 ( (1) (1) (1) (1) Biolog System (1) spp. Identification (No.) (1) (1) (1) C. C. afermentans C. C. amylocolatum C. runs nice CDC Group E (Actinomyces C. C. ammoniagenes C. diphtheriae jeikeium C. C'aseobacterpolymorpbus Rliodococcus equi ( C. C. afermentans C. bovis Rhodococcus Actinomyces odontolyticus (5) (1) (2) (1)

(4) ) rRNA Similarity Identification (No.) (1 nishiomyaensis C. pseudotuberculosis/ulceransC. C. C. xerosis Coryneform bacteria C. pilosum/vitarumen Table 10-Continued R. R. equi M. M.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 belong to C. bovis. The 16S rDNA of this organism demonstrated the highest degree

of similarity (95%) with Brevibacterium helvolum.

Four strains were identified as Listeria spp. Members of this genus have been

previously isolated from cases of bovine mastitis (as reviewed in Watts, 1988). These

organisms, while constituting a small number of isolates in the current and previous

studies (as reviewed in Watts, 1988), assume a greater importance due to the zoonotic

potential of this organism. Mastitis bacteriology laboratories should be capable of

differentiating Listeria spp. from the corynebacteria so as to properly manage animals

infected with these organisms.

A total of 87 strains were selected for rRNA gene similarity studies. Of these.

50 (75.5%) were identified as C. bovis and all exhibited distinct lipophilism. C. bovis

was the only lipophilicCorynebacterium spp. identified in this study and all strains

were positive for G-galactosidase. The Biolog system identified 27 (54.0%) strains as

C. bovis, 20 (40.0%) as C. jeikeium, 1 (2.0%) as Corynebacterium accolens, 1 (2.0%)

as a Corynebacterium spp., and 1 (2.0%) as a Corynebacterium CDC group G. In

contrast, the API Coryne system identified 44 (88.07c) as C. bovis, 1 (2.07c) as C.

jeikeium, 1 (2.0%) as Corynebacterium group ANF (Corynebacterium afermentans),

and 1 (2.07c) strain was not identified. These data indicate that the majority of

lipophilic Corynebacterium isolated from bovine IMI are C. bovis. However, neither

commercial system provides an acceptable level of identification of this organism.

Corynebacterium amylocolatum was the next most frequently isolated

organism with 15 strains (Table 10). This organism is a non-lipophiiic

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 Corynebacterium that is frequently isolated from the skin of humans (Coyle &

Lipsky, 1990; Funke et al., 1997). Whether this organism is an inhabitant of the skin

of cows or is transferred to cows via the milking process is unknown. The Biolog

system correctly identified 11 (73.3%) strains of this organism while the API Coryne

system failed to identify any of these strains correctly.

Based on rDNA analysis, five strains (strain nos. 4, 31,44, 151, 227) were

identified as C. pseudotuberculosis or C. ulcerans while four were identified as C.

xerosis (strain nos. 5, 6, 14, 144). Previous taxonomic studies (Funke, 1997; Pascual

et al., 1995) have indicated that the 16S rDNA of C. ulcerans shares a high similarity

with the one of C.pseudotuberculosis and should be placed in this species.

Corynebacterium pseudotuberculosis, C. ulcerans, and C. xerosis have been

previously isolated from bovine mastitis (Watts, 1988). The Biolog system failed to

correctly identify any of these strains while the API Coryne system identified only a

single strain of C. pseudotuberculosis and of C. xerosis (Table 10). Three strains of

these five exhibited only 95% rDNA similarity with either C. pseudotuberculosis or

C. ulcerans indicating that they are closely related to these organisms. These strains

could not be differentiated from either C. pseudotuberculosis or C. ulcerans based on

phenotypic characteristics.

The rDNA analysis identified strains 7, 8, 13, 18, 26, and 141 as C.

ammoniagenes, a glutamate producing Corynebacterium species. This organism is

part of the normal skin flora for humans and is not considered to be medically

significant (Funke et al., 1997). Strain 80 demonstrated 95-96% similarity with

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 Corynebacterium vitarumen or Corynebacterium pilosum. Corynebacterium

vitarumen was originally isolated from the rumen of cattle andC. pilosum has been

isolated from the urinary tract and vagina of cattle (Jones & Collins, 1986). While the

latter organism has been isolated from urinary tract infections in cattle (Jones &

Collins, 1986), this is first time either species has been reported as a cause of mastitis.

The low similarity this strain exhibits with either C. vitarumen or C. pilosum indicates

that this strain may represent a new species.

