Search
Author
Title
Vol.
Issue
Year
1st Page

# Journal of Animal Science - Article

1. Vol. 86 No. 14_suppl, p. E173-E187

Accepted: Aug 30, 2007
Published: December 5, 2014

2 Corresponding author(s):
View
Share

doi:10.2527/jas.2007-0447

# Food animal-associated Salmonella challenges: Pathogenicity and antimicrobial resistance1

1. S. L. Foley2 and
2. A. M. Lynne
1. National Farm Medicine Center, Marshfield Clinic Research Foundation, Marshfield, WI 54449

## Abstract

Salmonellosis is a worldwide health problem; Salmonella infections are the second leading cause of bacterial foodborne illness in the United States. Approximately 95% of cases of human salmonellosis are associated with the consumption of contaminated products such as meat, poultry, eggs, milk, seafood, and fresh produce. Salmonella can cause a number of different disease syndromes including gastroenteritis, bacteremia, and typhoid fever, with the most common being gastroenteritis, which is often characterized by abdominal pain, nausea, vomiting, diarrhea, and headache. Typically the disease is self-limiting; however, with more severe manifestations such as bacteremia, antimicrobial therapy is often administered to treat the infection. Currently, there are over 2,500 identified serotypes of Salmonella. A smaller number of these serotypes are significantly associated with animal and human disease including Typhimurium, Enteritidis, Newport, Heidelberg, and Montevideo. Increasingly, isolates from these serotypes are being detected that demonstrate resistance to multiple antimicrobial agents, including third-generation cephalosporins, which are recommended for the treatment of severe infections. Many of the genes that encode resistance are located on transmissible elements such as plasmids that allow for potential transfer of resistance among strains. Plasmids are also known to harbor virulence factors that contribute to Salmonella pathogenicity. Several serotypes of medical importance, including Typhimurium, Enteritidis, Newport, Dublin, and Choleraesuis, are known to harbor virulence plasmids containing genes that code for fimbriae, serum resistance, and other factors. Additionally, many Salmonella contain pathogenicity islands scattered throughout their genomes that encode factors essential for bacterial adhesion, invasion, and infection. Salmonella have evolved several virulence and antimicrobial resistance mechanisms that allow for continued challenges to our public health infrastructure.

### INTRODUCTION

Infections by Salmonella enterica are a significant public health concern around the world. In the United States alone, it is estimated that there are approximately 1.4 million cases of Salmonella infections resulting in 17,000 hospitalizations and 585 deaths each year (Mead et al., 1999; Voetsch et al., 2004). The number of cases of salmonellosis increased steadily from the mid 1940s through the late 1980s. Since then, the number of cases has decreased slightly, with a total of more than 35,000 cases reported in 2005 (CDC, 1993; McNabb et al., 2007). It is estimated that for every case diagnosed and reported to the Centers for Disease Control and Prevention (CDC), there are approximately 38 that are not reported (Mead et al., 1999). Salmonella is responsible for an estimated 26% of all infections caused by foodborne pathogens in the United States, with 95% of human salmonellosis cases associated with the consumption of contaminated of food products (Mead et al., 1999). The impact of Salmonella infections on the US economy is significant, costing an estimated $2.3 billion to$3.6 billion due to loss of work, medical care, and loss of life (Buzby et al., 1996; Frenzen et al., 1999).

### BACKGROUND

Salmonellosis can manifest in a number of disease syndromes including gastroenteritis, bacteremia, typhoid fever, and focal infections (Darwin and Miller, 1999). Typhoid fever is a unique disease manifestation associated with an infection by Salmonella enterica serovar Typhi; it is associated with human-to-human transmission and is not covered in this review, which focuses on foodborne Salmonella. The most common manifestation of nontyphoid salmonellosis is mild to moderate gastroenteritis, consisting of diarrhea, abdominal cramps, vomiting, and fever. Typically, symptoms of gastroenteritis develop within 6 to 72 h after ingestion of the bacteria (CDC, 2001; Pegues et al., 2005). The symptoms are usually self-limiting and typically resolve within 2 to 7 d. In a small percentage of cases, septicemia and invasive infections of organs and tissues can occur, leading to diseases such as osteomyelitis, pneumonia, and meningitis (Cohen et al., 1987). People who are very young, very old, or immunocompromised are most susceptible to these severe manifestations of salmonellosis, which typically require antimicrobial therapy (Benenson et al., 1995). The recommended drugs for Salmonella septicemia are fluoroquinolones or ceftriaxone. Fluroquinolones are not approved for use in children under the age of 16 due to concerns about cartilage damage; therefore, ceftriaxone is an important line of therapy for this age group (Gilbert et al., 2004).

