AN ABSTRACT OF THE THESIS OF
Jennifer Lenart for the degree of Master of Science in Microbiology presented on
June 28. 2001.
Title: Growth and Development of Tetracycline Resistant Chiamydia suis
Abstract Approved:
Redacted for Privacy
Daniel D. Rockey
Tetracycline is a front line antibiotic for the treatment of chiamydial
infections in both humans and animals, and the emergence of tetracycline resistant
(tetR) Chiamydia is of significant clinical importance. Recently, several tetR
chlamydial strains have been isolated from swine (Sus scrofa) raised in production
facilities in Nebraska. Here, the intracellular development of two C. suis tet'
strains, R19 and R27, is characterized through the use of tissue culture and
immunofluorescence. The strains grow in tetracycline up to 4 .tg/m1, while a
tetracycline sensitive (tetS) C. suis swine strain (S45) and a C. trachomatis strain of
the human serovar L2 (LGV-434) grow in tetracycline up to 0.1 .tg/m1. Although
inclusions form in the presence of tetracycline, many contain large aberrant RBs
that do not differentiate into infectious EBs. The percentage of inclusions
containing typical developmental forms decreases with increasing tetracycline
concentrations, and at 3 pg/mI of tetracycline, 100% of inclusions contain aberrant
RBs. However, upon removal of the tetracycline, the aberrant RBs revert to typical
RBs and a productive developmental cycle resumes. In addition, inclusions were
found that contained both C. suis R19 and C. trachomatis L2 after sequential
infection, demonstrating that two biologically distinct chiamydial strains could both
develop within a single inclusion.
© Copyright by Jennifer Lenart
June 28, 2001
All Rights Reserved
Growth and Development of Tetracycline Resistant
Jennifer Lenart
A THESIS
submitted to
Oregon State University
In partial fulfillment
of the requirements for the
degree of
Master of Science
Presented June 28, 2001
Commencement June 2002
Chiamydia suis
Master of Science thesis of Jennifer Lenart presented on June 28. 2001
APPROVED:
Redacted for Privacy
or Professor, represeiting Mifrobiology
Redacted for Privacy
of the Department of
Redacted for Privacy
Dean of the
raluate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to
any reader upon request.
Redacted for Privacy
Author
ACKNOWLEDGMENT
There are many people I wish to thank for helping me through the last
several years. First and foremost, I would like to thank my major professor, Dan
Rockey. Thank you for all the guidance, both scientific and personal, and for
always having faith in my ability. Thank you also to my conunittee members, JoAnn Leong, Dennis Hruby, and my graduate representative Mary Slabaugh, for all
your help and time. Thank you to Dr. Arthur Andersen, who generously provided
the chiamydial strains for me to study. I would also like to recognize the Eckelman
foundation for funding my degree. I owe a big thank you to all my lab mates over
the years, Wendy Brown, Theresa Scholz, Ryan Griffiths, Wasna Viratyosin,
Jennie Mayo, Eric Werth and Rob Blouch, who helped me in more ways then they
can imagine. I want to thank my friends, Fabiane Carter, Melissa Foley, Ruth
Milston, Shaun Clements, Chris Whipps, Kris Spinning and Grant Barnes for
helping me stay sane. Last but not least, I would like to thank all my family, Mom
and Chase, Dad and Lauren, Chris, Carrie, Will, Rob, and my grandparents for all
their love and support.
TABLE OF CONTENTS
Page
I.
INTRODUCTION
II. LITERATURE REVIEW
1
5
Chiamydial Diseases
5
Chiamydial Developmental Cycle
7
Recurrent and Chronic Chiamydial Infections
11
Treatment of Chiamydial Infections
12
Chlamydial Antimicrobial Resistance
13
Tetracycline and Its Mode of Action
14
Uptake of Tetracycline
19
Tetracycline Resistance
19
Resistance Mechanisms and Nomenclature
20
Regulation of Tetracycline Resistance Genes
23
Transfer of Tetracycline Resistance
25
Antibiotic Use in Livestock
25
III. MATERIALS AND METHODS
29
Cell Culture
29
Chlamydial Infections
29
Production of Infectious Chlamydiae in the Presence of Tetracycline
30
Protein Radiolabeling
31
TABLE OF CONTENTS, Continued
Page
Chiamydia trachomatis L2 and Chiamydia suis R19 Fusion Experiments
32
Immunofluorescence Studies
32
C6-NBD-Cer Labeling
33
Induction of Tetracycline Resistance
34
Delayed Tetracycline Addition
34
Production of Peptide Antibody Against R19 MOMP
35
Enumeration of Infected Cells
35
IV. RESULTS
37
Chlamydial Resistance Patterns
37
Quantitative and Qualitative Analysis of Chlamydial Development
39
Aberrant RB Reversion to EB Upon Tetracycline Removal
42
Protein Synthesis in the Presence of Tetracycline
45
R19 and L2 Development Within a Single Inclusion
45
C6-NBD-Cer Trafficking
47
Induction of Tetracycline Resistance
47
Delayed Tetracycline Addition
50
V. DISCUSSION
BIBLIOGRAPHY
54
60
LIST OF FIGURES
Page
Figure
1
Mode of action of typical tetracyclines
18
2
Tetracycline resistance patterns of several strains
38
3
Temporal analysis of EB production
40
4
Decreased growth of R19 and the effects of tetracycline
41
5
Inclusion morphology
43
6
Production of EBs upon removal of tetracycline
44
7
Overall protein production in response to tetracycline
46
8
L2 and R19 growth within a single inclusion
48
9
C6-NBD-Cer trafficking to the R19 inclusion
49
10
Induction of tetracycline resistance
51
11
Delayed tetracycline addition
52
LIST OF TABLES
Page
Table
Tetracycline and its derivatives
15
Antibiotics approved by the FDA for use in livestock
27
Growth and Development of Tetracycline Resistant Chiamydia suis
CHAPTER I
INTRODUCTION
The chlamydiae are important pathogens that cause a wide variety of
significant diseases in both humans and animals. In humans, Chiamydia
trachomatis is the causative agent of trachoma, the leading cause of preventable
blindness worldwide, as well as the cause of the most commonly reported sexually
transmitted bacterial infection (26, 93). Chiamydia pneumoniae causes pneumonia
and has recently been linked to atherosclerosis (56). Several chlamydial species
have been recognized as important veterinary pathogens that cause diverse clinical
syndromes. Chlamydiae have been isolated from swine (Sus scrofa), these strains
being associated with pneumonia, enteritis, conjunctivitis, pericarditis, perinatal
mortality, and reproductive disorders. The majority of these swine strains are
believed to be C. trachomatis-like organisms, and have recently been reclassified as
C. suis (29). Chiamydia pecorum and C. abortus have also been identified in
swine. The two resistant strains examined here are very similar to C. trachomatis
and have only recently been reclassified as C. suis (18, 29, 81). Although these
strains are referred to using their new species name, all other strains mentioned in
2
this paper are classified under the old classification system which recognizes four
species: trachomatis, psittaci, pneumoniae and pecorum.
Chlamydiae are obligate intracellular pathogens that develop within a
nonacidified vacuole termed an inclusion. Within the inclusion, chlamydiae
undergo a unique biphasic developmental cycle that consists of two functionally
and morphologically distinct developmental forms. Elementary bodies (BBs) are
infectious and are involved in extracellular survival and transmission. Shortly after
entry, EBs differentiate into noninfectious reticulate bodies (RBs), which are
metabolically active and divide by binary fission. After several rounds of
replication, RBs redifferentiate back into EBs, the cells lyse, and new infections
can result.
As early as 1950, RBs were detected that existed in an altered, aberrant state
(104). Since then, persistent chiamydial infections have been established in
numerous cell culture systems using a variety of strains. In these infections, the
chlamydiae deviate from the normal developmental cycle, remaining viable but
persisting in a nonproductive state of growth. Aberrancy can be induced by
exposure to antimicrobial agents such as penicillin, immunological agents such as
interferon-gamma, or through nutrient deprivation. These conditions generally
delay maturation of the RB and block RB-to-EB transitions. It has been proposed
that chlamydiae exploit this aberrant growth stage to persist in clinical infections
and possibly exacerbate the disease process (13).
3
Uncomplicated acute chiamydial infections are generally cured with proper
antibiotics, although the ability to effectiyely treat chronic or persistent infections is
not well understood (27). However, acute chlamydial infections are often
asymptomatic and escape detection and treatment. This is thought to lead to severe
complications such as pelvic inflammatory disease, salpingitis and ectopic
pregnancy in humans (49), and wasting syndrome and abortions in animals (20,
89). Many antibiotics are effective in treating chlamydial infections. But, in
practice, chlamydial infections are primarily treated with tetracycline or a
tetracycline derivative. The Center for Disease Control (CDC) recommends treating
individuals with either a seven day course of doxycycline (a tetracycline derivative)
or a single dose of azithromycin (3). Livestock infections are most commonly
treated with tetracycline. In addition, livestock feed has been supplemented with
tetracycline for the past 50 years to ward off infections and promote growth (37).
