PREVALENCE OF MRSA AND ANTIMICROBIAL RESISTANCE OF STAPHYLOCOCCUS ...

147 Pages·2010·1.58 MB·English

by  Matthew Hill

ABSTRACT

Title of Document: PREVALENCE OF MRSA AND
ANTIMICROBIAL RESISTANCE OF
STAPHYLOCOCCUS AUREUS IN
MARYLAND GROUND MEAT PRODUCTS

Twain Brown, Nicole Dupuy, Kourtney Johnson,
Alina Kelman, Edward Kestler, William
Richbourg, Daniel Shafer, Yee-Ann Soong

Directed By: Dr. Jianghong Meng, DVM, MPVM, Ph.D.
Department of Nutrition and Food Science

The aim of this study was to evaluate the risk of exposure to antimicrobial-
resistant Staphylococcus aureus from food-grade raw ground meat products in
Maryland. Samples of ground beef (n = 198), pork (n = 300), and turkey (n = 196),
were collected by random sampling from March-August, 2008. All isolates were
tested for resistance to methicillin and confirmed S. aureus isolates (n = 200) were
tested for susceptibility to 21 additional antimicrobials. Overall, turkey- and pork-
derived isolates were more likely to be resistant to commonly used antimicrobials.
One isolate from pork was confirmed to be the USA100 strain of MRSA and was
resistant to 10 antibiotics. In addition, antibiotic-resistant non-S. aureus isolates were
characterized and may represent a source for the transfer of resistance genes to S.
aureus. Our findings suggest that meat production practices may impact the
prevalence and antimicrobial resistance of S. aureus in ground meat.
PREVALENCE OF MRSA AND ANTIMICROBIAL RESISTANCE OF
STAPHYLOCOCCUS AUREUS IN MARYLAND GROUND MEAT PRODUCTS

By

Team Antibiotic Resistance

Twain Brown
Nicole Dupuy
Kourtney Johnson
Alina Kelman
Edward Kestler
William Richbourg
Daniel Shafer
Yee-Ann Soong

Thesis submitted in partial fulfillment of the requirements for the Honors Gemstone
Program, University of Maryland, College Park
2010

Advisory Committee:
Dr. Jianghong Meng, DVM, MPVM, Ph.D., Mentor
Dr. Vincent Lee, Ph.D., Discussant
Dr. Patrick McDermott, Ph.D., Discussant
Dr. Jie Zheng, Ph.D., Discussant
Dr. Kevin McIver, Ph.D., Discussant
© Copyright by
Team Antibiotic Resistance
Twain Brown, Nicole Dupuy, Kourtney Johnson, Alina Kelman, Edward Kestler,
William Richbourg, Daniel Shafer, Yee-Ann Soong
2010
Acknowledgements
We would like to thank our mentor, Dr. Jianghong Meng, and our librarian, Thomas
Harrod, for their expertise and continual support. We would also like to thank Dr.
Patrick McDermott, Dr. Jie Zheng, and Yi Li for their important contributions to our
project. Further, we acknowledge the training and advice provided by Sherry Ayers,
Althea Glenn, and Dr. Shaohua Zhao at the FDA. This study was made possible by
funding from the Joint Institute for Food Safety and Applied Nutrition (JIFSAN) of
the University of Maryland and the U.S. Food and Drug Administration as well as
from the Gemstone Program. We would like to thank the Gemstone staff for their
continual guidance and support.
iv
Table of Contents