There are a number of different Salmonella that are capable of causing human disease. Historically, Salmonella were divided into separate species based on the results of serotyping; for each serotype (serovar), there was a separate species (Brenner et al., 2000). Serotyping uses differences in the polysaccharide portion of lipopolysaccharide layer (O antigen) and the filamentous portion of the flagella (H antigen) present on the surface of Salmonella to separate strains into distinct serotypes (Voogt et al., 2002; CDC, 2006). Currently, there are over 2,500 identified Salmonella serotypes. Most serotypes share a very high degree of genetic similarity, and because of this similarity, the Salmonellae are now divided into just 2 species, Salmonella enterica and Salmonella bongori. Over 99% of the serotypes are grouped into the species S. enterica, which contains all of the major serovars that are pathogenic to humans (Brenner and McWhorter-Murlin, 1998; Brenner et al., 2000; Popoff et al., 2001). Even with the changes in speciation, the serotype name remains the main taxonomic descriptor for Salmonella isolates. Serotyping is performed by mixing a suspension of Salmonella with a series of antisera specific for a variety of O and H surface antigens. Following incubation, the mixtures are observed for agglutination, and the specific agglutination profile is used to determine the serotype of an isolate (Brenner et al., 2000; Voogt et al., 2002). The particular serotypes are designated using the Kauffmann-White scheme used by public health organizations (Popoff et al., 2001; CDC, 2006).

There are a number of commonly identified serotypes of Salmonella associated with human infections in the United States (Table 1), with the most common serovars being Typhimurium, Enteritidis, Newport, Heidelberg, and Javiana (CDC, 2005). In other parts of the world, there are some differences in the predominant serovars associated with disease. In the European Union, Enteritidis is the predominant serovar. In many parts of Asia, Choleraesuis is one of the top serovars, whereas in the United States, there are very few human cases reported of salmonellosis caused by Choleraesuis (only 13 in 2005; Chiu et al., 2004; CDC, 2006; de Jong and Ekdahl, 2006). Salmonellosis is most often attributed to the consumption of contaminated foods such as poultry, beef, pork, eggs, milk, seafood, nut products, and fresh produce. Direct contact with infected animals may also serve as a source for Salmonella infections (Tauxe, 1991; Benenson et al., 1995). When the common serovars from human infections are compared with those most commonly isolated from different food-animal sources (Table 1), there are a number of instances of overlap (CDC, 2005). Of the top 10 most-common serovars causing human infections, 8 are also in the top 10 most-identified serovars from at least 1 of the major food-animal species. Because of the overlap in serotypes, there is a likelihood of pathogen spread through the food supply. Consumers in the United States have continued to eat greater amounts of meat and poultry over the past century. Per capita consumption of poultry products has increased more than 6.5-fold since 1910, whereas beef and pork consumption has increased approximately 20% (Buzby and Farah, 2006). With this increase in consumption of meat and poultry comes increased potential for exposure to Salmonella through these food commodities. Because of the potential for pathogen exposure through the food supply, the US government and food producers have developed improved programs for pathogen reduction and for response following a potential contamination of the food supply to protect consumers (Swaminathan et al., 2001; Rose et al., 2002).