However, the introduction of antibiotics into animal feeds has encouraged the
selection of resistant organisms (92).
The emergence of tetracycline resistant (tetR ) chlamydiae, in both the
human or animal population, is of great concern. There are nine documented cases
of human C. trachomatis isolates recovered in clinical settings that exhibited
resistance to tetracycline or a tetracycline derivative. In 1990, Jones et al. (53)
collected five isolates that were resistant to multiple antibiotics including
tetracycline, doxycycline, erythromycin, sulfamethoxazole, and clindamycin. A
second human tetracyclineR C. trachomatis isolate was recovered in France in 1997
'ii
(59). This isolate was resistant to tetracycline but sensitive to all other antibiotics
tested including azithromycin, erythromycin, ofloxacin and pristinamycin.
Recently, Somani et al. (90) reported the isolation of three urogenital isolates that
were resistant to doxycycline, azithromycin, and ofloxacin. The mechanisms
responsible for these resistant phenotypes are not known. All of the above tetR
chlamydia were documented after treatment failure or suspected treatment failure
with tetracycline and most isolates were shown to lose their resistance properties
after several passages in tetracycline-free media (53, 59).
Eight tet' C. suis strains were isolated from swine that resided on farms in
either Nebraska or Iowa (7). Resistance appeared stable, as it was retained after 10
to 15 passages in tetracycline-free media. Six of these eight strains were also
resistant to sulfadiazine. Here we further characterize two of the eight resistant C.
suis swine strains, R19 and R27, initially identified by Andersen and Rogers (7).
These strains were isolated from pigs on a Nebraska farm and exhibit resistance to
both tetracycline and sulfadiazine at concentrations up to 4 pg/mI and 20 pg/mi,
respectively. Although inclusions formed in the presence of tetracycline, they often
contained large aberrant RBs that retained the ability to differentiate into EBs upon
removal of the antibiotic. These strains traffick the fluorescent lipid C6-NBD-Cer,
and can develop within an inclusion occupied by a strain of the human C.
trachomatis serovar L2.
hi
CHAPTER II
LITERATURE REVIEW
Chiamydial Diseases
The chlamydiae are important pathogens of humans and animals causing a
wide variety of significant diseases. There are four species within the genus
Chiamydia, although there has been a recent proposal to amend the classification
system to include nine distinct species based on DNA sequence analysis (29).
Chiamydia trachomatis is composed of two biovars that are transmitted human-tohuman, often through sexual contact. They include the biovar trachoma (14
serovars) and the biovar lymphogranuloma venereum (LGV, 4 serovars). All 18
serovars can cause trachoma, sexually transmitted infections, forms of arthritis,
neonatal inclusion conjunctivitis and pneumonia (86). Trachoma is the leading
cause of preventable blindness worldwide, with approximately 400 million people
infected, of whom 6 million people are blinded by the disease (100). Chiamydia
trachomatis is the most commonly reported bacterial sexually transmitted infection
in the United States, and untreated infections can lead to severe complications such
as salpingitis, ectopic pregnancy, epididymitis, and sterility (49). An estimated 90
million new cases occur every year worldwide (2), 4 million of these in the United
States (1). The annual costs of chlamydial infections and their consequences in the
United States alone are more than $2 billion (5). There are also C. trachomatis
strains that infect animals, namely mice and swine. In swine, these infections have
been linked to conjunctivitis and abortions (81, 99).
Chiamydia pneumoniae is also a major human pathogen. Studies show that
more than 60% of adults worldwide have antibodies against this organism,
indicating exposure. Chiamydia pneumoniae is an important cause of respiratory
disease in young adults and can lead to severe complications in older
immunocompromised individuals (85). Respiratory disease ranges from pharyngitis
and otitis media to bronchitis and pneumonia. Approximately 12 out of every 1,000
cases of pneumonia are caused by C. pneumoniae (35). Chiamydia pneumoniae
infection can also be involved in pharynitis, sarcoidosis, pericarditis, endocarditis,
sinistis, bronchitis and myocarditis (35, 36). Chiamydia pneumoniae infection has
recently been linked to athlerosclerosis, although this association remains to be
characterized (56). Coronary specimens of 90 heart surgery patients were
examined for presence of chlamydia, and 79% of experimental tissue showed
evidence of C. pneumoniae infection, as compared to 4% in control tissues (71). It
is not clear if the chlamydial organisms are merely taking advantage of the
damaged tissue as a niche to colonize, or if they are mediating the tissue damage
and eliciting autoimmune disease (25, 36). Although primarily a disease of humans,
C. pneumoniae has also been shown to cause severe disease in some animal
populations, including horses and koalas (86).
7
Chiamydia psittaci is primarily a disease of animals, namely psittacine birds
(parrots). Other avian species are commonly infected, including pigeons and
turkeys, and as many as 130 species are known to be sources (68). In addition, C.
psittaci infections occur in numerous other animal populations, including rodents,
lizards, sheep, swine, and cows. Human infections do occur, in the form of a rare,
yet serious, pneumonia termed psittacosis. Humans that contract C. psittaci
generally work with birds or care for them as pets, and infection is acquired by the
respiratory route. Complications do occur and include an enlarged spleen,
meningitis, myocarditis, and possibly endocarditis (86).
Chiamydia pecorum is a new species that infects only animals and is not
well characterized. It was originally classified as a C. psittaci strain, but Fukushi
and Hirai (33) separated it as the causative agent of abortions, polyarthritis and
conjunctivitis in sheep. Since then, C. pecorum infections have been recognized in
bovine, ovine and swine species (34).
Chiamydial Developmental Cycle
The chlamydiae are obligate intracellular pathogens that reside inside a nonacidified vacuole termed the inclusion. Within the inclusion, chlamydiae undergo a
unique biphasic developmental cycle that consists of two functionally and
morphologically distinct developmental forms. Elementary bodies (EBs) are
infectious and are involved in extracellular survival and transmission. EBs are
approximately 0.3 ..zm in diameter and display no measurable metabolic activity.
Within two hours of entry, EBs differentiate into noninfectious reticulate bodies
(RBs), which are metabolically active and divide by binary fission. RBs are larger,
about 1 jim in diameter, and begin to accumulate 8-12 h postinfection. As the
numbers of RBs increase, the inclusion membrane expands to accommodate the
multiplying chlamydiae. After about 18 h, depending on the strain, RBs
asynchronously redifferentiate back into EBs. The host cells lyse anywhere from
36-72 h postinfection, leading to infection of neighboring cells (39).
The chlamydiae only grow within the inclusion, inside of eukaryotic cells.
The inclusion provides an environment in contact with the nutrient rich host
cytoplasm, where the chlamydiae have access to all intermediates of metabolism.
Thus, unlike other bacteria, chlamydiae do not have to synthesize all these products
themselves. This is believed to have allowed for a reduction in overall genome size.
Hatch et al. (44, 45) have shown that chlamydiae are capable of limited DNA,
RNA and protein synthesis in vitro as long as they are provided with the
appropriate building blocks. The chlamydiae are auxotrophic for three of the four
ribonucleotides: ATP, GTP and UTP (66). Chlamydia obtain amino acids (43),
nucleotides (44, 66) and lipids (42, 105) from host cells. However, chlamydia
contain all the machinery necessary for transcription and translation including
polymerases, ribosomes, tRNAs, and regulatory factors (96).
Chlamydiae face many challenges in order to productively infect cells and
propagate. First, the EB must attach and enter the eukaryotic cell. EBs have a very
high affinity for eukaryotic cells, as they are internalized 10-100 times more
efficiently than are latex beads or Esherichia coli (38). This is believed to occur
through receptor mediated endocytosis, although the receptors and ligands involved
have yet to be identified. In fact, the process is so efficient that it has been termed
"parasite-specified endocytosis" to demonstrate the belief that EBs trigger their
own uptake by cells that are not professional phagocytes (19). The chlamydiae, in
general, invade at mucosal surfaces and exhibit host range preferences. However,
in vitro, many different strains have been shown to productively infect numerous
cell types and hosts. For example, C. trachomatis L2, a strain initially cultured
from a human, has been shown to productively infect Chinese hamster ovary cells
(CHO), mouse fibroblast cells (McCoy), rat kangaroo cells (Ptk2), and fish cells
(EPC) (Daniel Rockey, personal communication).
Once internalization has occurred, the chlamydiae must avoid the host
cellular defenses, namely the lysosomal pathway. There are three main strategies
employed by organisms to avoid lysosomal destruction. Some organisms, such as
Rickettsia and Listeria, are taken into the endocytic vesicle but then escape and
replicate within the cytoplasm. Other organisms, such as Coxiella, have adapted to
replicate and survive within the harsh acidic environment of the lysosome (31). The
chlamydiae, like many pathogens, use a third strategy, living in a vacuole that
avoids fusion with the lysosome completely (28). The events leading to the evasion
10
of lysosomal fusion are not clear. However, most pathogens use varying
mechanisms to achieve this end result, as evidenced by markers found within the
vacuolar membranes (31).
