CHARACTERIZATION OF SALMONELLA ENTERICA SEROVAR AGONA SLAUGHTER ISOLATES FROM THE ANIMAL ARM OF THE NATIONAL ANTIMICROBIAL

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1 CHARACTERIZATION OF SALMONELLA ENTERICA SEROVAR AGONA SLAUGHTER ISOLATES FROM THE ANIMAL ARM OF THE NATIONAL ANTIMICROBIAL RESISTANCE MONITORING SYSTEM ENTERIC BACTERIA (NARMS): 1997 THROUGH by APHRODITE DOURIS (Under the Direction of David Stallknecht) ABSTRACT Salmonella enterica serovar Agona is an enteric pathogen isolated from food animals. Salmonella Agona has routinely appeared on the list of top10 most commonly isolated Salmonella serovars from both humans and animals. This underlines the importance of S. Agona as a potential source of food-borne illness in humans. This thesis represents a phenotypic (antimicrobial susceptibility profiles) and genotypic (pulsed field gel electrophoresis, plasmid analysis and integron analyses) characterization of S. Agona slaughter/processing isolates from cattle, chickens, turkeys, and swine submitted to the National Antimicrobial Resistance Monitoring System from 1997 through. Data from these studies suggest a shift in prevalence among sources, an increase in antimicrobial drug resistance to six of the 19 antimicrobials tested, relatedness between pulsed field gel electrophoresis patterns and

2 antimicrobial resistance profiles, and an increased prevalence of large plasmids and class 1 integrons among multiple drug resistant isolates compared to pan-susceptible isolates. INDEX WORDS: Salmonella enterica serovar Agona, food animals, cattle, swine, poultry, food-borne disease, antimicrobial drug resistance, pulsed field gel electrophoresis, plasmid, and class 1 integron

3 CHARACTERIZATION OF SALMONELLA ENTERICA SEROVAR AGONA SLAUGHTER ISOLATES FROM THE ANIMAL ARM OF THE NATIONAL ANTIMICROBIAL RESISTANCE MONITORING SYSTEM ENTERIC BACTERIA (NARMS): 1997 THROUGH by APHRODITE DOURIS B.S., The College of William and Mary, 1995 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2005

4 2005 Aphrodite Douris All Rights Reserved

5 CHARACTERIZATION OF SALMONELLA ENTERICA SEROVAR AGONA SLAUGHTER ISOLATES FROM THE ANIMAL ARM OF THE NATIONAL ANTIMICROBIAL RESISTANCE MONITORING SYSTEM ENTERIC BACTERIA (NARMS): 1997 THROUGH by APHRODITE DOURIS Major Professor: Committee: David Stallknecht Corrie Brown Paula J. Fedorka-Cray Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2005

6 DEDICATION I dedicate this thesis to my son, Dmitri Banks Palmer. iv

7 ACKNOWLEDGEMENTS I would like to thank my committee members David Stallknecht, Corrie Brown, and Paula Fedorka-Cray for giving me the opportunity to complete this degree. I would also like to express my appreciation to my supervisor, Charlene R. Jackson, for her expert advice. And last but not least I would like to thank my friends and family who gave me the emotional support to keep on trying until I reached my goal. v

8 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS...v LIST OF TABLES... viii LIST OF FIGURES...x CHAPTER 1 Introduction...1 Reference List Literature Review of Salmonella...5 Preface...5 A General Description...6 A Historical Perspective...6 Taxonomy...7 An Introduction to Salmonella enterica serovar Agona...8 Implications for Human Health...10 Salmonella spp. and S. Agona in Food Animals...11 Reference List Literature Review of Antimicrobial Drugs...20 A General Description of Antimicrobial Drug Classes...20 Antimicrobial Drug Use in Food Animals...24 Antimicrobial Drug Resistance...28 Reference List...36 vi

9 4 Characterization of Salmonella enterica serovar Agona Slaughter Isolates from the Animal Arm of the National Antimicrobial Resistance Monitoring System Enteric bacteria (NARMS): 1997 through...43 Abstract...45 Introduction...46 Materials and Methods...50 Results...52 Discussion...57 Reference List...63 Acknowledgements Detection of Plasmids and Class 1 Integrons in Salmonella enterica serovar Agona Isolated from NARMS Slaughter Samples Collected in Years 1997 through Abstract Introduction Materials and Methods Results Discussion Reference List Acknowledgements Conclusion Reference List vii

10 LIST OF TABLES Page Table 4.1: Resistance Breakpoint (BP) and Concentrations of Antimicrobial Drugs Tested Table 4.2: Distribution by year of total number of all Salmonella and S. Agona isolates from NARMS slaughter samples...74 Table 4.3: Distribution (number and percent) of S. Agona recovered from NARMS slaughter samples by animal source and collection year Table 4.4: Resistance patterns of S. Agona slaughter isolates regardless of source: Table 4.5: Resistance of S. Agona slaughter isolates by source: Table 4.6: Resistance of S. Agona isolates from cattle slaughter samples: Table 4.7: Resistance of S. Agona isolates from turkey slaughter samples: Table 4.8: Resistance of S. Agona isolates from swine slaughter samples: Table 4.9: Resistance of S. Agona isolates from chicken slaughter samples: Table 4.10: Number of Antimicrobials S. Agona Isolates were Resistant to Table 4.11: Number of S. Agona slaughter isolates with resistance to > 5 antimicrobials: Table 4.12: Source of S. Agona slaughter isolates with MDR to > 5 antimicrobials...85 Table 4.13: Antimicrobial Resistance Profiles from S. Agona Slaughter Isolates with MDR to > 5 Antimicrobials and Occurs in >1 Isolate from 1997* viii