The DNA of two strains exhibited 987e similarity with a coryneform 16S

rDNA gene and were identified only as a Corynebacterium species. Further

taxonomic studies are needed to properly classify these two strains. Two strains were

identified as Rhodococcus equi by both the Biolog and API Coryne systems. Based

on rRNA gene data, one strain was identified as a R. equi (100% similarity) but one

strain exhibited only 96% similarity with Microbacterium nishiomyaensis.

Two new corynebacterial species isolated from ovine mastitis,

Corynebacterium camporealis and Corynebacterium mastitidis, have been recently

described (Fernandez-Garayzabal, 1997; Fernandez-Garayzabal, 1998).

Corynebacterium camporealis does not require lipids while C. mastitidis is a

lipophilic species (Fernandez-Garayzabal, 1997; Fernandez-Garayzabal, 1998). None

of the strains in the current study was identified as either species. This may indicate

that these organisms have a narrow host range limited to sheep. However, this study

represents a limited number of isolates and a larger survey is needed to rule out these

organisms as a cause of bovine mastitis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 These results confirm the first hypothesis of the study. That is,C. hovis, as

identified using current methods is a heterogeneous group of organism comprised of

other Corynebacterium species as well as bacteria belonging to other genera. Neither

of the two commercial systems provides an adequate level of identification for the

coryneform bacteria encountered in this study. However, presumptive identification

o f C. bovis could be performed utilizing Gram reaction, cellular morphology, catalase

production, nitrate reduction, and B-galactosidase production. Direct sequencing of

I6S ribosomal RNA gene is the most accurate method and should be employed in

epidemiological studies.

Antimicrobial Susceptibility of C. bovis and C. amvlocolatum Isolated From Bovine Mammary Glands

No information is available on thein vitro activity of antimicrobial agents

commonly used to treat bovine mastitis against strains of C.hovis. Pankey et al.

(1985) determined that treatment with antimicrobial agents administered at the end of

lactation (referred to as “drying ofF’ a cow) containing either cloxacillin or penicillin

were effective in eliminating experimentally induced C. bovis infections. This

indicated that the strain used was susceptible to (3-lactam antimicrobial agents. Thus,

the purpose of this study was to determine the in vitro activity of antimicrobial agents

used to treat bovine mastitis as well as other agents available in veterinary medicine

using the quantitative MIC method against isolates of C. bovis.

The lack of in vitro antimicrobial susceptibility data may be due to the

difficulty in cultivating this organism as it fails to grow without lipid supplementation

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 of the basal medium (Funke et al., 1997). Previous studies (Funke et al., 1997,

Soriano et al., 1995) determining the antimicrobial susceptibility of lipophilic

corynebacteria isolated from humans such as C. jeikeium have recommended the

addition of rabbit serum or 0.1 % Tween 80. However, the lipid requirements for C.

jeikeium appears to be lower than those of C.bovis as the former organism will grow

on blood supplemented media while C. hovis grows very poorly if at all. Results

from the previous study indicated that acceptable growth of C. bovis could be

achieved by supplementing the basal medium with 1% Tween 80. Thus, we chose to

use Mueller-Hinton broth supplemented with 1% Tween 80 for the test medium.

Although this supplementation has not been recommended by NCCLS, the test results

for the individual antimicrobial agents with the quality control organisms fell within

guidelines published by NCCLS (1999). This indicates that supplementation with

1.0% Tween 80 did not affect the test results of the broth microdilution method.

The MIC results obtained with 46 strains of C. hovis are summarized in Table

11 and Figures 2-16. All of the antimicrobial agents tested except tilmicosin were

active against the strains of C. bovis. Phylogenetic studies (as reviewed in Funke et

al., 1997) have determined that C. jeikeium is the organism most closely related toC.

bovis and the antimicrobial susceptibility of 43 strains of this organism has been

determined by Soriano et al. (1995). In the present study, the type strain o f C.

jeikeium (ATCC 43734) was included. The MIGJ0 for the 6-lactams antimicrobial

agents, ampicillin, oxacillin, cephalothin, and ceftiofur against C.bovis ranged from

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 Table 11

Summary of Minimum Inhibitory Concentrations (MIC) for 46 Strains of Corynebacterium bovis Isolated From Bovine Mammary Glands (all values in pg/ml)

Antimicrobial Summary of MIC data

Agent MIC*, M ICy„ Range Ampicillin 0.25 0.25 <0.06-0.25 Oxacillin 1.0 4.0 0.125-8.0