Once the chlamydiae avoid degradation by the phagosome-lysosome fusion,
they must create a unique intracellular niche that the host will not attack, where
they can replicate. This is accomplished by existing in a parasitophorous vacuole
termed the inclusion. The inclusion is not acidified and contains no lysosomal
markers (57) nor does it have the ability to fuse with secondary lysosomes (106).
The presence of C6-NBD-ceramide, a marker for the Golgi apparatus, in the
inclusion indicates that the inclusion interacts with the host cell vesicular
trafficking pathway (41). The inclusion membrane contains chiamydial specific
proteins termed inclusion membrane proteins, or Incs (10). Incs have been
identified in all chlamydial genomes sequenced (80). Although the function of
these Inc proteins has not been elucidated, they exist at the interface between the
inclusion and the host cell cytoplasm, which suggests they may mediate an
interaction between the organisms and the host cell.
Chlamydia can deviate from the typical developmental cycle when growth
conditions are stressed. As early as 1950, RBs were detected that existed in an
altered, aberrant state (104). In these infections, the chlamydiae diverge from the
normal developmental cycle, remaining viable but persisting in a nonproductive
state of growth. It has been proposed that chlamydiae exploit this aberrant growth
stage to persist in clinical infections and possibly exacerbate the disease process.
11
Aberrant RBs mediate persistent infections because the organisms are not cleared
during standard antibiotic therapy (13). Once the antibiotic is removed, the RBs
may return to their typical developmental form and start a new infectious cycle.
Aberrancy may be induced, in vitro, by exposure to a variety of factors. These
include exposure to antimicrobial agents such as penicillin, immunological agents
such as interferon-gamma (12), or through nutrient deprivation (24). These
conditions generally delay maturation of the RB and block RB-to-EB transitions
(103).
Recurrent and Chronic Chlamydial Infections
As stated previously, serious sequalae result from untreated chlamydial
infections. If not cured, these can include sterility or blindness. One of the main
problems in effectively eliminating patients of chlamydia is the high frequency of
reinfection. Commonly, reinfection is due to reexposure among sexually active
populations. However, the high rate of recurrent infection may also be due to
persistent infections that fail to be cleared. These infections are believed to lead to
chronic human disease. Chiamydia pneumoniae is currently associated with several
chronic diseases including coronary artery disease, multiple sclerosis, Alzheimer's
disease, diabetes, lung cancer and stroke. Chiamydia trachomatis has recently been
associated with cervical cancer (103). With reinfection and chronic infections on
12
the rise, it is becoming increasingly important to make rapid, accurate diagnoses of
chiamydial infections, and to treat them effectively until completely eliminated.
Treatment of Chiamydial Infections
Historically, chiamydial infections are easily treated. However, as antibiotic
resistance becomes an increasing problem, the antibiotics available for use in a
clinical setting are reduced. Although chlan-iydiae are sensitive to the penicillin
family of antibiotics in vitro, these drugs are not used to treat clinical infections.
Therapeutically, penicillin does not clear a chiamydial infection, and may actually
exacerbate the disease (14). This is likely due to the fact mentioned above;
penicillin induces aberrant forms of chlamydiae that revert to wild type organisms
upon removal of the drug. According to the 1998 CDC guidelines, uncomplicated
C. trachomatis infections should be treated with either azithromycin (1 g orally in a
single dose) or doxycycline (100 mg orally twice a day for 7 days) (3). Clinical
trials indicate that either treatment is equally efficacious, although the single dose
of azithromycin should be used whenever patient compliance is in question.
Follow-up tests are generally not required, unless symptoms persist or re-exposure
is documented. For the treatment of pregnant women, erythromycin is
recommended.
13
Chiamydial Antimicrobial Resistance
There are increasing reports of clinical persistent infections (98). Antibiotic
resistance has been steadily increasing over the past decade. More organisms are
becoming resistant, and the spectrum of resistance is also increasing. Single
organisms are commonly becoming resistant to multiple antibiotics. In this age of
increasing resistance, it is becoming more probable that clinical persistent
infections are due to resistant strains. Chiamydia trachomatis has historically been
very sensitive to tetracyclines, macrolides, and fluoroquinolones. However, there
have been increasing reports of resistance, especially to tetracycline and
azithromycin.
There are currently nine human C. trachomatis isolates that have been
reported to be resistant to tetracycline. In 1990, Jones et al. reported the first
resistant C. trachomatis isolates from five patients, one of which exhibited
resistance to tetracycline between 4 and 8 ig/ml (53). These isolates were resistant
to multiple antibiotics including doxycycline, erythromycin, sulfamethoxazole, and
clindamycin, but they remained sensitive to rifampin, ciprofloxacin and ofloxacin.
A second report was published in 1998 by Lefévre et al. (59) and included a C.
trachomatis strain isolated in France that was resistant to tetracycline at a
concentration of 64 tg/ml, while control strains were sensitive at less than 0.25
jig/ml of tetracycline. Finally, Somani et al. (90) reported three urogenital isolates
that exhibited resistance to doxycycline, azithromycin and ofloxacin. Hence, there
14
are several independent reports of resistant C. trachomatis, and the existence of
strains with resistance to multiple antibiotics leads to the possibility of a strain
emerging that could not be treated clinically. There have been no reports of
resistant C. pneumoniae, but there has not been extensive testing as of yet, and the
possibility of resistance developing in this species is a concern (94). Without a
promising vaccine for chlamydia, antimicrobial resistance will undoubtedly be an
increasing important problem.
Tetracycline and Its Mode of Action
The tetracycline family of antibiotics contains at least eight derivatives
(Table 1) (62), the first of which, chlortetracycline, was discovered in 1947 (77).
Tetracyclines have been widely used since the 1950's, and for several decades
tetracyclines were the most prescribed antibiotics in the United States to treat both
humans and animals. Tetracyclines are second only to penicillins in the tons used
each year throughout the world (77). They are used for very broad purposes
including the treatment of infections with common pathogens, prevention of
periodontal disease, the treatment of acne, and are used in the livestock and fish
farming industry to ward off infections and promote growth (60, 77). There are
several reasons that the tetracyclines are considered an ideal pharmacological drug.
They are active against most common pathogens, both intracellular and
15
retracycne
.
Chovtet,acrdne
Uinocyde
o CH,
N(CH
OH
NCH
OH
Doxycychne
0
OH
CH, OH
0
N(CH),
çNH.
OH
0
0
04,
OH
0
Anhyd'oteracydne
Anhydsochoc
tetracycline
PC
II1I(I)CONI4,
N(CHj
6- TPxatetracyclne
o
0
ci
CH,
OH
0
-..
)....
Chetocardin
4-Ep.iahydrOchlO
tetracycline
L. .-L
N(CH}1
COHHx
Table 1: Tetracycline and its derivatives. Structures were reprinted with
permission from the American Society of Microbiology.
16
extracellular, and both gram-positive and gram-negative. They also have few side
effects, show good oral absorption, cause few allergic reactions, and are relatively
inexpensive (69, 87). These properties have allowed widespread use of these
antibiotics to treat clinical infections in humans and animals. As with any product,
there are also some limitations to the utility of tetracyclines. The tetracycline
derivatives that are widely used have a mode of action that is bacteriostatic, not
bactericidal. This is not optimal, as some of the organisms may be able to
reestablish growth once treatment is ceased. Tetracyclines also hinder skeletal
growth and thus may not be used by pregnant women or small children (95). In
addition, multiple doses of the antibiotic are generally needed, so patient
compliance may be a problem (92). However, the advantages far outweigh the
limitations, and thus tetracyclines continue to be widely used clinically.
Tetracyclines have additional non-antibacterial properties. They are antiinflammatory as well as immunosuppresive (52). They have been shown to
suppress antibody production in lymphocytes, reduce phagocytic function, reduce
leukocyte and neutrophil chemotaxis, inhibit lipase and collagenase activity, and
act as a sclerosing agent (77). Interestingly, Hatsu et al. (46) have discovered a new
tetracycline that has anti-tumor activity. These properties have encouraged the use
of tetracycline for non-infectious conditions such as arthritis and recurrent thyroid
cysts (77). Using tetracyclines for these conditions should be kept to a minimum,
however, because the normal flora is continually exposed to the drug, which
encourages antibiotic resistance.
17
Typical tetracyclines, which include tetracycline, chiortetracycline,
minocycline, and doxycycline, reversibly inhibit protein synthesis. They bind
mainly to the S7 ribosomal protein on the 30S ribosomal subunit, and also interact
wealdy with other sites on both subunits (77). Once bound, they prevent the
attachment of aminoacyl-tRNAs (Figure 1). The precise mechanism of inhibition
remains unclear. It is possible that the binding of the tetracyclines causes a
conformational change in the ribosome, which causes the tRNA binding site to
become unavailable. It is also thought that binding of the tetracycline to the
ribosome may alter its chemical reactivity and cause it to not function properly.