11 Table 4.14: Antimicrobial Resistance Profiles from S. Agona Slaughter Isolates with MDR >5 Antimicrobials and Occurs in >1 Isolate from 1997* Table 5.1: Resistance Breakpoint (BP) and Concentrations of Antimicrobials Tested Table 5.2: Small Plasmids Recovered from Pan-Susceptible S. Agona Slaughter Isolates Table 5.3: Small Plasmids Recovered from MDR S. Agona Slaughter Isolates Table 5.4: Large Plasmids Recovered from Pan-Susceptible S. Agona Slaughter Isolates and Class 1 Integrons Detected in Pan-Susceptible S. Agona Slaughter Isolates Table 5.5: Large Plasmids Recovered from MDR S. Agona Slaughter Isolates and Class 1 Integron Detection in MDR S. Agona Slaughter Isolates Table 5.6: MDR profiles, Large Plasmids Recovered, and Class 1 Integrons Detected from MDR S. Agona Slaughter Isolates ix

12 LIST OF FIGURES Page Figure 4.1: Cluster analysis of all 482 S. Agona isolates...89 Figure 4.2: MDR pattern A...99 Figure 4.3: MDR pattern B Figure 4.4: MDR patterns A and B Figure 5.1: Cluster analysis of pan-susceptible S. Agona isolates Figure 5.2: Cluster analysis of pentaresistant S. Agona isolates Figure 5.3: 25 Pan-susceptible S. Agona isolates Figure 5.4: 35 S. Agona isolates resistant to 5 or more antimicrobials Figure 5.5: 60 S. Agona isolates including pan-susceptible and isolates resistant to 5 or more antimicrobials x

13 CHAPTER 1 Introduction Salmonella enterica serovar Agona is an enteric zoonotic pathogen that has been isolated from both food animals and humans with salmonellosis. Salmonella Agona has been one of the top 15 Salmonella serovars isolated from both humans and animals since its introduction into the United States. Contaminated animal feed was implicated as the source of introduction (3). Food animal production has become progressively more integrated in the U.S. and other developed countries. More animals housed in closer quarters and under increased stress has lead to amplified levels of prevalence for some enteric pathogens, including Salmonella. Food-borne illness occurs through consumption of contaminated foodstuffs. Risk factors for illness include increased consumption of foods prepared outside of the home, increased vulnerable human populations (young, elderly and immune compromised), and improperly handling/storing/cooking foods in the home. Despite significant efforts, the prevalence of enteric pathogens in foods, at some level, continues to occur. At the farm, producers have been forced to look for ways to decrease zoonotic pathogen loads in their animals. Antimicrobial drug use is common in the food animal industry, providing economic benefits through disease prevention and growth promotion. A side effect of the increased agricultural use of antimicrobial drugs has been the observance of antimicrobial drug resistant enteric zoonotic pathogens and fear of the emergence of a super pathogen that defies all treatments. 1

14 Specific Salmonella serovars such as S. Typhimurium DT104 and S. Newport have caused concern in the public health sector. This is due in part to increased virulence as well as the development of resistance to five or more antimicrobials. In the European Union, S. Agona isolates from poultry and human origin have been identified as having the same antimicrobial resistance profiles and similar genotypic characteristics as multiple drug resistant strains of S. Typhimurium DT104 and S. Newport (1,2,4-6). Similar studies on the antimicrobial drug resistance profiles and genotypic characteristics of S. Agona isolated in the U.S. are not available. This thesis examined S. Agona slaughter isolates (cattle, chickens, turkeys, swine) submitted to the National Antimicrobial Resistance Monitoring System from 1997 to. Isolates antimicrobial drug resistance profiles and pulsed field gel electrophoresis patterns were analyzed. Furthermore, a subset of isolates was chosen and the presence of small plasmids, large plasmids, and class 1 intergrons was determined. This information was compared with the antibiograms and PFGE patterns for relatedness. 2

15 Reference List 1. Boyd, D., A. Cloeckaert, M. Chaslus-Dancla, and M. Mulvey.. Characterization of Variant Salmonella Genomic island 1 Multidrug Resistance Regions from Serovar Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46: Boyd, D., G. Peters, A. Cloeckaert, K. Boumedine, M. Chaslus-Dancla, H. Imberechts, and M. Mulvey.. Complete nucleotide Sequence of a 43- Kilobase genomic island Associated with the Multidrug Resistance Region of Salmonella enterica Serovar Typhimurium DT104 and Its Identification in Phage Type DT120 and Serovar Agona. Journal of Bacteriology 183: Clark, G., A. Kaufmann, E. Gangarosa, and M. Thompson Epidemiology of an International Outbreak of Salmonella Agona. Lancet ii: Cloeckaert, A., K. Boumedine, G. Flaujac, H. Imberechts, I. D'hooghe, and M. Chaslus-Dancla.. Occurence of a Salmonella enterica Serovar Typhimurium DT104-Like Antobiotic Resistance Gene Cluster Including the flor Gene in S. enterica Serovar Agona. Antimicrob Agents Chemother 44: Doublet, B., D. Boyd, M. Mulvey, and A. Cloeckaert The Salmonella genomic island 1 is an integrative mobilizable element. Molecular Microbiology 55:

16 6. Doublet, B., P. Butaye, H. Imberechts, D. Boyd, M. Chaslus-Dancla, and A. Cloeckaert Salmonella Genomic Island 1 Multidrug Resistance Gene Clusters in Salmonella enterica Serovar Agona Isolated in Belgium in Antimicrob Agents Chemother 48:

17 CHAPTER 2 Literature Review of Salmonella Preface This literature review is broken into two chapters Salmonella and Antimicrobial Drugs. Salmonella is a topic that has been studied since it was first described in the 19 th century. A plethora of literature is available on Salmonella and much of it is beyond the scope of this review. The Salmonella section is a brief overview of general characteristics and topics related to Salmonella. Information gathering on antimicrobials started much later than for Salmonella. Currently, research is being done using cutting edge technology to gain clearer insight into the acquisition, mechanisms, dissemination and public health consequences of antimicrobial drug resistance. Policies that control the veterinary and human distribution and use of antimicrobials are fluid, changing as more literature is published. The objective of this section is to provide a description of the relevant classes of antimicrobials, antimicrobial drug use in food animals and the potential consequences of resistance as it impacts both human and animal health. Throughout the text within both chapters are references to and examples from literature focused on Salmonella enterica serovar Agona. The intention is to allow the reader to gain a familiarity with why and how the organism of interest, S. Agona, is significant and relevant. Additionally, this literature review may allow for the reader to better understand the implications of this research. 5

18 A General Description Salmonella spp. are small, facultative anaerobes, non-sporing, gram-negative rods belonging to the Enterobacteriaceae family (11,17,19). The primary habitat of Salmonella spp. is the intestinal tract of animals (11,17,19). Members of Salmonella include motile variants by peritrichous flagella, nonflagellated variants, and nonmotile strains due to dysfunctional flagella (11,17,19). They are chemoorganotrophic and have the ability to use both respiration and fermentation to metabolize nutrients (11,17,19). Optimal growth occurs at 37 C. A contemporary biochemical definition of a typical Salmonella isolate is one that would produce acid and gas from glucose in triple sugar iron (TSI) agar medium and would not utilize lactose or sucrose in TSI or in differential plating media such as brilliant green, xylose lysine deoxycholate, and Hektoen enteric agars. Furthermore, salmonellae readily produce an alkaline reaction from the decarboxylation of lysine to cadaverine in lysine iron agar, they can generate hydrogen sulfide gas in TSI and lysine iron media and they do not hydrolize urea (11). A Historical Perspective A 19 th century French clinical pathologist first documented the link between human intestinal ulceration and a contagious agent that was later identified as typhoid fever (11). Further investigations by Europeans led to the isolation and characterization of typhoid bacillus and to a serodiagnostic test for its detection (11). In the United States, contemporary work by Salmon and Smith in 1885 led to the isolation of Bacillus cholerae-suis from swine with hog cholera (11). In 1900 Lignieres coined the term Salmonella to include all the related typhoid and paratyphoid organisms being discovered 6

19 (11). The beginning of the 20 th century witnessed great advances in the serological detection of somatic (O) and flagellar (H) antigens within the Salmonella group. In 1926, White first proposed an antigenic scheme for classifying Salmonella (11,17). This scheme was later expanded by Kauffmann in 1941 and subsequently called the Kauffmann-White scheme, which currently recognizes more than 2,300 serovars (11,17,19). It is still in use today and expanding. Taxonomy There are over 2,324 Salmonella serovars that are grouped into two species S. enterica and S. bongori. Approximately 2,000 Salmonella serovars have been placed in S. enterica, which has further been divided into 5 subspecies (19). Epidemiologically salmonellae can be placed into three groups: 1. Serovars that infect humans only. 2. Hostadapted serovars. 3. Unadapted serovars (no host preference). The scope of this paper will focus on Salmonella with no host preferences, which are pathogenic for humans and animals; this includes most foodborne serovars (19). Often, these are referred to a zoonotic pathogens. Currently, identification of Salmonella spp. includes serotyping, a serological confirmation. This technique is complex and labor intensive involving the agglutination of bacterial surface antigens with Salmonella-specific antibodies. These include O lipopolysaccharides (LPS) on the external surface of the bacterial outer membrane, H antigens associated with the peritrichous flagella, and Vi, the capsular antigen, present only in S. typhi, S. Paratyphi C and S. Dublin (11). The goal of the serological testing procedure is to determine the complete antigenic formula of a Salmonella isolate. 7

20 Currently, under international agreement, a new serovar is named after the place where it was first isolated (19). Genetic variability, due to bacterial mutations and conjugative intra- and intergenic exchanges of plasmids encoding biochemical traits, is reducing the number of typical Salmonella biotypes (11). This has resulted in an ever evolving taxonomic system and a continually greater reliance on molecular techniques vs. biochemical testing for detection and identification. An Introduction to Salmonella enterica serovar Agona This thesis work focuses on a single Salmonella serovar, Salmonella enterica serovar Agona. Salmonella Agona is an unadapted serovar and therefore shows no host preference infecting animals and humans. S. Agona has maintained enough of a presence in food animals and among humans with food-borne salmonellosis to warrant a closer look. Furthermore, there is a lack of current literature focusing on the characteristics of S. Agona in the United States. Salmonella enterica serovar Agona was first described in 1961 when it was reported to have been recovered from cattle in Ghana (16). In Salmonella Agona emerged as a public health problem in the United States, the United Kingdom, the Netherlands and Israel (9). The cause of that international human outbreak was traced back to contaminated Peruvian fishmeal, a common source of protein in animal feeds (9). Prior to outbreak, the United States had only two documented human infections from S. Agona; however, by 1972, S. Agona was the 8 th most commonly isolated serotype accounting for more than 500 human cases of infection (9). In 1974, S. 8

21 Agona was reported to have a common presence in eutrophic regions of a feshwater lake in the US and the source was thought to be run off from poultry operations (10). This finding re-enforced the belief that S. Agona had established itself in the environment and would be a continuing concern for public health in the United States. Since gaining worldwide recognition, S. Agona has increasingly been in the forefront as a serotype of concern for both humans and animals. From 1997 through The Centers for Disease Control and Prevention (CDC) Salmonella Surveillance Annual Summary included S. Agona on the top 20 lists for the most frequently reported Salmonella serotypes from human sources, the most frequently reported Salmonella serotypes from clinical nonhuman sources, and the most frequently reported Salmonella serotypes from non-clinical nonhuman sources (2-4). Salmonella Agona was reported as the 7 th most commonly isolated Salmonella from human sources in Canada during the years (20) and was the 5 th most commonly isolated Salmonella from nonhuman sources in Canada during those same years (20). From 1997 through, the animal arm of the National Antimicrobial Resistance Monitoring System Enteric Bacteria (NARMS) included S. Agona as one of the top 15 Salmonella serotypes recovered from animals ( The fact that S. Agona is routinely included on lists of the most commonly isolated serovars from both human and food animal sources underlines the importance of this pathogen as a potential source of food-borne illness in humans. 9