Cephalothin 0.5 0.5 <0.06-64.0 Ceftiofur 0.125 0.5 <0.06-64.0 Penicilllin+Novobiocin 0.125 0.5 <0.06->64.0 Erythromycin <0.06 <0.06 <0.06->64.0 Tilm icosin 1.0 >32.0 0.5->32.0 Clindamycin 0.125 0.25 0.125-0.5 Pirlimycin 0.125 0.25 <0.06->64.0 Tetracycline 0.25 0.25 0.125->32.0 Florfenicol 1.0 2.0 1,0->32.0

Enrofloxacin 0.125 0.25 <0.03->32.0 Sarafloxacin 0.25 0.5 0.25->32.0 Danofloxacin 0.125 0.25 0.06->32.0

Premafloxacin 0.015 0.015 <0.0078->8.0

0.25 to 4.0 |Jg/ml, whereas the value for C. jeikeium ranged 16.0 to >64.0 pg/m l. In

contrast, Soriano et al. (1995) reported that the MIC™ values for clinical strains of C.

jeikeium against ampicillin, oxacillin, cephalothin, and cefuroxime were >256.0

M g/ml.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44

16 14 12 10 8 6 4 2 0 0.06 0.12; 0.25 0.5 2 4 H 16 32 64

M R/m l

□ C. bovis B C. amylocolatum

Figure 2. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Ampicillin.

20 ■ s s 55 i 10

0.06 0.125 0.25 0.5I 2 4 8 16 32 64

M g /m l

□ C. bovis B C. amylocolatum

Figure 3. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Oxacillin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45

35 32 30

2 25 - haa 20 - ■v: 15 z 10 10 - 5 1 1 0 I l k 0.0ft 0.125 0.25 32 HR/ml

| □ C. hovis I C. amylocolatum 1

Figure 4. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Cephalothin.

20 IK

'S3 15 s ll 1 10 35 10 z 5 l 0 1 0.00 0.125 0.25 0.5 I 2 4 X 10 32 A4 MR/ml

□ C. bovis B C. amylocolatum

Figure 5. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Ceftiofur.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46

20 -I 19

15 14 s n 55 10 e 3 1 :! I H JLL. 0.06 0.125 0.25 0.5 I 2 4 X 16 32 64

MR/ml

□ C. bovis B C. amylocolatum

Figure 6. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against a Combination of Penicillin and Novobiocin.

20 19

14 s» 15 3 i io

3 illli n i 0.06 0.125 0.25 0.5 I 2 4 0 16 32 64

M R/m l

□ C. bovis ■ C. amylocolatum

Figure 7. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Erythromycin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47

0.06 0.125 0.25 0.5 1 2 4 H 16 32 64 Mgfad

□ C bovis M C. amytocolatuin

Figure 8. Distribution of MIC Values for 46 C. hovis and 13 C. amylocolatum Against Tilmicosin.

30 -I 27 25 - s 20 - 1H

X 15 e 10 Z 10 5 o 0.06 0.125 0.25 0.5 I 2 4 0 16 32 64 Mg/ml

□ C. bovis M C. amylocolatum |

Figure 9. Distribution of MIC Values for 46 C. hovis and 13 C. amylocolatum Against Clindamycin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48

35 -I 30

0.06 0.125 0.25 0.5 1 2 It 16 32 644 MR/ml

□ C. bovis B C. amylocolatum

Figure 10. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Pirlimycin.

30 -|

■ T i S un

j a . 0.06 0.125 0.25 0.5 1 4 8 16 32 64

M g /m l

□ C. bovis M C. amylocolatum

Figure 11. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Tetracycline.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49

25 22 n 20 20 S i s I 15 H 55 s io H z 5 H 1 1 0 rzi J m . 0.06 0.125 0.25 0.5 1 2 4 X 16 32 64

M R/m l

□ C. bovis ■ C. amylocolatum

Figure 12. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Florfenicol.

35 32 30 S■Si 25 -

2 2 0 ’j i 15 z 10 5 4 4 5 -

o -1 Ql lit. 0.03 0.06 0.125 0.25 0.5 16 32 64

M R /m l

□ C. bovis M C. amylocolatum

Figure 13. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Enrofloxacin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50

40 35 * 30- 1 25- 53 20 z 10

320.06 0.125 0.25 0.5 2 4 It 16320.06 64

MR/ml

□ C. hovis B C. amylocolatum

Figure 14. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Sarafloxacin.

35 - 30 - J) S

20 -

z 10 -

0.06 0.125 0.25 0.5 2 4 8 16 32 64

M g /m l

□ C. bovis B C. amylocolatum

Figure 15. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Danofloxacin.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51

30 i 25 - 20

53 15

z 10 -

J=L 0.0078 0.015 0.03 0.00 0.125 0.25 0.5 1 4 8 MR/ml

□ C. bovis U C. amylocolatum

Figure 16. Distribution of MIC Values for 46 C. bovis and 13 C. amylocolatum Against Premafloxacin.