The second set of tetracyclines, termed atypical tetracyclines, include
anhydrotetracycline, anhydochiortetracycline, and 6-thiatetracycline. These
derivatives appear to be bactericidal and act through interfering with membrane
permeability, causing cell damage and lysis. The function of atypical tetracyclines
was shown through studies that measured -galactosidase release from the
cytoplasm upon exposure to the tetracyclines (75). These derivatives do not appear
to inhibit protein synthesis through binding of the ribosome (77). These atypical
tetracyclines are not used therapeutically, however, because they do not selectively
affect prokaryotic cell membranes and thus have severe toxic side effects on the
host (87).
Ribosorne in the absence of tetracycline
mR1A
Ribosonie in the presence of tetracycline
Figure 1: Mode of action of typical tetracyclines.
19
Uptake of Tetracycline
Tetracyclines enter a bacterial cell by passive diffusion through hydrophilic
pores in the outermost membrane. Passage through the inner cytoplasmic
membrane is an energy-dependent active transport process (77). The passive
diffusion can be explained by binding to cell components such as proteins or
phospholipids (8). The mechanism associated with the energy dependent phase is
controversial, as no carrier protein has been identified (9). A model has been
proposed by Nikaido and Thanassi (72), which explains tetracycline accumulation.
Briefly, in gram-negative organisms, tetracycline is believed to pass through the
outer membrane chelated to a metal cation through the OmpC or OmpF porin. The
cationic metal-tetracycline complex accumulates in the periplasm, where the
complex most likely dissociates yielding free tetracycline. The tetracycline is then
driven through the cytoplasmic membrane by a pH gradient that exists due to the
higher pH and metal concentration in the penplasm.
Tetracycline Resistance
The frequency of antibiotic resistance has continued to increase despite
efforts in recent years to improve awareness and reduce antibiotic use. Many
bacteria are resistant to particular antibiotics, but increasingly, organisms are being
20
discovered that are resistant to many different classes of antibiotics. A particular
resistance genotype is not unique to the organism in which it was discovered, as
resistance genes can be transferred from one organism to another. This transfer can
occur through conjugation between cells, transformation of naked DNA, or through
transduction via viruses. In any case, there is currently an increasing trend of
antibiotic resistance in both scope and severity.
Tetracycline resistance is well documented, and the efficacy of tetracyclines
is limited to only a few clinical infections, as most organisms have emerged with
resistance. The first tetracycline resistant organism, Shigella dysenteriae, was
isolated in 1953, just 6 years after the discovery of the first tetracycline (30).
Research on tetracycline resistant organisms began in the 1950's. Since 1960,
investigators have determined that resistance is primarily due to the passage of
resistant detenninants and not through chromosomal mutations (76, 78). Research
in the past few decades has focused on the specific mechanisms associated with the
resistant phenotype.
Resistance Mechanisms and Nomenclature
There are four main mechanisms of resistance to tetracycline: ribosome
protection, efflux proteins, reduced uptake, and enzyme inactivation (92). The
genes responsible for resistance have been divided into classes based on DNA-
21
DNA hybridization studies. These classes are designated by a capital letter after the
word Tet. When only a single gene or protein is in a class, the class is generally
named after that gene.
Ribosome protection may be the most widespread mechanism of resistance
(83) and is found in Campylobacter, Streptococcus, Enterococcus, Bacteroides and
many other gram-negative and gram-positive organisms. There are nine recognized
classes of determinants responsible for this resistance including Tet M, 0, P, Q, S,
T, W, tet and otrA (61). These determinants encode for a cytoplasmic protein of
approximately 72.5 kDa that is similar in sequence to elongation factors Tu and G
(84, 97). The similarities in sequence suggest that the ribosomal protection proteins
may function either as tetracycline resistant elongation factors themselves, or bind
the ribosome and block binding of tetracycline (77). However, experiments using
radiolabeled tetracycline demonstrated that both resistant and sensitive ribosomes
bound the tetracycline equally well over a range of concentrations. This gives
support to the likely mechanism of the determinants themselves acting as
elongation factors (77, 97). Another supporting piece of evidence for this
mechanism of action includes the demonstration that TetM has GTPase activity
(16). However, Burdett et al. (17) has pointed out that is not certain if the similarity
between the resistance proteins and elongation factors indicates a similar
mechanism of action since EF-Tu and EF-G are not involved in steps in protein
synthesis that are sensitive to tetracycline. In general, ribosome protection does not
confer high levels of tetracycline resistance when expressed in E. coli (15).
22
Efflux proteins are the best studied tetracycline resistance proteins and are
encoded by determinants of 20 classes including Tet A, B, C, D, B, F, G, H, I, J, K,
L(plasmid), L(chromosomal), P, V, Y, Z, otrB, tcr3, and Tet3O (61). Efflux genes
are found in gram-positive and gram-negative genera including Staphylococcus,
Enterococcus, Bacillus, Clostridium, Vibrio, Haemophilus, and Enterobacteriaceae
(92). Each efflux gene encodes for a 46 kDA membrane bound efflux protein (77).
These proteins comprise two structurally symmetrical halves, each with six
transmembrane segments (87). However, some gram-positive organisms contain
proteins with 14 membrane spanning regions instead of 12 (21). Most of the protein
exists in the lipid bilayer with hydrophilic loops protruding into the periplasmic and
cytoplasmic regions (77). The pumps are energy dependent antiporters, which
exchange a proton for a tetracycline-cation complex against a concentration
gradient (107). This reduces the intracellular concentration of tetracycline by
pumping the antibiotic out of the cell and removing its access to the ribosomal
target. Kaneko et al. (54) demonstrated that the energy for this active transport is
derived from the pH gradient across the cytoplasmic membrane, and is not
dependent on the electrical potential. It has also been demonstrated that a metal
cation is required for transport (107). Efflux proteins have been shown to be
inducible (55, 74), and confer high levels of tetracycline resistance.
There is some debate as to whether or not reduced uptake of tetracycline is
truly a distinct mechanism of resistance. However, some gram-negative organisms
have demonstrated this type of resistance (23). Cohen et al. (22) have shown that
23
multiple antibiotic resistance of some E. coli is due to a change in OmpF as well as
other outer membrane proteins. Altering the porins can limit the diffusion of
tetracycline into the periplasm and can decrease susceptibility 6 to 18-fold (92).
Strains that acquire this type of resistance are resistant to multiple antibiotics, in
addition to tetracycline.
The final class of tetracycline resistance involves enzyme modification, and
the resistance is coded for by the class Tet X, found in Bacteroides (92). The gene
product was shown to be transposon encoded, and the resulting protein is a 44 kDa
cytoplasmic protein that chemically modifies tetracycline in the presence of oxygen
and NADPH (91). Sequence analysis has shown that this protein shares amino acid
homology with several NADPH-requiring oxidoreductases (91). The Bacteroides
species in which it was discovered is a strict anaerobe, so the requirement of
oxygen for this mechanism is puzzling. The clinical significance of this resistance
mechanism is unknown, although there is speculation that interaction with
hemoglobin or other oxygen containing molecules could allow for function of this
protein in the human body (92).
Regulation of Tetracycline Resistance Genes
The expression of many tetracycline resistance genes is subjected to
regulation. The best studied regulation system involves the efflux protein of the
24
class Tet B and a repressor, termed Tet R(B). In this system, the repressor is bound
to the operator of both Tet B and Tet R(B) in the absence of tetracycline. This
blocks transcription of both products. The Tet R(B) gene however, has a very high
affinity for tetracycline, so as tetracycline enters the cell, the repressor binds to the
tetracycline instead of to the operators and transcription occurs for both the
tetracycline resistance gene and the repressor. Thus, Tet R(B) functions as an
inhibitor only when there is no longer any tetracycline present in the cell (48).
Gram-positive efflux genes, such as Tet K and Tet L, use the mechanism of
attenuation to regulate the transcription of the resistance genes. In short, the mRNA
transcript contains two ribosome binding sites (RBS 1 and RBS2). In the absence of
tetracycline, the ribosomes first bind RBS I causing a hairpin loop to form, which
hides RBS2 and does not allow ribosomes to bind and therefore translate the
message into protein. When tetracycline is present, the ribosomes tend to stall and a
more stable looped structure forms which does not hide RBS2. Therefore, when
tetracycline is present, the resistance gene can be translated properly (50). A third
mechanism of regulation appears to occur at the level of transcription, but neither a
repressor nor a stem loop structure appear to be involved. Wang and Taylor (102)
showed that a 400bp region directly upstream of the coding sequence of Tet 0 was
needed for full expression of the gene. However, the function of this region has not
been elucidated.
25
Transfer of Tetracycline Resistance
There is evidence for extensive horizontal transfer of tetracycline resistance
genes throughout many bacterial genera based on population surveys of resistance
genes (92). This is not surprising, as the majority of tetracycline determinants are
associated with either conjugative or mobilizable elements (67). The gram-negative
efflux determinants are found on transposons inserted into a wide range of plasmids
(67). The gram-positive efflux genes are associated with mobilizable plasmids (77).
The ribosome protection genes are generally found on conjugative plasmids or in
the chromosome (77). Some determinants such as Tet M and Tet Q are associated
with conjugative chromosomal elements that code for their own transfer
mechanism and often can mobilize other genetic elements such as plasmids (82).