22 Implications for Human Health The rate of acute gastroenteritis in the U.S. was estimated to be 0.72 episodes per person per year resulting in 195 million episodes nationally (14). The United States has 1.4 million cases of illness, 15,000 hospitalizations and 400 deaths attributed annually to Salmonella infections (14). England has 9.4 million cases of gastroenteritis, which include 106,800 cases of nontyphoidal Salmonella infections (14). Human Salmonella infections can lead to several clinical conditions, including enteric (typhoid) fever, uncomplicated enterocolitis (also referred to as Food-Poisoning Syndrome), and systemic infections by non-typhoid microorganisms (11,17,19). Human infections with nontyphoid salmonellae commonly result in enterocolitis, appearing 8 to 72 hours after ingestion of the invasive pathogen (8,11). This disease is generally self-limiting. Nonbloody diarrheal stools, vomiting, nausea, headache, chills, muscular weakness, moderate fever and abdominal pain usually subside within 5 days of onset (11,17,19). Treatment is limited to supportive therapy. The use of antibiotics is discouraged due to their results of prolonging the carrier state (up to 5% of recovering patients may become carriers) which includes the intermittent excretion of salmonellae (8,11,19). The very young, elderly and the immune compromised may be at risk for a nontyphoid infection to degenerate into a systemic infection and possibly lead to various chronic conditions. Septicemia occurs in 6% of confirmed cases of human salmonellosis annually in the U.S. (15) ( Furthermore, Salmonella induced chronic conditions include aseptic reactive arthritis, Reiter s syndrome, and ankylosing spondylitis (11,19). 10

23 Globalization of the food supply has presented new challenges for food safety and has contributed to the international public health problem of foodborne disease. Salmonella spp. are the leading cause of foodborne bacterial diseases in humans (11). Ireland reported 17.2 million cases of gastroenteritis due to food transmission (14). In the United States 95% of nontyphoidal Salmonella infections are the result of foodborne transmission (22). Furthermore, nontyphoidal Salmonella accounts for 31% of all deaths from foodborne illnesses of known pathogens in the United States (22). FoodNet surveillance areas reported preliminary incidence for 2004 laboratory-diagnosed cases of infection for Salmonella to be 6,464. The overall incidence per 100,000 persons was 14.7 (5). Comparing with 2004 preliminary data showed an 8% decline in overall Salmonella infections (5). However, this was a modest decline in comparison to other foodborne bacterial pathogens. The problem of human salmonellosis from the consumption of contaminated food remains a public health concern worldwide and the global increase is considered real, not the result of enhanced surveillance (11). Salmonella spp. and S. Agona in Food Animals Salmonella is a zoonotic enteropathogen that commonly resides in the intestinal tract of food animals, in animal waste, and the environment that is in contact with the animal waste. The characteristics of integrated farming, large numbers of animals living within a small space with periods of great stress, are the ideal setting for maintaining a cycle of Salmonella infections. Since the 1940 s and the beginning of modern farming, there has been a rapid increase in the isolation of the non-host specific Salmonella serovars from humans and animals (24). Across geographical areas and over time there is 11

24 considerable variation in the prevalence and type of serovars recovered from animals. Although salmonellosis is likely underreported, surveillance data allows broad comparisons for the identification of trends, reservoirs, and routes of transmission of Salmonella serovars. Poultry: Poultry and poultry products have been reported as the main sources of non-host specific Salmonella infections in humans (24). The estimated annual cost of poultry-asscociated cases of salmonellosis in the United States is between $64 million and $114.5 million (8). From , poultry was responsible for 10% of the foodborne outbreaks that were traced back to their origins (8). Factors that contribute to the prevalence of poultry as a source for human salmonellosis include modern agricultural practices, Salmonella contaminated animal feed, processing procedures that promote the spread or growth of Salmonella, an increasingly susceptible human population including the young, elderly and immune compromised, and improper food handling and preparation (8). Salmonella infected poultry can develop a carrier state or are clinically ill, although this is infrequent. Poultry can be a large reservoir and source of contamination for human, animals and the environment. Poultry acquire Salmonella infections through horizontal and vertical transmission. Sources of horizontal transmission include hatching, aerosols, litter, water, feed, rodents, stress, sewage, and housing (8,24). Vertical transmission is from the hen to the egg through an infection of the ovary and/or oviduct (8,24). Among chickens in the U.S., S. Enterititis and S. Typhimurium are the two serovars that are attributed to the highest human health concerns based on the percent 12

25 prevalence of these serovars isolated from humans (24). However, each country has its own dominant serovars in poultry. The United States turkey industry spends over $10 million annually on Salmonella prevention at the breeder flock hatchery level in an attempt to control morbidity and mortality losses from Salmonella infections (23). The Centers for Disease Control and Prevention (CDC) reported that from 1973 to 1983, turkeys were the source for 9% of reported human salmonellosis infections (18). In 1984, Canada reported that 70% of all poultry associated salmonellosis outbreaks were due to turkey (18). Meanwhile, outbreaks of human salmonellosis (due to S. Agona) in the U.K. have been reported from pre-cooked turkey meat (25,27). The prevalence of salmonellosis in turkeys depends on serovar, age of birds, dose, route of infection, and stress (18). Unlike broilers, Salmonella infections cause heavy loss among young pullets (18). Salmonella Paratyphoid infections are considerably more prevalent in turkeys than in any other avian species and can result in pullet loss, decreased fertility, decreased hatchability, decreased egg production, unpipped eggs and dead embryos (18). : Salmonella infection is a cause of mortality and morbidity in cattle (28). are an important source for S. Typhimurium DT104, S. Newport AmpC and S. Agona (6,7,21,26,29). In a Canadian study, S. Agona accounted for 18% of Salmonella recovered from beef cattle (1). From 1973 to 1983, beef accounted for 19% of human salmonellosis in the U.S. (28). Furthermore, in the United States contaminated raw beef caused a S. Agona outbreak in humans (4). Seventy-five percent of large dairy herds in the western U.S. are infected with Salmonella (28). Adult cattle that have recovered from salmonellosis can be active carriers and excretors of Salmonella (28). 13