The macrolides (erythromycin and tilmicosin) and lincosaminides

(clindamycin and pirlimycin) inhibit protein synthesis in bacterial cells by binding to

the same site on the ribosome (Auckenthaler et al., 1986; Hermans. 1986: Yao &

Moellering, 1995). As a result, resistance to a macrolide often confers resistance to a

lincosaminide and vice versa (Auckenthaler et al., 1986; Hermans, 1986; Yao &

Moellering, 1995). In the present study, the MICyovalues for erythromycin,

tilmicosin, clindamycin, and pirlimycin were <0.06, >32.0, 0.25, and 0.25,

respectively. This is markedly different from the MICW values obtained for C.

jeikium which were >256.0 pg/ml for both erythromycin and clindamycin. The

reason for the decreased activity of tilmicosin is unknown but this compound appears

to be the least active against C. bovis of this group of compounds. Whether this

reduced activity is due to poor penetration ofC. bovis or differences in the ribosomal

binding site is unclear.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 Tetracycline and florfenicol are approved for use in veterinary medicine. An

intrammary infusion product containing tetracycline has been available in the past.

Florfenicol is approved for use in the treatment of bovine respiratory disease but not

mastitis. Both compounds were much more active against strains of C.hovis when

compared to values reported for C.jeikeium by Soriano et al. (1995). For example,

the MICyo for tetracycline was 0.25 pg/ml for C.hovis compared to the 64.0 pg/ml

reported for C. jeikeium (Soriano et al., 1995).

The fluoroquinolones are widely used in human and veterinary medicine to

treat a variety of diseases (Yao & Moellering, 1995). Of these, the second-generation

fluoroquinolones (enrofloxacin, sarafloxacin, and danofloxacin) demonstrated good

activity with MICyovalues ranging from 0.25 to 0.5 pg/ml (Table 11). In contrast, the

expanded-spectrum fluoroquinolone (Watts et al., 1997). premafloxacin was much

more active with an MICW value of 0.015 pg/m l (Table 11). Again, the reported

values for strains ofC. jeikeium tended to be much higher with an MlCw value of

64.0 pg/ml (Soriano et al., 1995).

Corynebacterium amylocolatum is a normal resident of healthy human skin

and is one of the most frequently isolated corynebacterium from this body site (as

reviewed in Funke et al., 1996; Funke et al., 1997). As C. amylocolatum is easily

misidentified as either Corynebacterium striatum or Corynebacterium minutissimum,

it is believed that some of these strains isolated from sick humans were, in fact, C.

amylocolatum (Funke et al., 1996). Funke et al. (1996) determined the antimicrobial

susceptibility of 101C. amylocolatum strains isolated from human clinical sources. In

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 the present study, we tested 13 strains isolated from bovine IMI (Table 12 and

Figures 2-16). In contrast to the bovine strains, the human strains tend to be much

more resistant to antimicrobial agents.

Table 12

Summary of Minimum Inhibitory Concentrations (MIC) for 13 Strains of Corynebacterium amylocolatum Isolated From Bovine Mammary Glands (all values in pg/ml)

Antimicrobial Summary of MIC data

Agent MIC,,, MIG,,, Range Ampicillin 0.125 0.25 <0.06-0.25 Oxacillin 0.5 2.0 0.5-4.0 Cephalothin 0.25 0.5 0.13-0.5 Ceftiofur 0.125 0.5 <0.06-64.0 Penicilllin+Novobiocin <0.06 0.125 <0.06-0.125 Erythromycin <0.06 0.13 <0.06->64.0 Tilmicosin 4.0 32.0 2.0->32.0 Clindamycin 0.25 0.5 0.25-64.0 Pirlimycin 0.25 0.25 0.125->64.0 Tetracycline 0.25 16.0 0.125-32.0 Florfenicol 32.0 32.0 1.0->32.0 Enrofloxacin 0.125 0.25 0.06-0.25 Sarafloxacin 0.25 0.5 0.125->32.0 Danofloxacin 0.125 0.25 0.06-0.5

Premafloxacin 0.015 0.015 <0.0078->8.0

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 For example, the M IQ o values for the human strains with ampicillin, oxacillin,

cephalothin, and ceftriaxone (a third generation cephalosporin) were >64.0 pg/ml for

all these agents (Funke et al., 1996), while the MICyo values for the bovine strains

were 0.25, 2.0, 0.5, 0.5 pg/ml (ceftiofur value), respectively.