Low levels of tetracycline have been shown to increase the transfer of these
transposons (101).
Antibiotic Use in Livestock
Soon after antibiotics became available for treatment in humans, they were
used by veterinarians in animal populations. This began as early as World War II,
when penicillin was administered to sick dairy animals. Sulfa drugs were also used
at this time. After the war, streptomycin, tetracyclines, and chloramphenical
26
became available in injectible forms to be used by veterinarians. In the early 1950s
however, the use of antibiotics in the livestock industry skyrocketed when
antibiotics were introduced into livestock feed (37). Antibiotics are used for three
main purposes in livestock production: as therapeutics for managing clinically
apparent diseases, as prophylactics at subtherapeutic levels, and as growth
promoters (FDA). The United States Food and Drug Administration now monitors
the use of antibiotics and regulates which ones may be used, but the diversity and
sheer number of antibiotics approved for use is alarming (Table 2).
In the United States, the main antibiotics used in feeds are the tetracyclines
and penicillin. Chlortetracycline and oxytetracycline are commonly used, and in
fact chiortetracycline was the very first additive in 1950. Originally the
tetracyclines were added at low levels, but as it became cheaper to manufacture and
thus purchase these antibiotics, their use increased. The additional growth
promoting benefits of tetracycline addition stimulated further use in the livestock
industry (37). In the 1950's and 1960s, antibiotics were used extensively and
unnecessarily (32). The use of these drugs lead to a decreased emphasis on
husbandry and hygienic practices that had previous controlled infections. Thus,
antibiotics became a part of a balanced approach to control infections in most
species of animals.
There are increasing reports of antibiotic resistant organisms isolated from
livestock. This raises a problem in the human population as well, as these resistant
organisms can be passed to humans through the consumption of meat and milk.
27
Dairy and Beef
Cattle
Hogs
Sheep
Chickens and
Turkeys
Amoxicillin
Ampicillin
Bacitracin
Ceftiofur
Chiortetracycline
Dihydrostreptomycin
Erythromycin
Furamazone
Gentamycin
Lacalocid
Monensin
Neomycin
Oxytetracycline (oral)
Oxytetracycline
(injection)
Penicillin
Streptomycin
Tetracycline
Tilmicosin
Tylosin
Sulfabromomethazine
Sulfachioropyridazine
Sulfadimethoxine
Sulfaethoxypyridazine
Sulfamethazine
Sulfamethoxine
Amoxicillin
Ampicillin
Apramycin
Arsanilate acid
Arsanilic acid
Bacitracin
Bambermycins
Carbadox
Chiortetracycline
Efrotomycin
Erythromycin
Gentamycin
Lincomyciri
Neomycin
Oleandomycin
Oxytetracycline
Penicillin
Roxarsone
Spectinomycin
Streptomycin
Sulfachloropyridazine
Sulfaethoxypyridazine
Sulfamethazine
Sulfathiazone
Tetracycline
Tiamulin
Tylosin
Virginiamycin
Chlortetracycline
Erythromycin
Neomycin
Oxytetracycline
Penicillin
Streptomycin
Bacitracin
Bambermycins
Chiortetracycline
Erythromycin
Fluoroquinolones
Gentamycin
Neomycin
Novobiocin
Oleandomycin
Oxytetracycline
Penicillin
Roxarsone
Spectinomycin
Streptomycin
Tetracycline
Tylosin
Virginiamycin
Table 2: Antibiotics approved by the FDA for use in livestock. Antibiotics,
chemotherapeutics and sulfonamides approved for use by the United States Federal
Drug Administration in 1998. These compounds can be used for growth promotion
and feed efficiency, therapeutic purposes, or both.
The occurrence of antimicrobial resistance is increasing in the human
pathogens Campylobacter and E. coli derived from animals (6). The media has
given much attention to the pathogenic E. coli 0157:H7 that is often found in beef
and can cause serious disease in humans. This particular strain has also exhibited
increased antimicrobial resistance, adding an additional health risk to the human
population. Schwarz et al. (88) demonstrated tetracycline resistance in
Staphylococcus spp. from numerous domestic animals including cattle, cats, dogs,
ducks, guinea pigs, horses, mink, pigeons, pigs, rabbits, and turkeys. Roughly 30%
of the tested organisms exhibited resistance, and 67% of these were resistant to
antibiotics other than tetracycline. This trend has lead to increased concern
regarding consumption of meat and milk products from animals raised on feeds
supplemented with antibiotics.
29
CHAPTER III
MATERIALS AND METHODS
Cell Culture
The human epithelial cell line, HeLa 229 (CCL 2.1; American Type Culture
Collection, Rockville, Md.) was cultured in Minimal Essential Medium (MEM)
supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 10 jig/mI
gentamicin (all reagents from Gibco; Bethesda, MD). All cells were cultured at
37°C in 5%
CO2.
Chlamydial Infections
HeLa cells were grown to 2 x105 cells on sterile 12 mm glass coverslips in 24 well
trays. EBs were diluted in Hank's Balanced Salt Solution (HBSS-Gibco) and cell
monolayers were inoculated. Plates were centrifuged at 750xg for lh at room
temperature (RT). The inocula were removed and replaced with fresh medium, with
or without the indicated concentration of tetracycline (Sigma; St. Louis, MO) and
the infection proceeded for 50 h. At 50 hours post infection (hpi), cells were either
30
fixed for 10 minutes in 100% methanol and stored in phosphate-buffered saline
(PBS), or prepared for other experiments as described below.
Production of Infectious Chlamydiae in the Presence of Tetracycline
Experiments were designed to quantify the number of infectious EBs present in an
infected monolayer of cells. At 50 hpi, infected cells were lysed with 0.5 ml sterile
water for 1 mm on ice. One-half milliliter of HBSS were added and the lysate was
mixed. Three hundred microliters were placed on monolayers of HeLa cells grown
on sterile 12 mm diameter glass coverslips at 2 x105 cells per well, and infections
were performed as described in the previous section. At 50 hpi, cells were fixed in
100% methanol for 10 mm and stored in PBS. To quantify EB production over
time, infections were performed similarly and at appropriate timepoints indicated in
Figure 2, cells were lysed and used to infect new monolayers of HeLa cells. Fifty
hours post infection, cells were fixed for 10 mm in 100% methanol and stored in
31
Protein Radiolabeling
HeLa cells were grown to 1.2 x106 cells per well in 6 well trays. Chiamydia
trachomatis serovar L2 or C. suis strain R19 EBs were diluted in HBSS and added
to the HeLa cell monolayers at a multiplicity of infection (MOl) of 2. Plates were
centrifuged at 750xg for 1 h at RT. The inocula were removed and replaced with
fresh medium, supplemented with emitine at 2 pg/mi, with or without the
appropriate concentration of tetracycline. The infection was allowed to proceed for
24 h. At 24 hpi, 35S-methionine & cysteine (1.25 Ci/mol; Amersham; Piscataway,
NJ) was diluted to 70tCi/ml in methionine- and cysteine-free RPMI 1640 media
(ICN Biomedicals; Aurora, Ohio) supplemented with 0.5% (vol/vol) FBS and 10
.ig/ml gentamicin, and added to each well. Cells were incubated for 5 h, after which
cells were lysed in sample buffer (1% sodium-dodecyl-sulfate, 50 mlvi Tris-HC1 pH
6.8, 1% 2-mercaptoethanol, 10% glycerol), boiled 5 mm, and electrophoresed
through a 12.5% SDS-acrylamide gel. After electrophoresis, the gel was incubated
in 10% acetic acid, 45% methanol for 30 mm, and then equilibrated in 1% glycerol.
Gels were dried and placed onto X-ray film for 4 h at RT.
32
Chiamydia trachomatis L2 and Chiamydia suis R19 Fusion Experiments
HeLa cells were grown to 2 x105 cells on sterile 12 mm glass coverslips in 24 well
trays. Cells were infected with C. suis strain R19 diluted in HBSS at an MOl of 1
and the plate was centrifuged at 750xg for 1 h at RT. The inocula were removed
and replaced with fresh MEM. After a 16 h culture period, cells were infected with
L2 at an MOl of 2 in HBSS. The plate was placed on a rocker platform for 1 h at
RT. The inocula were removed and replaced with fresh MEM. Cells were fixed 46
hours after the initial Ri 9 infection with 100% methanol for 10 minutes and stored
in PBS.
Immunofluorescence Studies
Infected and mock-infected cells fixed with methanol were incubated in 2% bovine
serum albumin (BSA)-PBS at RT for 20 minutes on a rocker platform. This
solution was aspirated from each well, primary antibody was added in 2% BSAPBS, and the plate was rocked for 1 h at RT, followed by three washes with PBS. A
secondary antibody conjugated to the appropriate fluorophore was added in 2%
BSA-PBS and incubated in the dark for 1 h at RT. Cells were washed three times
with PBS and coverslips were inverted onto a drop of Vectashield mounting
medium (Vector Laboratories; Burlingame, Calif.) on a microscope slide.
