facial microbiota and antibiotic resistance

87 Pages·2017·2.06 MB·English

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Electronic Thesis Collection
Winter 12-16-2016
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Kayla R. Stricklan
Pittsburg State University, [email protected]
Follow this and additional works at: https://digitalcommons.pittstate.edu/etd
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Stricklan, Kayla R., "FACIAL MICROBIOTA AND ANTIBIOTIC RESISTANCE: A COMPARATIVE STUDY
OBSERVING THE PRESENCE OF RESISTANCE GENES AND DIMINISHED SUSCEPTIBILITY" (2016).
Electronic Thesis Collection. 221.
https://digitalcommons.pittstate.edu/etd/221
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FACIAL MICROBIOTA AND ANTIBIOTIC RESISTANCE: A COMPARATIVE
STUDY OBSERVING THE PRESENCE OF RESISTANCE GENES AND
DIMINISHED SUSCEPTIBILITY

A Thesis Submitted to the Graduate School in Partial Fulfillment of the
Requirements for the Degree of Master in Science

Kayla Stricklan

Pittsburg State University

Pittsburg, Kansas

December 2016
FACIAL MICROBIOTA AND ANTIBIOTIC RESISTANCE: A COMPARATIVE
STUDY OBSERVING THE PRESENCE OF RESISTANCE GENES AND
DIMINISHED SUSCEPTIBILITY

Kayla Stricklan

APPROVED:

Thesis Advisor:____________________________________________________
Dr. Peter Chung, Biology

Committee Member:________________________________________________
Dr. Anu Ghosh, Biology

Committee Member:________________________________________________
Dr. Mandy Peak, Biology

Committee Member:________________________________________________
Dr. Barbara McClaskey, Nursing
FACIAL MICROBIOTA AND ANTIBIOTIC RESISTANCE: A COMPARATIVE
STUDY OBSERVING THE PRESENCE OF RESISTANCE GENES AND
DIMINISHED SUSCEPTIBILITY

An Abstract of the Thesis by
Kayla Stricklan

The looming threat of increased antibiotic resistance leading to a post-
antibiotic world is progressively becoming a reality. A significant portion of the
fight against antibiotic resistance development is slowing down the prescribing of
antibiotics for unnecessary conditions while simultaneously encouraging stricter
adherence to antibiotic course completion. One of the often-unnecessary
conditions is acne vulgaris. Though the disease may be unsightly and
discouraging, it does little to affect overall health of the individual with the
exception of psychosocial affects. Utilizing facial swabs from 144 participants,
samples were tested for the colonization of Propionibacterium acnes,
Staphylococcus aureus and Staphylococcus epidermidis as well as the presence
of resistance genes Cerm(X) and Tet(M) using polymerase chain reaction and
gel electrophoresis. Amplification of the samples portrayed that
Propionibacterium acnes was the most prevalent microbe present, even from
samples obtained from students with minimal blemishes. The second most
common was Staphyloccocus epidermidis and few samples tested positive for
Staphylococcus aureus. Amplification of resistance genes demonstrated fair
amounts of resistance present, even in participants who had not taken antibiotics
in the last two years. Out of the 144 initial samples, 60 isolated samples were
utilized for susceptibility testing and the first set determined a slight decrease in
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diameter of the resulting zone of inhibition when exposed to clindamycin,
doxycycline and tetracycline, but not rifampin.
A subsequent 42 samples were collected for susceptibility testing using
direct colony suspension. Results from testing demonstrated a significant
decrease in the zones of inhibition between those who had not taken antibiotics
and those who had taken two types of antibiotics for general illnesses and acne.
The differences between tetracycline, clindamycin, rifampin and doxycycline
were 4.64, 6.34, 11.86 and 7.66-millimeters, respectively with significant p-values
between rifampin and doxycycline suggesting that the development of resistance
is more prevalent in rifampin than previously determined.