Similar results are also seen forC. amylocolatum with the macrolides and

lincosaminides as the MIGm values for the human strains were >64.0 pg/ml for both

erythromycin and clindamycin (Funke et al., 1996) compared to 0.13 and 0.5 pg/ml

for these same agents with the bovine strains (Table 12). Again, the results obtained

for C. amylocolatum for tilmicosin were similar to those observed withC. bovis as

tilmicosin was much less active (M IG o = 32.0 pg/ml) compared to erythromycin,

clindamycin, or pirlimycin (Table 11). Based on these data, tilmicosin would not be

an appropriate compound for treatment of bovine mastitis caused by C. hovis or C.

amylocolatum. Both tetracycline and florfenicol were much less active against the C.

amylocolatum strains tested than against C. bovis (Table 11 and 12). The MIC90

value for ciprofloxacin was also >64.0 for the human strains (Funke et al.. 1996)

compared to 0.25 with enrofloxacin (a ciprofloxacin analogue) for the bovine strains.

The distribution of MIC values for the C. bovis and C. amylocolatum strains

tested are presented in Figures 2-16. While the MIC values for C. bovis tended to be

one to two dilutions higher for most compounds tested, the distributions for the two

species tended to be similar.

The differences in antimicrobial susceptibility between the bovine strains of

C. bovis and C. amylocolatum compared to the similar human species reflect the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55 differences in antimicrobial exposure of the two host species. Infections caused by

Corynebacterium species other than C. diphtheriae in humans such as C. jeikeium are

usually contracted during hospitalization in immunocompromised patients receiving

antimicrobial therapy. In contrast, dairy cattle receive antimicrobial therapy only

because of infection and organisms have limited or no prior exposure to antimicrobial

agents before treatment.

Phylogenetic Studies on C. bovis

No phylogenetic studies have been conducted using strains of

Corynebacterium species derived solely from bovine mastitis. Previous studies

(Pascual et al., 1995; Ruimy el al., 1995) have been limited to comparisons of the

type strain and have only included a single C. bovis reference strain. In the present

study, 50 strains were identified as C. hovis based on similarity values of >98% with

sequences for the type strain. Since no other phylogenetic studies on the

corynebacteria have examined a large number of strains of C. bovis. we examined the

phylogenetic relationships of these strains to the C.bovis type strain and other

members of the C. urealyticiun group (C. urealyticum, C. bovis, C. jeikeium, C.

variabilis). Previous studies by Pascual et al. (1995) and Ruimy et al. (1995)

indicated that while C. bovis was a member of the C. urealyticum group and most

closely related to the lipophilic speciesC. jeikeium, bootstrap values were low

indicating that this relationship was not robust. Initial phylogenetic analyses in the

present study using the neighbor-join method indicated that all strains fell within this

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56 group (bootstrap value = 100.0%; data not shown). For that reason, we limited our

phylogenetic analyses of the test strains to the type strains of theC. urealyticum

group (Figure 17).

All 50 strains identified in the present study as C. bovis demonstrated a high

similarity (>98%) to the type strain sequence (C. hovis NCTC 3224) using BLAST

searches. The rRNA gene sequence for C. bovis NCTC 3224 (EMBL accession no.

X84444) was most closely related to the rRNA gene of the type strain ofC. jeikeium.

These results agree with results of previous studies on the phylogeny of

Corynebacterium species (Fernandez-Garayzabal, 1997; Fernandez-Garayzabal,

1998; Pascual et al., 1995; Ruimy et al., 1995). Interestingly, the 16S rRNA gene

sequences for C. bovis ATCC 7715 (EMBL accession no. D38575) did not fall within

this cluster. These results are interesting as NCTC 3224 and ATCC 7715 are

supposedly the same strain. Since the sequence for these two strains were deposited

by two different investigators working with type strains from two different sources

(Pascual et al., 1995; Takahashi et al., 1994), these data suggest that these two strains

should not be considered synonymous.

The results indicate that the test strains fell into two large clusters (bootstrap

value = 76%). One cluster contained 9 strains (cluster containing strain 65 through

68) while the other cluster was subdivided into two subclusters. However, the

separation of these two clusters was not robust (bootstrap value = 22%).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57

• 5 1 • 4 1 2.34 62 - 225 - 1 72 * * Cb3224 * * Cjk - 34 - 1 50 - 1 36 - Curea *216 •Cb 7715 ■ 89 • 1 01

69 1 85 ■ 52 231 1 39 66 l------67 64 - t - 39 ■ 38 50 55 1 84 70 226 ■114 ■ 96 53 232 — 236 96 i— r■------^ 1 40 1 43 33 ■ c ± 1 1 5 - 32 68 54 - 233 £ 238 - 230 197 1 59 97 65 36