33
Fluorescent and differential interference contrast (DIC) images were collected and
examined on a Leica DMLB microscope equipped with appropriate fluorescence
filters. Images were collected digitally using a Spot Camera (Diagnostic
Instruments; Sterling Heights, MI) and processed through Adobe Photoshop 5.0
(Adobe software; San Jose, CA).
C6-NBD-Cer Labeling
Chlamydial infections of HeLa cells with R19 were performed as described above.
At 25 hpi, media was removed and cells were washed three times with HBSS.
Three hundred microliters of the fluorescent C6-NBD-Cer (Molecular Probes;
Eugene, OR) complexed with 0.034% defatted bovine serum albumin (dfBSA) in
MEM (42) were added to each well. Infected cells were incubated with the
dfBSAIC6-NBD-Cer complex for 20 mm at 37°C, washed twice with HBSS, and
incubated for 1.5 h in MEM at 37°C in 5% CO2. The coverslips were then mounted
on a microscope slide and examined on a Leica DMLB microscope equipped with
appropriate fluorescence filters.
34
Induction of Tetracycline Resistance
HeLa cells were grown to 2 x105 cells on sterile 12 mm glass coverslips in 24 well
trays. Cells were infected with C. suis strain R19 diluted in HBSS at an MOl of 1
and the plate was centrifuged at 750xg for 1 h at RT. The inocula were removed
and replaced with fresh MEM containing either no tetracycline, or 0.25 .ig/ml of
tetracycline. At 6 hpi, media was removed and replaced with 0.25, 4, 6, 8, or 10
jig/mi of tetracycline for each of the initial tetracycline treatments (0 and 0.25
jig/mi). At 50 hpi, cells were fixed in 100% methanol for 10 mm and stored in PBS.
Cells were labeled with anti-HSP6O antibody as described above and enumerated.
Delayed Tetracycline Addition
HeLa cells were grown to 2 x105 cells on sterile 12 mm glass coverslips in 24 well
trays. Cells were infected with C. suis strain R19 diluted in HBSS at an MOl of 1
and the plate was centrifuged at 750xg for 1 h at RT. The inocula were removed
and replaced with fresh MEM containing 0, 1, 2, 3, 4, 5, 10, or 15 jig/mI of
tetracycline. Duplicate wells received no tetracycline for the first six hours
following infection, at 6 hpi fresh MEM containing 0, 1, 2, 3, 4, 5, 10, or 15 jig/mi
of tetracycline was added. At 50 hpi, infected cells were lysed with 0.5m1 sterile
water for 1 mm on ice. One-half milliliter of HBSS were added and the lysate was
35
mixed. Three hundred microliters were placed on monolayers of HeLa cells grown
on sterile 12 mm diameter glass coverslips at 2
x105
cells per well, and infections
were performed incubated for 50 hours in the absence of tetracycline. At
50
hpi,
cells were fixed in 100% methanol for 10 mm and stored in PBS. Cells were
labeled with anti-HSP6O antibody as described above and enumerated.
Production of Peptide Antibody Against R19 MOMP
Rabbit antisera against a peptide (CGAGKVEDKGSAGELC) present in the strain
R19 major outer membrane protein (MOMP) was produced by Genemed Synthesis,
Inc. (San Francisco, CA). The peptide was linked to keyhole limpet hemocyanin
and administered in Complete Freund's Adjuvant. Three subsequent booster
injections were given with Incomplete Freund's Adjuvant, followed by ELISA
analysis to confirm the specificity and relative concentration of the antibody.
Enumeration of Infected Cells
When appropriate, average inclusion forming units per milliliter (IFUs/ml) were
calculated from triplicate wells. Within each well, 10 fields of view under the 40X
objective of a Leica DMIL inverted microscope were counted and averaged. The
36
surface area for a field of view (FOV) on the 40X objective was calculated as
0.00196cm2. The surface area for the tissue culture tray well was calculated as 2
cm2. Thus, there are 1019 FOV/ well. Therefore, the average number of inclusions
per FOV was multiplied by 1019 to estimate the number of inclusion in the entire
well. This number was then divided by the number of j.il of EBs added to each well
and then multiplied by 1000 to get the number of inclusions per milliliter of EBs.
Triplicate wells were enumerated in this way and the average titer of these three
wells was recorded and standard deviations were calculated.
37
CHAPTER IV
RESULTS
Chiamydial Resistance Patterns
HeLa cells infected with chiamydial strains were cultured in the presence of
varying concentrations of tetracycline for 50 h and examined by fluorescence
microscopy for inclusion development. The C. trachomatis serovar L2 was highly
sensitive to tetracycline, as reported previously (58, 51). Rare inclusions were
observed in extremely low concentrations of tetracycline (0.1 jtg/ml and below),
but development was completely inhibited at 0.25 ig/m1 and above (data not
shown). These results were consistent with the tetS C. suis strain, S45. In contrast to
these results, two C. suis strains, R19 and R27, exhibited approximately forty times
the resistance to tetracycline. Both strains formed inclusions in tetracycline
concentrations up to 4 .ig/m1 and were completely inhibited at 5 g/m1 (Figure 2).
R19 produced 1.8% inclusions in 4 .tg/ml relative to culture in the absence of
tetracycline, while R27 produced 14.4%. Strains R19 and R27 were very similar in
resistance properties and phenotypic characteristics, and all further studies were
conducted using R19.
108
io6
:L2
ios
S45
IFUs/mi
iO4
R19
R27
iO3
02
1
101
100
o
1
2
3
4
Tetracycline Concentration (.tg/m1)
Figure 2: Tetracycline resistance patterns of several strains. Infected cells were
fixed at 50 hpi and inclusion forming units were enumerated using
immunofluorescent microscopy and a monoclonal antibody against HSP6O. The
human C. trachomatis serovar, L2, was sensitive to tetracycline at 0.25 .tgIml.
Similar results were observed with the sensitive C. suis strain, S45. The two
resistant C. suis strains, R19 and R27, formed inclusions in tetracycline at 4
.tg/ml and no growth of R19 and R27 was observed at 5 .tg/mI of tetracycline.
Values are averages of three wells.
39
Quantitative and Qualitative Analysis of Chlamydial Development
Experiments were performed to examine chiamydial growth and the
production of BBs in infected monolayers under varying concentrations of
tetracycline. Mature chlamydial inclusions are heavily laden with typical
developmental forms, including both RBs and EBs. Under these conditions, if the
inclusion is lysed, infectious EBs can be harvested that will productively infect new
cells. An atypical inclusion, however, contains large, aberrant RBs that do not
mature to infectious EBs. Thus, "output experiments" can be used to evaluate the
production of infectious EBs during different culture conditions. In preliminary
output experiments, maximal EB production was shown to occur between 40 and
50 hpi (Figure 3). Experiments were then conducted that compared inclusion
formation and the production of mature, infectious EBs in the presence of
increasing concentrations of tetracycline. Although the numbers of inclusions
formed decreased with increasing concentrations of tetracycline, inclusions
developed up to 4 .ig/ml of tetracycline (Figure 4). The production of EBs is also
inversely proportional to tetracycline concentration, and at higher concentrations of
tetracycline (3 and 4 .ig/ml) no EBs were present in the initial infection, as
inclusions did not form in an output infection. These results indicate that 100% of
the development was aberrant (Figure 4).
These results were confirmed by microscopic analysis. Monoclonal
antibody directed against chlamydial heat shock protein (HSP6O) was used as a
35
2
is
10
20
25
32
40
48
60
72
Hours Post Infection
Figure 3: Temporal analysis of EB production. HeLa cells were infected with
C. suis R19 and EBs were harvested at timepoints indicated. Cells were lysed
and the lysate was used to infect new monolayers of HeLa cells. At 50 hpi, cells
were fixed and inclusion forming units were enumerated using
immunofluorescence with antibodies directed against HSP6O. Values are
averages of three wells and standard deviations are represented with positive
error bars.
41
0
1
2
3
4
Tetracycline Concentration (.ig/ml)
Figure 4: Decreased growth of R19 and the effects of tetracycline. Infected cells
were fixed at 50 hpi and total numbers of inclusions were enumerated using
immunofluorescence with antibodies against HSP6O. These numbers, indicated
with black bars, include both typical and aberrant inclusions. The hatched bars
represent the number of inclusions formed after cells were lysed at 50 hpi and used
to re-infect new cells and cultured in the absence of tetracycline. At 3 and 4 tg/ml,
100% of the RBs were aberrant and no EBs were present. Values are averages of
three wells and standard deviations are represented with positive error bars.
42
probe for fluorescent microscopic analysis of methanol-fixed C. suis S45- and R19infected HeLa cells. Strain S45 inclusions appeared similar to published L2
inclusions (Figure 5A and B). The majority of R19 inclusions were typical when
grown in the absence of tetracycline (Figure 5C and D), although approximately 15% of the inclusions contained aberrant RBs. As the concentration of tetracycline
was increased, an increasing percentage of inclusions appeared smaller,
morphologically irregular, and contained large aberrant RBs (Figure 5E and F).