26 : Porcine Salmonella infections include S. cholerasuis (host adapted) and S. Typhimurium as well as other serovars. Although adult pigs are usually asymptomatic carriers of Salmomella, in the U.S. there are two clinical manifestations of porcine salmonellosis, entercolitis which is mainly due to infection with S. Typhimurium and septicemia which is mainly due to infection with S. choleraesuis (13). Porcine populations prone to salmonellosis are intensively reared 6-12 week old weaned pigs (13). In 1968, 74.2% of Salmonella isolates recovered from swine in the U.S. were S. cholerasuis (13). Salmonella cholerasuis is uncommon in Europe and prevalence in the U.S. has decreased, however, it remains a concern for the U.S. pork industry (13). In 1997, S. Typhimurium and S. Derby were the most common Salmonella isolated from pork (13). In 1995, S. Agona was the second most reported serovar from the U.S. pork industry and in S. Agona was also the second most reported serovar from German pork industries (13). In Mexico, S. Agona was one of the top 10 serovars isolated from raw pork and has been the causative agent in human illness (12). Multiple drug resistant S. Typhimurium DT104 has also been recovered from swine (6,13). The following values show percent prevalence of Salmonella on pork carcasses from a variety of countries: Greece 28%, Canada 17.5%, The Netherlands 21%, U.S. 6.5%, Belgium 27% (13). 14

27 Reference List 1. Abouzeed, Y., H. Hariharan, C. Poppe, and F. Kibenge.. Characterization of Salmonella isolates from cattle, broiler chickens and human sources on Prince Edward Island. Comparative Immunology, Microbiology and Infectious Diseases 23: Anonymous.. Salmonella Surveillance: Annual Sumary, US Department of Health and Human Services, CDC, Atlanta, Georgia. 3. Anonymous.. Salmonella Surveillance: Annual Summary, US Department of Health and Human Services, Atlanta, Georgia. 4. Anonymous Salmonella Surveillance: Annual Summary, US Department of Health and Human Services, Atlanta, Georgia. 5. Anonymous Preliminary FoodNet Data on the Incedence of Infection with Pathogens Transmitted Commonly Through Food Sites, United States, 2004, p Baggesen, D., D. Sandvang, and F. Aarestrup.. Characterization of Salmonella entrica Serovar Typhimurium DT104 Isolated from Denmark and Comparison with Isolates from Europe and the UNited States. J Clin Microbiol 38:

28 7. Berge, A., J. Adaska, and W. Sischo Use of Antibiotic Susceptibility Patterns and Pulsed-Field Gel Electrophoresis to Compare Historic and Contemporary Isolates of Multi-Drug-Resistant Salmonella enterica subsp. enterica Serovar Newport. Applied and Environmental Microbiology 70: Bryan, F. and M. Doyle Health Risks and Consequences of Salmonella and Campylobacter jejuni in Raw Poultry. Journal of Food Protection 58: Clark, G., A. Kaufmann, E. Gangarosa, and M. Thompson Epidemiology of an International Outbreak of Salmonella Agona. Lancet ii: Cook, W., R. Champion, and D. Ahearn Isolation of Salmonella enteritidis Serotype Agona from Eutrophic Regions of a Freshwater Lake. Applied Microbiology 28: D'Aoust, J Salmonella Species, p In M. Doyle, L. Beuchat, and T. Montville (eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington D.C. 12. Escartin, E., Lozano JS, O. Rodriguez, N. Gonzales, and J. Torres Incidence and level of Salmonella serovars in raw pork obtained from Mexican butcher shops. Food Microbiology 12: Fedorka-Cray, P., J. Gray, and C. Wray.. Salmonella Infections in Pigs, p In C. Wray and A. Wray (eds.), Salmonella in Domestic Animals. CABI, New York, NY. 16

29 14. Flint, J., Y. Van Duynhoven, F. Angulo, S. Delong, P. Braun, K. Martyn, E. Scallan, M. Fitzgerald, G. Adak, P. Sockett, A. Ellis, G. Hall, N. Gargouri, H. Walke, and P. Braam Estimating the Burden of Acute Gastroenteritis, Foodborne Disease, and Pathogens Commonly Transmitted by Food: An International Review. Clinical Infectious Diseases 41: Giles, W., A. Benson, M. Olson, R. Hutkins, J. Whichard, WInokur, and P. Fey DNA Sequence Analysis of Regions Surrounding bla CMY-2 from Multiple Salmonella Plasmid Backbones. Antimicrob Agents Chemother 48: Guinee, P., E. Kampelmacher, and H. Willems Six new Salmonella types isolated in Ghana (S.volta, S.agona, S. wa, S. techimani, S. mampong, S.tafao. Antonie Van Leeuwenhoek 27: Guthrie, R Salmonella. CRC Press, Inc., Boca Raton, FL. 18. Hafez, H. and S. Jodas.. Salmonella Infections in s, p In C. Wray and A. Wray (eds.), Salmonella in Domestic Animals. CABI, New York, NY. 19. Jay, J.. Foodborne Gastroenteritis Caused by Salmonella and Shigella, p In Modern Food Microbiology. Aspen Publication, Gaithersburg, Maryland. 17