Abbreviations for Type strains: Cb3224, C. bovis NCTC 3224; Cb7715, C. bovis ATCC 7715; Curea, C. urealyticum DSM 7109; Cvar, C. variabilis NCDO 2097

Figure 17. Unrooted Tree Depicting the Phylogenetic Relationships Among Strains of C. bovis and Other Members of the Corynebacterium urealyticum Group. The tree was constructed using the Neighbor- joining method comparing approximately 1360 nucleotides. The bar represents percent sequence divergence.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58 In order to further explore the genetic diversity within the species, similarity scores

for all strains compared to sequence data for the type strain sequences were calculated

using the RDP II database online analysis tools (Table 13). The RDP II database

calculates a similarity coefficient by identifying the number of unique oligomers that

match known organisms and dividing this value by the lowest number of unique

oligomers in either the test or matching organism. This strategy differs from the

algorithm utilized by NCBI as all 50 strains identified as C. bovis had BLAST

similarity values of 98% or higher to theC. bovis type strain. Thus, the RDP II

similarity coefficient values were only used to examine the genetic diversity within

the strains identified as C. hovis using BLAST searches. The RDP II similarity

coefficient values for the strains identified as C. hovis ranged from .883 to .992 when

compared to the C. bovis type strains. These data indicate that significant genetic

diversity exists among the C. bovis strains tested. Moreover, the similarity coefficient

values for C. bovis ATCC 7715 and C. bovis NCTC 3224 were only .95 I which is

further evidence that these two strains should not be considered synonymous.

The possibility that the individual clusters may represent specific subtypes or

possibly subspecies of C. bovis was examined by comparing API Coryne biocodes

and biochemical data. Based on the large number of shared biocodes between the

clusters or RDP similarity values, the individual clusters do not represent subtypes or

subspecies. These data indicate that C. bovis is a well-defined species but that

considerable phenotypic variation exists within the species.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 59 Table 13

Similarity Coefficients of rRNA Sequences forC. hovis Strains to C. hovis ATCC 7715 and C. hovis NCTC 3224 (Calculated Using the RDP Taxonomy Browser)

Similarity Coefficient: Strain No. C. hovis NCTC 3224 C. hovis ATCC 7715 NCTC 3224 1.0 .951 ATCC 7715 .951 1.0 32 .987 .961 33 .977 .948 34 .988 .963 36 .992 .966 38 .986 .964 39 .978 .960 41 .988 .947 50 .960 .936 51 .992 .966 52 .969 .921 53 .992 .969 54 .989 .969 55 .989 .966 62 .957 .940 64 .992 .969 65 .992 .969 66 .992 .969 67 .976 .963 68 .989 .973 69 .903 .883 70 .915 .908 89 .989 .960 96 .974 .951 97 .992 .969 101 .897 .875 114 .987 .954 1 15 .972 .951 136 .979 .957 139 .983 .969 140 .984 .967 143 .963 .947 150 .938 .947 159 .992 .969 172 .945 .940

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60

Table 13 - Continued

Similarity Coefficient: Strain No. C. bovis NCTC 3224 C. bovis ATCC 7715 184 .978 .966 185 .977 .957 196 .980 .966 197 .992 .969 216 .967 .986 225 .971 .951 226 .992 .969 230 .992 .968 231 .962 .942 232 .992 .969 233 .987 .969 234 .973 .953 236 .989 .966 238 .992 .963

Repetitive elements in bacterial genomes have been described and are a useful

tool for differentiating strains within a species (Koeuth et al., 1995; Versalovic et al..

1994; Tyler et al., 1997). These repetitive sequences include the 33 to 40 bp

repetitive extragenic palindrome (REP) sequences; the 124 to 127 bp enterobacterial

repetitive intergenic consensus (ERIC) sequences, and the 154 bp BOX elements of

Streptococcus pneumoniae (Koeuth et al., 1995; Versalovic et al.. 1994: Tyler et al.,

1997). In the present study, the genetic diversity of C. bovis was determined using

rep-PCR with BOX primers. Results of these studies are presented in Figures 18 and

19.

These data indicate that the same major band patterns were observed for the

C. bovis wild type strains and C. bovis ATCC 7715. C. bovis ATCC 13722 yielded a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61 band pattern different from all the C. bovis strains tested further indicating that this

organism is not aC. bovis. These results conform to those of Sanza-Parra et al.

(1999) as these workers used rep-PCR with ERIC primers as well as ARDRA for

species identification and typing of C. bovis. Sanza-Parra et al. (1999) determined

that the ERIC PCR consistently yielded a species specific band pattern and concluded

that this technique may be useful for identification of C. bovis. A comparative study

using both ERIC and BOX primers is needed to determine which primer is most

accurate for identification of C.bovis.