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TABLE OF CONTENTS

CHAPTER PAGE

I. INTRODUCTION 1

Antibiotic Resistance: A Global Problem 1
Propionibacterium acnes: History and Microbiological Implications 3
Acne and Antibiotic Resistance Development 4
Antibiotic Resistance Mechanisms in Microorganisms 6
Acne Specific Antibiotics and Resistance Development 7
Staphylococcus aureus and Staphylococcus epidermidis 13
Proposed Thesis Project 15

II. MATERIALS AND METHODS 17

Sample Collection 17
Gram Staining Bacterial Samples 18
Media Preparation 19
Microbial Species Identification and Kirby Bauer Susceptibility 20
Molecular Species Identification 22
Molecular Resistance Gene Amplification 23
Gel Electrophoresis 24
Subsequent Sampling 25
Mueller Hinton Antimicrobial Susceptibility Replication 25
Statistical Analysis 26

III. RESULTS 28

Initial Sampling and Amplification 28
Resistance Gene Amplification 29
Susceptibility Testing 30
Morphology Confirmation 35
Subsequent Susceptibility Testing 36

IV. DISCUSSION 39

V. REFERENCES 45

APPENDICES 50

Appendix A: Initial Survey of Antibiotic Use 51
Appendix B: Initial Survey of Specific Antibiotics Prescribed 51
Appendix C: Susceptibility, Morphology and Species Identification 52
Appendix D: Results of Each Amplification in Group Zero 53
Appendix E: Results of Each Amplification in Group One 54
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Appendix F: Results of Each Amplification in Group Two 56
Appendix G: Results of Each Amplification in Group Three 56
Appendix H: Common Morphologies-Gram Stains 58
Appendix I: Comprehensive Results for Doxycycline and Rifampin 59
Appendix J: Propionibacterium acnes Primer Samples 61
Appendix K: Propionibacterium acnes Primer Samples 62
Appendix L: Staphylococcus aureus Primer Samples 63
Appendix M: Staphylococcus aureus Primer Samples 64
Appendix N: Staphylococcus epidermidis Primer Samples 65
Appendix O: Staphylococcus epidermidis Primer Samples 66
Appendix P: Cerm(X) Resistance Gene Primers 67
Appendix Q: Cerm(X) Resistance Gene Primers 68
Appendix R: Tet(M) Resistance Gene Primers 69
Appendix S: Tet(M) Resistance Gene Primers 70
Appendix T: BD BBL Sensi Disc Susceptibility Chart 71
Appendix U: Tetracycline and Clindamycin ZOI Organized by Category 72
Appendix V: Rifampin and Doxycycline ZOI Organized by Category 75
Appendix W: Comprehensive Gram Staining Results 77
Appendix X: Informed Consent 78

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LIST OF TABLES
TABLE PAGE
I. Division of Participants Categorically………………………..16
II. List of Primers Utilized for Species Identification……….…..23
III. Comprehensive Survey of Bacteria Amplified by PCR…….28
IV. Percentage of Antibiotic Resistance Genes…………….…..30
V. Clindamycin ZOI Compared to Presence of Cerm(X)…..….30
VI. Tetracycline ZOI Compared to Rresence of tet(M)………....32
VII. Comprehensive Results of Susceptibility Testing…….….....37

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LIST OF FIGURES
FIGURE PAGE
I. Comparison of ZOI Between Categories in First
Susceptibility Testing………………………………………….35

II. Comparison of ZOI Between Categories in Second Replication
of Susceptibility Testing………………………………………..36

III. Comparison of ZOI for Tetracycline/Clindamycine………….38

IV. Comparison of ZOI for Rifampin/Doxycycline………….……38
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CHAPTER I

INTRODUCTION

Antibiotic Resistance: A Global Problem

Antibiotic use is on a continuous rise while the development of new
antibiotics has reached a drastic halt. Combine both phenomenon with bacteria
that are perpetually evolving and the world is facing a global crisis. Antibiotics
have been likened to a four-edged sword against bacteria because they are
capable of benefitting both the individual and the community by preventing the
spread of illness but these benefits come at the cost of communal antibiotic
resistance development and the collateral damage affecting one’s microbiota
(Yatsunenko et al. 2012). Although antibiotics have existed in nature for
hundreds of millions of years, they have become a cornerstone of medicine in the
decades since the Second World War (Davies, 2010). It is widely known that
Alexander Fleming ignited the search for competing antibiotics with his discovery
of penicillin in 1928. Since then, the magnitude at which antibiotics are both
prescribed and utilized has exploded and may be attributed to the perception that
antibiotics are entirely safe (Blaser, 2016). However, looking specifically at
agriculture, evidence suggests that the use of small doses of antibiotics promotes
growth in livestock and affects metabolic development (Zimmerman, 1986).
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