30 20. Khakhria, R., D. Woodward, W. Johnson, and C. Poppe Salmonella isolated from humans, animals and other sources in Canada, Epidemiol Infect 119: Lindqvist, N., A. Siitonen, and S. Pelkonen.. Molecular Follow-Up of Salmonella enterica subsp. enterica Serovar Agona Infection in and Humans. J Clin Microbiol 40: Mead, P., L. Slutsker, V. Dietz, L. McCaig, J. Bresee, C. Shapiro, P. Griffin, and R. Tauxe.. Food-Related Illness and Death in the United States. Emerging Infectious Diseases 5: Pomeroy, B., K. Nagaraja, L. Ausherman, I. Peterson, and K. Friendshuh Studies of Feasibility of Producing Salmonella Free s. Avian Diseases 33: Poppe, C.. Salmonella Infections in the Domestic Fowl, p In C. Wray and A. Wray (eds.), Salmonella in Domestic Animals. CABI, New York, NK. 25. Synnott, M., M. Brindley, J. Gray, and J. Dawson.. An outbreak of Salmonella agona infection associated with pre-cooked turkey meat. Commun Dis Public Health 1: Threlfall, E., J. Frost, L. Ward, and B. Rowe Increasing spectrum of resistance in multiresistant Salmonella typhimurium. The Lancet 347:

31 27. Threlfall, E., M. Hampton, L. Ward, and B. Rowe Application of Pulsed-Field Gel Electrophoresis to an International Outbreak of Salmonella agona. Emerging Infectious Diseases 2: Wray, C. and R. Davies.. Salmonella Infections in, p In C. Wray and A. Wray (eds.), Salmonella in Domestic Animals. CABI, New York, NY. 29. Zhao, S., S. Qaiyumi, S. Friedman, R. Singh, S. Foley, D. White, P. McDermott, T. Donkar, C. Bolin, S. Munro, E. Baron, and R. Walker.. Charactrization of Salmonella enterica Serotype Newport Isolated from Humans and Food Animals. Journal of Clinical Microbiology 41:

32 CHAPTER 3 Literature Review of Antimicrobial Drugs A General Description of Antimicrobial Drug Classes Aminoglycosides are bactericidal drugs that are divided into three subclasses: narrow-spectrum, extended-spectrum, and miscellaneous aminoglycoside antibiotics. Narrow-spectrum aminoglycosides, such as streptomycin, are active against aerobic and gram-negative bacteria. Expanded-spectrum aminoglycosides are active against several gram-positive bacteria in addition to gram-negative aerobic bacteria. Antibiotics in this subclass include kanamycin, gentamicin and amikacin. Apramycin is considered a miscellaneous aminoglycoside since its chemical structure is slightly different from a typical aminoglycoside, yet is similar enough to be included in this class. Aminoglycosides are most effective against rapidly multiplying organisms. At therapeutic concentrations they are bactericidal, however, at subtherapeutic concentrations aminoglycosides are bacteriostatic. They have several modes of action and are effective with short exposure times. These drugs interfere with protein synthesis at the membrane-associated bacterial ribosome; this involves crossing both the cell wall and the cell membrane (23,33). Cephalosporins are a class of β-lactam antibiotics that are similar to penicillins. The subclasses are divided according to generations of cephalosporins. The newer generations have broader ranges of activity. First-generation cephalosporins are very 20

33 active against gram-positive bacteria but less active against gram-negative and anaerobic bacteria. These drugs are inactivated by β-lactamases. An example of a first-generation cephalosporin is cephalothin. Second-generation cephalosporins, which include cefoxitin, are active against gram-negative and gram-positive bacteria and are β-lactamase resistant. However, these antibiotics are ineffective against enterococci, Pseudomonas aeruginosa, Actinobacter spp. and obligate anaerobes. Third-generation cephalosporins include ceftiofur (a veterinary use only drug) and ceftriaxone (a human use only drug). They are active against a wide array of gram-negative bacteria but are only moderately effective against gram-positives. These drugs are highly resistant to β-lactamase enzymes. Thirdgeneration cephalosporins are capable of penetrating the blood-brain barrier and are therefore useful for treating bacterial meningitis. The mode of action for cephalosporins is similar to penicillins, they bind to penicillin-binding proteins located beneath the cell wall which results in an interference with the actions of transpeptidase and other cell wall enzymes (23,33). Chloramphenicol is a unique antimicrobial agent that is highly effective, well tolerated, and effective across a broad spectrum. Florfenicol is a thiamphenicol derivative of chloramphenicol. Chloramphenicol s mode of action includes inhibiting protein synthesis by binding to the 50 S subunit of the 70 S ribosome and impairing peptidyl transferase activity. Chloramphenicol is bacteriostatic except if used at high concentrations when it sometimes becomes bactericidal. Additionally, chloramphenicol inhibits protein synthesis in both prokaryotic and eukaryotic (mitochondrial) ribosomes (23,33). While still in use in human medicine, it has been banned from veterinary use (32). 21