Aberrant RB Reversion to EB Upon Tetracycline Removal
Previous research has shown that atypical inclusions can revert back to
normal inclusions upon removal of the stressor (13). To test if this would also be
the case upon removal of tetracycline, infections were carried out for 24 h in the
presence of tetracycline, and then the tetracycline was removed and the infections
were allowed to proceed for 26 more hours. At 50 hpi, parallel wells were either
fixed for enumeration of inclusions, or used for output experiments. In agreement
with Figure 3, when C. suis strain R19 is cultured in the presence of tetracycline for
the entire course of infection, inclusions formed at all concentrations although no
EBs were produced at 3 and 4 .tg/m1 of tetracycline (Figure 6A). In contrast, if the
drug was removed after 24 h, and replaced with medium lacking tetracycline,
43
Figure 5: Inclusion morphology. Chiamydia suis strain S45 formed typical
inclusions when grown in the absence of tet, as depicted in panels A and B.
C. suis strain R19 predominately formed typical inclusions when grown in the
absence of tet (panels C and D). These inclusions are heavily laden with
developmental forms. When grown in 2 pg/mi of tet, however, the majority of
the inclusions contained only a few large aberrant RBs (panels E and F). Panels A, C, and E represent differential interference contrast (DIC) micrographs.
Panels B, D, and F are fluorescent images labeled with antibodies against
HSP6O. The scale bar in Panel A represents 5 pm.
1 .OE08
1.OE+08
Al
1 .OE+07
I .OE+07
Cl)
I
1 .OE06
1 .OE+06
1 .OE+05
1 .OE+05
1 .OE+04
I .OE+04
1 .OE-i-03
I .OE03
I .OE+02
I.OE+02
1.OE+O1
1.OE+01
1 .OE+OO
1 .OE+00
0
2
3
4
0
2
3
4
Tetracycline Concentration (ig/m1)
Figure 6: Production of BBs upon removal of tetracycline. Infected cells were fixed at 50 hpi and total numbers of
inclusions were enumerated using immunofluorescence with antibodies against HSP6O. These numbers, indicated with
black bars, include both typical and aberrant inclusions. The hatched bars represent the number of inclusions formed
after cells were lysed at 50 hpi and used to re-infect new cells and cultured in the absence of tetracycline. Panel A
represents EB production when R19 is cultured for 50 h in the concentrations of tetracycline indicated. Panel B
represents EB production when R19 is cultured for the first 24 hours in the concentrations of tetracycline indicated, and
then in no tetracycline for the final 26 hours. Values are averages of three wells and standard deviations are represented
with positive error bars.
infectious EBs were produced in wells previously incubated in 3 and 4 ig/m1 of
tetracycline (Figure 6B).
Protein Synthesis in the Presence of Tetracycline
Chiamydia suis R19 infected HeLa cells were labeled with 35S-methionine
and cysteine to allow for visualization of total protein production. While L2 protein
production is completely inhibited in the presence of 1 .ig/m1 of tetracycline,
labeled chlamydial protein can still be detected during culture of R19 in the
presence of 1 jig/mI (Figure 7). Labeling is not observed at 2 and 3 jig/mi,
however, likely as a result of the decrease in total chlamydial numbers.
R19 and L2 Development Within a Single Inclusion
To test whether C. suis R19 and C. trachomatis L2, two biologically distinct
chiamydial isolates, fuse into one inclusion, HeLa cells were serially infected with
both strains. Cells were first infected with strain R19, and 16 h later, serovar L2
was added. The staggered infections eliminated the possibility that EBs from both
species entered a single cell in the same phagocytic event. After culturing, infected
cells were visualized using monoclonal antibodies specific to each strain's major
80
61
49
36
Figure 7: Overall protein production in response to tetracycline. Overall protein
production was examined through the use of radioactive methionine and cysteine.
Lane 1; uninfected HeLa cell control. Lane 2; L2 infected HeLa cells grown in the
absence of tetracycline. Lane 3; L2 grown in the presence of 0.5 .tg/ml of
tetracycline. Lane 4; L2 grown in the presence of 1 .tg/ml of tetracycline. Lane 5;
S45 infected HeLa cells grown in the absence of tetracycline. Lane 6; S45 grown in
the presence of 0.5 jig/ml of tetracycline. Lane 7; S45 grown in the presence of 1
Jg/ml of tetracycline. Lane 8; R19 infected HeLa cells grown in the absence of
tetracycline. Lanes 9(0.5 tg/ml), 10(1 jig/ml), 11(2 pg/mi), 12(3 pg/ml), 13 (4
pg/mi) and 14 (5 pg/ml) all represent R19 protein synthesis cultured in the
presence of tetracycline. Molecular mass standards are indicated in kDa.
47
outer membrane protein (MOMP). In repeated experiments, single inclusions were
found that contained both L2 and R19 EBs (Figure 8). This indicated that the two
separate vacuoles that initially contained these chlamydiae fused into one inclusion.
C6-NBD-Cer Trafficking
Chiamydia suis
R19 infected HeLa cells were labeled with C6-NBD-Cer, a
vital stain for the Golgi apparatus (63). Uninfected control cells exhibited diffuse
fluorescent staining, which faded within 2 h of removal of the label. The
chlamydiae accumulated the marker and remained fluorescent throughout the
remainder of their developmental cycle (Figure 9). Thus, the strains of C. suis
accumulate this label similarly to that seen with other chlamydiae (42, 79).
Induction of Tetracycline Resistance
Experiments were performed to examine the ability to induce greater levels
of tetracycline resistance after culture of chlamydiae in sub-inhibitory
concentrations of tetracycline for a short amount of time. When grown in the
absence of tetracycline for the first six hours and then challenged with higher drug
concentrations, inclusions were not observed in 6, 8, or 10 tg/ml of tetracycline
C
\
E
-
7G
j,
Figure 8: L2 and R19 growth within a single inclusion. HeLa cells sequentially
infected with C. suisRl9 (T=0 hours) and C. trachomatisL2 (T=16 hours) were
fixed with methanol and labeled with antibodies directed against C. trachomatis L2
and C. suisRl9. Panel A; DIC micrograph of an infected cell containing a single
inclusion 50 h following infection with R19. Panel B; fluorescent image of the cells
in Panel A, labeled with antisera against C. trachomatisL2 MOMP (red) and C.
suisRl9 MOMP (green). Panel C; DIC micrograph of infected cells fixed 32 h
following infection with R19. Panel D; fluorescent image of the cells in Panel C,
labeled with antisera against L2 IncA (red) and R19 MOMP (green). The nuclei of
cells in panel D are labeled blue with 4'6-diamino-2-phenylindole (DAPI; Sigma).
The scale bar in Panel A represents 5 jm for each panel.
Figure 9: C6-NBD-Cer trafficking to the R19 inclusion. HeLa cells infected with C.
suisRl9 were labeled with the fluorescent lipid marker C6-NBD-Cer and analyzed.
Panel A is a DIC micrograph of an infected cell containing two inclusions. Panel B
represents the labeling of the inclusions with the marker, indicating that the R19
chlamydiae traffick the marker from the Golgi apparatus. The scale bar in Panel A
represents 5 jsm.
50
(data not shown). This is in agreement with Figure 3. Similarly, no growth was
observed at 6, 8, or 10 tg/m1 of tetracycline when the cells were cultured in 0.25
ig/ml of tetracycline for the first six hours, indicating that induction of higher
levels of tetracycline resistance did not occur (Figure 10).
Delayed Tetracycline Addition
Experiments were performed to examine chlamydial growth and the production of
EBs in infected monolayers under varying concentrations of tetracycline, at varying
timepoints of tetracycline addition. Chiamydia trachomatis L2 only productively
infected cells in the absence of tetracycline, as expected, regardless of the time of
tetracycline addition (Figure 1 1A and B). When tetracycline was added to C. suis
R19 at 0 hpi, inclusions were seen in wells incubated in 0,1, 2, 3, and 4 tg/ml of
tetracycline, but no growth was observed in the wells incubated in 5, 10, or 15
.tg/ml of tetracycline (Figure 1 1C). When tetracycline was added 6 hpi, similar
growth was observed in the wells incubated in 0, 1, 2, and 3 tg/ml of tetracycline
(Figure 1 1D). However, the well incubated in 4 pg/mi of tetracycline showed a ten
fold increase in chlamydial inclusion forming units. In addition, the well incubated
in 5 .tg/ml of tetracycline showed limited growth when the tetracycline was not
added until 6 hpi. There was no growth in the wells incubated in 10 or 15 pg/mI of
tetracycline (Figure 1 1D). Therefore, delaying the addition of tetracycline until 6
51
Induction of Tetracycline Resistance
1 .OE+06
1.OE+05
1 .OE+04
00
Tet Concentration, jig/mi
(after initial culturing in 0.25 jig/rn! for 6 hours)
Figure 10: Induction of tetracycline resistance. Chiamydia suis infected cells were
cultured in 0.25 ig/ml of tetracycline for six hours, and then cultured in the
concentrations indicated above for 44 more hours. Infected cells were fixed at 50
hpi and total numbers of inclusions were enumerated using immunofluorescence
with antibodies against HSP6O. These numbers, indicated with black bars, include
both typical and aberrant inclusions. At 6, 8, and 10 ig/ml, no inclusions were
present. Values are averages of three wells and standard deviations are represented
with positive error bars.