Acknowledgements ...................................................................................................... iv
Table of Contents .......................................................................................................... v
List of Tables ............................................................................................................. viii
List of Figures .............................................................................................................. ix
Chapter 1: Introduction ................................................................................................. 1
1.1 Antibiotic Resistance: General Background ....................................................... 1
1.2 Use of Subtherapeutic Antibiotics in Livestock ................................................. 2
1.2.1 Antibiotic Use in Cattle ................................................................................ 4
1.2.2 Antibiotic Use in Poultry ............................................................................. 5
1.2.3 Antibiotic Use in Swine ................................................................................ 6
1.3 Methicillin-Resistant Staphylococcus aureus (MRSA) ...................................... 7
1.4 CA-MRSA and HA-MRSA: What is the difference? ....................................... 10
1.5 MRSA Contamination of Meat Products .......................................................... 11
1.6 Study Objectives ............................................................................................... 12
Chapter 2: Literature Review ...................................................................................... 15
2.1 Antibiotics Approved for Use in Livestock ...................................................... 15
2.1.1 Major Classes of Antibiotics Used in Animals .......................................... 18
2.2 Economic and Animal Health Benefits of Antibiotic Use in Animals ............. 19
2.3 Antibiotic Resistance Genes in the Farm Environment .................................... 22
2.4 Antimicrobial Resistant Pathogens Associated With Farm Animals ............... 23
2.4.1 Salmonella.................................................................................................. 24
2.4.2 Enterococci ................................................................................................ 26
2.4.3 E. coli ......................................................................................................... 28
2.5 Staphylococcus aureus: A Dangerous Pathogen ............................................... 29
2.5.1 Food Poisoning .......................................................................................... 31
2.5.2 Cutaneous Infections .................................................................................. 32
2.5.3 Staphylococcal Scalded Skin Syndrome ..................................................... 32
2.5.4 Impetigo ..................................................................................................... 33
2.5.5 Cellulitis ..................................................................................................... 33
2.5.6 Necrotizing Fasciitis .................................................................................. 34
2.5.7 Toxic Shock Syndrome ............................................................................... 34
2.5.8 Bacteremia ................................................................................................. 35
2.5.9 Pneumonia ................................................................................................. 36
2.5.10 Osteomyelitis ............................................................................................ 37
2.5.11 Endocarditis ............................................................................................. 37
2.5.12 Treatment ................................................................................................. 38
2.6 The Rise of Methicillin-Resistant Staphylococcus aureus (MRSA) ................ 38
2.6.1 Increase in the Incidence of CA-MRSA ..................................................... 39
2.6.2 MRSA in Food ............................................................................................ 40
2.6.3 MRSA as a Zoonotic Pathogen .................................................................. 40
2.6.4 MRSA: A Foodborne Pathogen ................................................................. 42
2.6.5 Methods of Screening for MRSA Contamination in Food ......................... 45
v
Chapter 3: Methodology ............................................................................................. 47
3.1 Materials: Media Preparation ............................................................................ 47
3.2 Random Sampling of Ground Meat Products ................................................... 49
3.3 Isolation and Identification of S. aureus from Ground Meat ............................ 51
3.3.1 Plating on Baird-Parker, a Selective Growth Medium .............................. 51
3.3.2 Gram Staining ............................................................................................ 52
3.3.3 Catalase and Coagulase Tests ................................................................... 53
3.3.4 Vitek Confirmation ..................................................................................... 54
3.4 Antimicrobial Susceptibility Testing ................................................................ 55
3.4.1 Minimum Inhibitory Concentration Assay for Methicillin......................... 58
3.4.2 Polymerase Chain Reaction Screen for mecA Gene .................................. 58
3.5 Strain Sub-Typing of MRSA by Pulsed-Field Gel Electrophoresis ................. 60
Chapter 4: Results ....................................................................................................... 63
4.1 Prevalence of S. aureus Contamination in Ground Meats ................................ 63
4.1.1 Prevalence of S. aureus Contamination by Meat Type .............................. 64
4.1.2 Prevalence of S. aureus Contamination by Grocery Store Chain ............. 66
4.2 Concentration of Bacteria in Meat: Colony Forming Units per Gram ............. 68
4.3 Antimicrobial Resistance in S. aureus Isolated from Ground Meat ................. 70
4.3.1 Differences in Antimicrobial Susceptibility of Beef, Pork, and Turkey
Isolates ................................................................................................................ 72
4.3.2 Multi-Drug Resistant S. aureus Isolates .................................................... 76
4.4 Methicillin-Resistant S. aureus Isolates from Ground Meat ............................ 78
4.4.1 PCR Confirmation of mecA-Positive Isolates Including MRSA Strain ..... 80
4.4.2 PFGE Typing of MRSA Isolate as USA100 ............................................... 81
4.4.3 Methicillin-Resistant Non-S. aureus Isolates ............................................. 82
Chapter 5: Discussion ................................................................................................ 84
5.1 Major Findings .................................................................................................. 84
5.1.1 Animal Husbandry Practices May Impact Antimicrobial Susceptibility ... 84
5.1.2 Potential for Horizontal Transfer of Resistance Genes in S. aureus ......... 87
5.1.3 Methicillin Resistance in Absence of mecA Gene ...................................... 89
5.1.4 Antimicrobial Resistance in Non-S. aureus Meat Isolates ........................ 90
5.1.5 Prevalence of MRSA in Retail Ground Meats Lower Than Expected ....... 90
5.1.6 Maryland Meat Consumers are Exposed to Multi-Drug-Resistant Bacteria
............................................................................................................................. 92
5.2 Data Analysis in Light of Previous Research ................................................... 92
5.3 Limitations ........................................................................................................ 97
5.4 Future Directions ............................................................................................ 101
5.5 Recommendations ........................................................................................... 105
Appendices ................................................................................................................ 110
Appendix A: Suspected S. aureus Isolates Tested at FDA for Confirmation (n = 215)
................................................................................................................................... 110
Appendix B: Standard MIC Values for Antimicrobials (S = Susceptible, I =
Intermediate, R = Resistant) ..................................................................................... 118
Appendix C: Non-S. aureus mecA+ Strains .............................................................. 119
Appendix D: Non-S. aureus mecA- Strains with Methicillin Resistance ................. 120
Appendix E: Vitek GP Card Substrates .................................................................... 122
vi
Glossary .................................................................................................................... 123
Bibliography ............................................................................................................. 126