34 Penicillins are a large and commonly used class of β-lactam antibiotics. There are many subclassifications of penicillin that are based on differences in antibacterial spectra. Broad-spectrum β-lactamase-sensitive penicillins include ampicillin and amoxicillin. Penicillins in this subclass are derived semisynthetically and are active against many gram-positive and gram-negative bacteria. These antibiotics are readily destroyed by the β-lactamases produced by many bacteria. Another subclass of penicillins, β-lactamaseprotected broad-spectrum penicillins, include naturally occurring and semisynthetic compounds that inhibit many of the β-lactamase enzymes produced by penicillin-resistant bacteria. These antibiotics are used in combination with broad-spectrum penicillins for a synergistic effect. The active penicillin is protected from enzymatic hydrolysis, resulting in full activity against previously resistant bacteria. Examples include clavulanatepotentiated amoxicillin and ticarcillin. Carbapenems, also a subclass of penicillins, includes one the most active drugs against a wide variety of bacteria, imipenem. Imipenem is derived from a compound produced by Streptomyces cattleya. Penicillin s mode of action is to impair the development of bacterial cell walls by interfering with transpeptidase enzymes responsible for the formation of the cross-links between peptidoglycan strands. Penicillin antimicrobials have minimal effect on already formed bacterial cell walls and therefore organisms must be actively multiplying or growing to be susceptible. Both gram-positive and gram-negative bacteria have these penicillinbinding proteins and the effects of penicillin are usually bactericidal. β-lactam antibiotics are able to promote phagocytosis at concentrations below minimal inhibitory concentrations (MIC) (23,33). 22

35 Quinolones are synthetic antimicrobial agents derived from carboxylic acid. Subclasses include quinolone carboxylic acids such as fluoroquinolones and Naphthydridine carboxylic acids such as quinolones. Ciprofloxacin and nalidixic acid are examples of fluoroquinolones and quinolones, respectively. Quinolone s mode of action focuses on the DNA gyrase which, when inhibited, reduces the ability of DNA to supercoil and leaves DNA nicks exposed to endonucleases that fragment the chromosomal DNA. Quinolones are bactericidal and fast acting (20 minutes). Furthermore, there can be a post-antibiotic effect in some bacteria that lasts 4-8 hours after exposure. Fluoroquinolones have significant antibacterial activity at extraordinarily low concentrations (23,33). Sulfonamides are divided into subclasses based on their use and duration of action. Standard use sulfonamides control systemic infections and include sulfamethoxazole. Sulfonamides mode of action is complex and results in the blocking of essential enzymes, which ultimately results in suppression of protein synthesis, metabolic pathways, growth and multiplication of organisms that cannot use preformed folate. Sulfonamides are bacteriostatic and are most effective in the early stages of an infection when the organism is rapidly multiplying. Often there is a latent period before the effects of sulfonamides are evident. A potentiated sulfonamide, when a diaminopyrimidine is used in combination with a sulfonamide, is trimethoprim-sulfamethoxazole. These combinations are able to create a cascade of blocked microbial enzyme systems, which result in bactericidal consequences (23,33). Tetracyclines are a class of broad-spectrum antibiotics including naturally occurring tetracyclines, semisynthetically derived tetracyclines, short-action tetracyclines 23

36 and long acting tetracyclines. The drug tetracycline is both semisynthetically derived and short-acting. Tetracyclines bind reversibly to 30 S ribosomes and inhibit protein synthesis, however, the exact site of the antimicrobial activity has not been clarified and there may be several mechanisms of action (23,33). Antimicrobial Drug use in Food Animals The modern agricultural practices in the food animal industry have created a new set of management concerns and interactions. There is often less space available resulting in closer animal to animal contact and therefore preventative health measures are often more beneficial than therapeutic ones. In 1991, 76 million pounds of antibiotics were used by humans and animals in the United States and 25% of that was used solely by the food animal industry (32). The food animal industry uses antimicrobials in three main ways, therapeutically to treat disease, prophylactically at subtherapeutic concentrations to prevent disease, and subtherapeutically for production enhancement. In 1985, 18 million pounds of antibiotics were used by the U.S. food animal industry and 90% of those antibiotics were used subtherapeutically (32). Subtherapeutic use of antimicrobials is believed by the food animal industry to increase production/performance, increase the efficient use of feeds for growth or production output and modify the nutrient composition of animal products. It is generally agreed that the use of antimicrobials at subtherapeutic concentrations results in a shorter duration of time needed for weight gain. Although there are several theories, it remains unknown how subtherapeutic antimicrobial treatment enhances weight gain (34). The mystery of antimicrobial benefits has not slowed down its application as 60% to 80% 24

37 of all U.S. livestock and poultry will receive antimicrobials in feed, water, or by systemic injections during their lifetime (32). Poultry: The majority of antimicrobials used in the integrated U.S. poultry industry are for prophylactic purposes. Administration to the entire flock through water, often referred to as mass-medication, is the only feasible way to treat poultry that are raised in such large numbers and in such close proximity to each other. The three most serious bacterial diseases affecting poultry are caused by Salmonella, E. coli, and Clostridium (32). At day of hatch chicks may be injected with either ceftiofur sodium or gentamicin sulfate (32). Other antimicrobials approved for use in poultry include penicillins, aminoglycosides, sulfonamides, cephalosporins and tetracyclines (26,32). Use of tetracyclines and penicillins as growth promoters are discouraged due to public health concerns about an increase in the number of tetracycline and penicillin resistant bacteria being observed (32). Furthermore, the FDA recently reversed approval for enrofloxacin, a fluoroquinolone, in poultry due to concerns of cross resistance to ciprofloxacin, a fluoroquinolone commonly used to treat human systemic infections (4). : There are 21 antimicrobials approved by the U.S. FDA for use in the swine industry. are often given subtherapeutic concentrations of antibiotics in their feed especially during times of stress such as at weaning or transportation (26). Antimicrobials commonly used by the swine industry include, but are not limited to, amoxicillin, ampicillin, apramycin, gentamicin, streptomycin, and tetracyclines. Tetracyclines and sulfonamides are two of the most frequently used antimicrobials in swine (26). 25