52
L2 EB production
Tetracycline added at 0 hpi
1 .OE+06
L2 EB production
Tetracycline added at 6 hpi
1.OE+06
B
Al
1 .OE+05
1 .OE+05
E
LL.
1 .OE+04
1 .OE+04
1.OE+03
1 .OE+03
C
C'
"
2
o
2
Tet Concentration (jig/mi)
Tet Concentration (jig/mi)
R19 EB production
Tetracycline added at 0 hpi
1.OE+06
R19 EB production
Tetracycline added at 6 hpi
1 .OE+06
cI
1 .OE+05
1 .OE+05
1 .OE+04
1 .OE+04
1 .OE+03
o
1 .OE+03
r4
cn
"
let Concentration (jig/mi)
2
c
2
let Concentration (jig/mi)
Figure 11: Delayed tetracycline addition. Cells were infected with either C.
trachomatis L2 or C. suisRl9, and exposed to varying concentrations of
tetracycline at either 0 or 6 hpi. At 50 hpi, cells were lysed, used to infect new cells
and cultured for an additional 50 h prior to fixation and enumeration. Panel A
represents L2 growth when tetracycline was added at 0 hpi. Inclusions only formed
when tetracycline was absent. The same is seen in Panel B, when tetracycline was
added to the L2 infections at 6 hpi. This indicates that the time of exposure did not
alter the sensitivity of L2. R19 inclusions were detected at 0, 1, 2, 3, and 4 Jg/m1 of
tetracycline when added at 0 hpi(Panel C), and 0, 1, 2, 3, 4, and 5 ig/m1 of
tetracycline when added 6 hpi (Panel D).
53
hpi resulted in an increase in inclusion forming units at the higher tetracycline
concentrations.
54
CHAPTER V
DISCUSSION
Tetracycline and its derivatives are currently the drugs of choice in treating
chiamydial infections because they are effective, relatively inexpensive, and have
low toxicity (92). Azithromycin is also used in clinical settings, but it is often more
costly (65). For the last several decades, the appearance of antibiotic resistant
organisms has been increasing, and many of the traditional antibiotics used to fight
pathogenic microorganisms are failing in clinical treatments. These data present an
initial characterization of tetR Chiamydia suis. There are currently nine reported tet'
C. trachomatis isolates, and eight C. suis isolates. The strains examined here are
resistant to both tetracycline and sulfadiazine. The emergence of tet' strains
isolated from livestock, raises the issue of antibiotic use in animal feeds (73).
Chlortetracycline was the first feed additive introduced in 1950 and has seen
widespread use over the past 50 years (37). Sulfa drugs were also used widely as
feed additives during the 1970's and early 1980's. With the emergence of several
veterinary pathogens with resistance to these antibiotics, and the associated human
health concerns with the consumption of milk and meats, the use of antibiotics is
now being reduced (4).
55
The swine strains characterized here, R19 and R27, are now classified as C.
suis (29), however they are very similar to C.trachomatis (81), and share several
characteristics. The inclusion morphology of S45 and R19 observed in the absence
of tetracycline is generally similar to L2 inclusions (40). Furthennore, this strain
trafficks a ceramide analog to the inclusion (Figure 9), as previously demonstrated
for other chlamydiae (42, 79). However, there are several traits unique to these
isolates. During culture of these organisms in the absence of tetracycline, large
aberrant forms are observed in approximately one to five percent of the inclusion
population. These forms are identical to those seen with the addition of
tetracycline. In addition, production of chlamydial proteins occurs in the presence
of tetracycline, as observed through radiolabeling and autoradiography (Figure 7).
Furthermore, C. suis R19 did not label with antibodies directed against C.
trachomatis inclusion membrane proteins (data not shown).
Several C. trachomatis isolates have been documented that exhibit
resistance to tetracycline or one of its derivatives (53, 59, 90). Jones et al. (53)
isolated five isolates resistant to tetracycline as well as doxycycline, erythromycin,
sulfamethoxazole, and clindamycin. They report that only a small population
exhibited resistance. This resistant population was not stable, as some isolates lost
resistance and others died upon passage in cell culture. Similarly, Somani et al.
(90) reported three isolates exhibiting multiple drug resistance. These isolates were
resistant to doxycycline, azithromycin, and ofloxacin. Only a small population of
organisms was reported to exhibit this resistance as well. The final isolate reported
56
by Lefevre et al. (59) was resistant to tetracycline and doxycycline, and was
sensitive to azithromycin, erythromycin, ofloxacin, and pristinamycin.
Approximately one percent of the population was resistant, and the isolate was lost
after several passages in tissue culture. C. suis strain R19, conversely, exhibited
stable resistance to tetracycline and sulfadiazine. It retains resistance after 10 to 15
passages in tetracycline-free media (7). In agreement with the other reports, the tetR
R19 strain had at least a hundred-fold decrease in inclusion numbers when exposed
to higher tetracycline levels ( 3 tg/ml).
Inclusions containing the tetracycline resistant swine strains were shown to
contain aberrant RBs that were enlarged and failed to differentiate into EBs when
grown in the presence of tetracycline. These aberrant RBs retain the ability to
differentiate into EBs upon the removal of tetracycline (Figure 6). This provides a
possible mechanism to facilitate persistence in vivo. Chlamydiae have been
previously shown to adapt to periods of stress by existing in a persistent,
nonculturable state. These stressors include nutrient deficiency, treatment with
13-
lactams or other antimicrobial agents, and treatment with cytokines such as
interferon-gamma (13). Persisting aberrant forms may mediate chronic infection
and be associated with exacerbated disease (11, 56, 70). The micrographs in Figure
5 panels E and F and the culture data in Figure 6 demonstrate that the aberrant
forms induced during the culture of strain R19 in higher concentrations of
tetracycline parallels chlamydial development observed in other stressful culture
57
conditions. This study represents the first characterization of tetracycline induced
aberrancy due to resistance.
Sequential infection of C. suis R19 and C. trachomatis serovar L2
demonstrated that inclusions formed by these strains will fuse and thus the
intracellular developmental forms can occupy the same intracellular vacuole
(Figure 8). This creates an environment where the two strains can interact.
Although stable gene transfer has not been documented in chlamydia, the growing
RB within an inclusion provides a likely environment where such exchange could
occur. The existence of chlamydial phage and plasmids allow opportunities for
genetic exchange that parallel common processes in other systems (47, 64).
Tetracycline resistance has been shown to be inducible depending on the
mechanism of resistance employed by the organism. Generally, efflux pumps can
be induced to handle higher tetracycline levels after initial exposure to low levels of
tetracycline (55,74). In addition, genes that are regulated using a repressor system
are generally inducible because low levels of tetracycline release the repressor and
allow transcription. Repeated attempts were made to induce higher levels of
tetracycline resistance in C. suis R19 (Figure 10), but the resistance profile
remained unchanged. It is not clear if the lack of induction was due to experimental
design or the mechanism of resistance. Further experiments are needed to test the
ability of the Ri 9 resistance to be induced to withstand higher levels of
tetracycline.
The level of tetracycline resistance was shown to vary depending on the
hours post infection the tetracycline was added. No change was seen if the
tetracycline was not added until 1, 2, 3, 4, or 5 hpi (data not shown). However,
when the tetracycline was not added until 6 hpi, an increase in inclusion numbers
was seen at both 4 and 5 pg/nil of tetracycline (Figure 11). The significance of this
finding is not clear. EBs are metabolically inactive and thus genes needed for
resistance may not be expressed early in the developmental cycle. It is possible that
those first six hours allow the chlamydiae to produce a needed protein to mediate
the resistance, and delaying addition of the antibiotic allows for more of the protein
to be accumulated prior to challenging with tetracycline. It is also possible that the
RBs are more resistant to the drug than are EBS, and at 6 hpi, the majority of
developmental forms consist of RBs. These theories are just speculation at this
point, and more experiments are needed to accurately interpret the significance of
this finding.
Overall, the results of these experiments characterize two tetracycline
resistant C. suis strains isolated from swine. The resistance was shown to be stable,
as it was retained after numerous passages in cell culture. Exposure to tetracycline
induced aberrant growth, which returned to normal upon removal of tetracycline.
Chiamydia suis R19 trafficked C6-NBD-Cer to the inclusion indicating an
interaction with the host cell exocytic pathway. In addition, C. suis R19 was shown
to be able to cohabitate an inclusion with C. trachomatis L2. Finally, if tetracycline
is not added until 6 hpi, an increase in C. suis R19 growth is evident.
59
Future research in this area will focus on the identification of candidate
genes associated with the resistance phenotype. This identification will allow an
expanded understanding of the origin of the resistance and allow rapid screening of
both veterinary and human populations for the presence of resistant chlamydial
strains.
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