32 33 34 36 38 39 41 50 51 52 53 54 55 61 62 64

23.1

4.4

2.3 Si'S SSX

1.0 + a.y « •- -*V »

0.6

0.3

Figure 18. BOX-PCR Based Fingerprinting of 16 C. bovis Strains Isolated From Bovine Mammary Glands.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 62 66 68 115 150 230 232 234 238 247

65 67 114 146 226 231 233 236 246

23.1

4.4

2.3

1.0

Legend. 146, contaminated; 246, C. bovis ATCC 13722, 247; C. bovis ATCC 7715

Figure 19. BOX-PCR Based Fingerprinting of 15 C. bovis Strains Isolated From Bovine Mammary Glands and Two Reference C. bovis Strains.

Transfer of C. bovis ATCC 13722 to the Genus Brevibacterium as Brevibacterium neaveae, sp. nov.

The taxonomic status of C.bovis ATCC 13722 has been in question for many

years (Jones & Collins, 1986). This organism, originally isolated from cow manure,

is not lipophilic and contains a diaminobutyric acid based peptidoglycan rather than

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63 the diaminopimelic acid based peptidoglycan considered characteristic of the true

corynebacteria (Jones & Collins, 1986). Previous investigators (as cited in Jones &

Colllins, 1986) have suggested that C. bovis ATCC 13722 is either a plant pathogenic

corynebacteria or belongs in a new taxon entirely. Results of this study are in

agreement with these findings as this strain displayed >99% similarity with a

sequence for a unidentified 16S RNA gene from an organism containing a

diaminobutyric acid cell wall (Uemori, 1999). The highest similarity (95%) of the

16S rRNA gene for this organism with a described species was demonstrated with the

16S rRNA gene of Brevibacterium helvolum. Phylogenetic analyses with strains

representing Brevibacterium, Clavibacter, Rathayibacter, and Curtobacterium

indicated that C. bovis ATCC 13722 was most closely related to members of the

genus Brevibacterium. A more detailed analysis indicated that while both C. bovis

ATCC 13722 and the unidentified 16S RNA sequence deposited by Uemori (1999)

should be placed in the genus Brevibacterium (Bootstrap values = 99% for each

organism), both organisms represent new species in this genus (Figure 20). Based on

these data we propose that C. bovis ATCC 13722 be transferred to the genus

Brevibacterium as Brevibacterium neaveae sp. nov.

Description ofBrevibacterium neaveae sp. nov.

B. neaveae is named after Frank. K. Neave, a British microbiologist involved

in developing the first mastitis control programs. This description is based on the

results of the presents study and data reported by Jones and Collins (1986) and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64 Cummins (1971). Cells are Gram-positive, non-motile, asporogenous bacilli that

exhibit coryneform morphology. Colonies are convex, with an entire margin, round,

smooth,

5 I______

______C. bovis ATCC 13722

______B. linens

______B. otiticlis

______B. casei

B. epidermidis

------B. iodmum

______B. helvolum

______Unidentified bacterium 16S RNA

______B. mcbrellneri

Figure 20. Unrooted Tree Depicting the Phylogenetic Relationship of C.bovis ATCC 13722 and Members of the Genus Brevibacterium. The tree was constructed using the Neighbor-joining method comparing approximately 1360 nucleotides. The bar represents percent sequence divergence.

greyish-white in color, and of butyrous consistency. Good growth at 37° C on

unsupplemented media. Lipid stimulated growth is not exhibited. The organism is

catalase and oxidase positive. Nitrate is reduced to nitrite. The organism does not

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 65 produce pyrazinamidase, pyrrolidonyl arylamidase, alkaline phosphatase, 13-

glucuronidase, G-galactosidase. a-glucosidase, N-acetyl-B-glucosaminidase. Esculin,

urease, and gelatin are not hydrolyzed. Acid in not produced from glucose,

ribose,xylose, lactose, sucrose, and glycogen. D-fructose, D-mannitol, D-mannose,

D-sorbitol, and xylitol are utilized. Peptidoglycan contains diamino-butyric acid

(DAB) or lysine. The cell wall contains rhamnose. The mol7o G+C is 73.7. Isolated

from cow manure. The type strain isB. neaveae ATCC 13722.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV

SUMMARY AND CONCLUSIONS

The results of this study indicate the diversity of coryneform bacteria isolated

from bovine mammary glands. However, the identification of these organisms

appears problematic for most mastitis bacteriology laboratories. Of 183 isolates

tested that had been previously identified as C. bovis, 13.7% were not coryneforms

and only 59% exhibited lipid stimulated growth. Thus, the current criteria utilized by

mastitis bacteriology laboratories are inadequate for identification ofC. bovis and

other coryneform bacteria. Based on the results of this study, the minimum

characteristics needed for identification of this organism include Gram-stain, cell

morphology, catalase production, nitrate reduction, G-galactosidase production, and

lipid stimulated growth on TSA supplemented with 1% Tween 80.