38 : In cattle the therapeutic use of antimicrobials is often necessary; pneumonia and diarrhea are major causes of calf mortality and calves are treated individually or medicated as a group (26). Furthermore, antimicrobials are administered in feedlots to control liver abscesses, accelerate weight gain, and prevent or treat respiratory disease outbreaks (26). In, 83% of cattle in feedlots received at least one antimicrobial in feed or water for prophylaxis or growth promotion (26). There are 17 antimicrobials that are U.S. FDA approved for use in cattle. These include, but are not limited to, amoxicillin, ampicillin, ceftiofur, gentamicin, streptomycin, and tetracycline (32). Approximately 50% of cattle feedlots use florfenicol, tetracyclines or some combination of these drugs, 38% use cephalosporins, 31% use penicillins, and 32% use fluoroquinolones for individual animal therapy (26). producers are being pressured to minimize the use of tetracyclines and penicillins as growth promoters due to public health concerns about the rise in resistance among bacteria to these classes of drugs (32). Furthermore, aminoglycosides are not approved for growth promotion in cattle and should not be administered to cattle in the U.S. that are intended for food (33). Concerns and Consequences: In developed countries, much of food animal production uses a broad range of antimicrobials and delivery mechanisms. The public has voiced concerns from the use of antimicrobials in food animals, particularly when the drugs are made available indiscriminately through feed or water or given at low concentrations for extended periods of time as with growth promotion. The widespread delivery of antimicrobials has brought into question the residual effects of antimicrobials not only in the meat products themselves but in the environment as a result of run off and waste disposal (34). Furthermore, the use of antimicrobials in food animals at low 26

39 concentrations for disease prevention or growth promotion has caused concern about antimicrobial drug resistance increasing in zoonotic enteropathogens. There is a fear that this use of antimicrobials is eliminating sensitive organisms and causing the development of a large gene pool of antimicrobial drug resistant organisms. This poses a potential threat to humans requiring treatment, especially the very young, very old, and immune compromised. In addition, this population of resistant enteropathogens may transfer resistance genes to commensal enteric bacteria, which may affect normally healthy individuals under certain circumstances, such as the development of resistant postsurgical infections. Interestingly, antimicrobial resistance of Salmonella has been reported less frequently in underdeveloped countries than in industrialized nations (22). The World Health Organization (WHO) has called for the halt of the use of antimicrobials as growth promoters if the antimicrobial belongs to a class of drugs used in human medicine (2,22). WHO objectives include decreasing the selective pressures for antimicrobial resistances and therefore expanding the useful life of presently available antimicrobials (9). Currently in the U.S., many antimicrobials and animal feeds with antimicrobials as additives are available over the counter (26). Also, pharmaceutical companies, retailers and some veterinarians make profit off of the prescription and sale of antimicrobials (26). Veterinarians in the U.S. also have the permission to prescribe extralabel uses for antimicrobials, with the exception of fluoroquinolones (23). Some countries such as Denmark and Sweden have prohibited the use of antimicrobials for growth promotion in food animals, required prescriptions from veterinarians for therapeutic/subtherapeutic uses, and have removed the economic incentives to write extra prescriptions (2,9,26). This has successfully decreased the amount of antimicrobials 27

40 being used on food animals in these countries without detrimental consequences to the farmers (2,26). Furthermore, it appears that in some cases resistant strains are in the decline. With the ever encroaching fears of antimicrobial drug resistance it is feasible that more countries will follow in Denmark and Sweden s footsteps. However, care should be taken before drawing sweeping conclusions from such a pro-active change in policy. Antimicrobial Drug Resistance Since the widespread use of antibiotics in humans began in the 1940 s, antimicrobial resistance has been observed in almost all bacterial species, against all classes of drugs and has been spreading into new clinical niches (21,22). There is a global trend towards sustained increases in the number of antibiotic-resistant Salmonella strains in humans and animals (3,12,13). Factors contributing to this trend appear to be the liberal administration of antimicrobial agents in human medicine and the widespread use of antibiotics to meet therapeutic, preventative and growth promotion needs in food animal production. Most Salmonella are sensitive to antimicrobial drugs; however, increases in resistance are being observed among specific serovars. Researchers have reported that of Salmonella recovered from retail meat in the US, 68% were resistant to tetracycline, 61% to streptomycin, 42% to sulfamethoxazole, and 29% to ampicillin (10). Animal NARMS data, which included slaughter, diagnostic and on farm samples, from showed a dramatic increase in percent of S. Agona isolates resistant to 8 of the antimicrobials tested ( Salmonella Agona, specifically, showed an increasing percent resistance to critical antimicrobials 28

41 such as ceftriaxone, a cephalosporin used to treat children with salmonellosis and nalidixic acid, a quinolone, a class of drugs that are being monitored due to their similarities with fluoroquinolones which includes the drug of choice for treatment of adult salmonellosis (1,10,37) ( Additionally, there is an increase in multiple drug resistance (MDR) and an increase in resistance determinants integrated into the Salmonella chromosome (22). The use of new antimicrobial drugs appears to lead to the development of new resistance attributes (22). Antimicrobial resistance can increases morbidity and mortality for humans and animals, resulting in social and economic consequences (1,2,21,26,27,32,37). Determining the true economic impact of antimicrobial drug resistance is a challenge due to the many variables and perspectives involved. However, The Institute of Medicine estimated the annual cost of human infections resulting from antimicrobial drug resistant organisms in the U.S. is $4 to $5 million (27). This estimate would increase greatly if the economic impact of antimicrobial drug resistance in food animals was also taken into account. Outside of the direct implications of failed treatment on an ill patient, and the economic loss to individuals and health care groups, antimicrobial drug resistant has significant societal implications that stretch worldwide. The emergence of a super pathogen that is capable of resisting all available treatments is cause for concern. Available drugs would become obsolete and the development of new drugs is costly ($800 million from development through the approval process) and time consuming (10 to 15 years) (34). Furthermore, there is concern that pathogens would quickly develop resistance to the newly marketed drugs. It is also disconcerting that developed nations are footing the bill for new drugs and undeveloped countries may 29