vii
List of Tables

Chapter 2:
Table 1: USDA Approved Antibiotics for Cattle, Hogs,
Chickens, and Turkeys......................................................................16
Table 2: Antibiotic Overlap Amongst Humans and Livestock.......................17

Chapter 4:
Table 1: S. aureus Positive Meat samples by Meat Type...............................64
Table 2: S. aureus Positive Meat Samples by Meat Type (Pork Loin)...........65
Table 3: S. aureus Positive Meat Samples by Grocery Chain........................67
Table 4: Prevalence of S. aureus Resistance to Profile of
Twenty Antibiotics............................................................................71
Table 5: MIC50, MIC90, and Percentage Resistant for
S. aureus Isolates..............................................................................74
viii
List of Figures
Chapter 2:
Figure 1: Level of Tetracycline Resistance in Swine
After Antibiotic Withdrawal............................................................21

Chapter 4:
Figure 1: CFU/g by Meat Type......................................................................69
Figure 2: S. aureus Resistance to Antibiotics by Meat Source......................73
Figure 3: Multiple-antibiotic Resistance Profile of S. aureus........................77
Figure 4: Methicillin MIC in S. aureus..........................................................79
Figure 5: PFGE Analysis of MRSA Isolate (409-1) .....................................82

ix
Chapter 1: Introduction

1.1 Antibiotic Resistance: General Background
Although the development of antibiotics throughout the twentieth century has
led to major advances in human and animal health, several factors have also led to
antibiotic resistance. One part of the problem is that the discovery of new antibiotics
has waned. Since 1970, there has only been one new class of antibiotics introduced,
and on average, research and development of antibiotics takes 10 to 20 years
(Lipsitch 2002). Therefore to maintain the efficacy of current antibiotics, humans
have turned to combining them.
At the same time, with the introduction of antibiotics into healthcare,
resistance has arisen. Up to 30% of Streptococcus pneumoniae and Haemophilus
influenzae, bacteria that cause the major infectious disease pneumonia, are resistant to
penicillin (Thornsberry 2002). Likewise, Shigella dysenteriae, a microbe which
causes diarrheal disease, has become resistant to every available drug except
ciprofloxacin within the past ten years (Green 2009). Ciprofloxacin belongs to a
group of antibiotics called fluoroquinolones, which were banned for the treatment of
poultry by FDA-CVM (the Food and Drug Administration Center for Veterinary
Medicine) in 2005 because of quickly developing microbial resistance to the drug as a
result of its use as a sub-therapeutic antibiotic in industry (FDA 2005). Similarly,
after penicillin was discovered, levels of resistant Staphylococcus aureus in hospitals
1