The accuracy of the commercial systems evaluated in this study varied among

the various Corynebacterium species. Of the two systems tested, the Biolog system

employs a larger number of tests and a more sophisticated, statistical based stimulated

growth on TSA supplemented with 1% Tween 80 rather than expensive commercial

systems.

These data confirm the first hypothesis of the study and fulfill the first

objective. That is, C. bovis, as presently identified is a heterogenous group of

organisms and that current methods for identification of C.bovis isolated from bovine

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 67 mammary glands are insufficient. Direct sequencing of the !6S rRNA gene proved

the most accurate system for identification of corynebacteria but remains too costly

and time-consuming for routine use in mastitis bacteriology laboratories at this time.

However, this method should be utilized for studies examining the epidemiology of

coryneforms from bovine sources.

This study represents the first quantitative assessment of the in vitro activity

of various antimicrobial agents against bovine strains ofC. bovis and C.

amylocolatum. Except for tilmicosin, all the agents tested demonstrated good in vitro

activity against these organisms. Based on the results of this study, the antimicrobial

susceptibility of the lipophilic corynebacteria such as C. bovis can be determined

using the broth microdilution method and Mueller-Hinton broth supplemented with

1 % Tween 80. A comparison of the results of this study to published data from

human corynebacteria suggests that comparable corynebacteria from humans have a

much higher level of antimicrobial resistance to a wide variety of antimicrobial

agents. It is most likely that the exposure of human skin flora during antimicrobial

therapy significantly contributes to the higher level of resistance seen in human

strains. Future studies on antimicrobial resistance in human corynebacteria may want

identification method. However, this system accurately identified only 54.0% of the

C. bovis strains tested. The API Coryne system correctly identified 88.0% o f C. bovis

strains. Routine identification of C.bovis strains by diagnostic laboratories can be

most efficiently done with conventional tests such as Gram-stain, cell morphology,

catalase production, nitrate reduction, B-galactosidase production, and lipid to include

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68 bovine strains as a baseline population. These data confirm the second hypothesis of

the study as the antimicrobial susceptibility of coryneform bacteria isolated from

bovine mastitis differs substantially from similar organisms isolated from human

sources.

Past studies on the classification of newCorynebacterium species have

included a single strain of C. bovis for comparison purposes. No phylogenetic studies

have included a large number of C. bovis strains isolated from bovine mammary

glands. Results of phylogenetic analyses on C.bovis in the current study indicate that

while C. bovis is a well defined species within the genus Corynebacterium.

significant diversity exists within the species. However, rep-PCR using BOX

primers yielded similar banding patterns for all the C. bovis strains tested. This

technique may be useful for differentiation of this organims from other corynefonns.

Results of this study confirmed the conclusions of previous studies (Cummins.

1971; Jones & Collins, 1986) which indicated that C. bovis ATCC 13722 was not a C.

bovis. Our results indicate that it should be reclassified as a Brevibacterium species.

This organism has been proposed to be transferred to the genus Brevibacterium as

Brevibacterium neaveae sp. nov.

Thus, the third hypothesis and final two objectives of the study were, at least

in part, fulfilled. There appears to be differences between C. bovis ATCC 7715 and

C. bovis NCTC 3224 as the similarity between deposited 16S rRNA gene sequences

for these reference strains is sufficiently different that these strains should not be

considered synonymous. Additionally, C. bovis ATCC 13722 was confirmed not to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69 be a Corynebacterium and is now proposed as a new species in the genus

Brevibacterium. The results of the 16S rRNA gene sequencing and rep-PCR indicate

that C. bovis is a well defined species within the genus Corynebacterium with only

minor differences observed among the strains tested.

In conclusion, this investigation represents the first study to utilize a

polyphasic approach to the identification, quantitative antimicrobial susceptibility

testing, and 16S rRNA gene sequencing of corynebacteria isolated from bovine

mammary glands. The results indicate that C. bovis is a well defined species within

the genus Corynebacterium. However, current procedures for identification of this

organisms fail to adequately delineate C. bovis from other coryneform bacteria. This

can be corrected by implementing a small number of routine conventional tests for

identification of the C. bovis isolated from bovine mammary glands.

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