## VIRULENCE AND PATHOPHYSIOLOGY

### Infection and Intestinal Attachment

The primary route of Salmonella infection in humans and other animal species is the fecal-oral transmission of the organism. The estimates of the number of organisms required to cause disease are quite variable, ranging from about 30 to more than 109 infectious organisms (Morgan et al., 1994; Vought and Tatini, 1998). This variability in infectious dose is due, in part, to the food matrix contaminated with Salmonella and to intrinsic factors of the infecting organisms (Giannella et al., 1972, 1973; Blaser and Newman, 1982). The infectious dose appears to be lower if the contaminated food that is consumed has a high fat content, such as cheese or ice cream (Vought and Tatini, 1998; de Jong and Ekdahl, 2006). In order to reach their sites of colonization, Salmonella must be able to survive the antimicrobial properties of the stomach, including the low pH and the presence of many organic acids. Salmonella have evolved mechanisms that allow for survival at low pH. Many acid shock proteins are associated with the acid tolerance response (ATR; Foster, 1991). Some of the major ATR regulatory factors include the RpoS σ-factor, PhoPQ, and Fur proteins (Bearson et al., 2006). The RpoS and PhoPQ proteins are important for the regulation of survival in the low pH environment created by inorganic acids, whereas Fur and RpoS are involved in the regulation of organic acid tolerance (Bearson et al., 1998). It appears that these regulatory proteins may play a role in the expression of multiple ATR proteins including molecular chaperones, cellular regulatory proteins, transcription and translation factors, envelope proteins, and fimbriae (Bearson et al., 1998, 2006). The exact mechanism that each protein plays in acid tolerance is not well understood; however, it is clear that multiple factors contribute to the overall process. Interestingly, there have been reports of other routes of infection besides fecal-oral that appear to lead to colonization of the gastrointestinal tract. In cattle and swine, the respiratory system and tonsils are potential sites of invasion by Salmonella (deJong and Ekdahl, 1965; Fedorka-Cray et al., 1995). The pathogens can then be transported to additional locations throughout the body by hematogenous or lymphatogenous spread. If the lungs are the initial site of colonization, Salmonella may be able to more easily enter the bloodstream due to the proximity of the circulatory system and lead to the development of septicemia (Fedorka-Cray et al., 1995).

Salmonella entering via the fecal-oral route that survive the low pH environment of the stomach are able to colonize multiple sites including the small intestine, colon, and cecum. Intestinal adhesion is mediated by fimbriae or pili present on the bacterial cell surface. There are many types of fimbriae associated with Salmonella that may play a role in colonization including type 1 fimbriae (Fim), long polar fimbriae (Lpf), thin aggregative or curli fimbriae, and plasmid-encoded fimbriae (Pef) (Darwin and Miller, 1999). The Fim bind to specific α-d-mannose receptors on the surface of multiple cell types. Type 1 fimbriae are made up of 7 genes (fimAICDHF). The major structural subunit is FimA, and FimH is the subunit that interacts with host cell surface receptors to facilitate attachment (Darwin and Miller, 1999; Duncan et al., 2005). Differences in the genetic sequence of fimH, and likely fimA, play a role in host cell tropism demonstrated by some pathogens (Duncan et al., 2005). The other types of fimbriae also appear to demonstrate host tissue specificity: Lpf binds to the surface of the Peyer’s patches and M cells, Pef binds to the villous intestine, and curli fimbriae likely bind to the small intestine (Baumler et al., 1996a,b; Sukupolvi et al., 1997; Althouse et al., 2003).

### Salmonella Pathogenicity Island-1 Type III Secretion System: Intestinal Invasion and Diarrhea Formation

Salmonella have evolved intricate measures to invade host cells following epithelial attachment. Following interaction with host cells, Salmonella can express a type III secretion system (T3SS), which facilitates endothelial uptake and invasion (Figure 1). The major T3SS regulatory protein is HilA, whose expression is mediated by a number of environmental factors important for cell survival (Lostroh and Lee, 2001). The T3SS is a complex of proteins that allows for the transfer of virulence factors directly into the host cells and is associated with at least 20 structural and regulatory proteins involved in cellular invasion (Marlovits et al., 2004; Galán and Wolf-Watz, 2006). The base structure of the T3SS complex spans the cell membrane and the cell wall of Salmonella, and a needle structure protrudes from the base that interacts with host cells. Within the base and needle structure is an inner rod that forms the conduit between the bacterial cytoplasm and the host cell membrane [see Galán and Wolf-Watz (2006) for a comprehensive review on T3SS structure]. On the cytoplasm side of the T3SS structure, there is a set of export machinery that contains an ATPase complex that facilitates the transport of effector molecules through the inner rod to a translocase structure in the host cell membrane.

The genes that encode the T3SS machinery are associated with Salmonella pathogenicity island 1 (SPI-1). Pathogenicity islands (PI) are genetic elements that carry genes encoding virulence factors, such as adhesion, invasion, and toxin genes (Hacker et al., 1990, 1997). The PI can be located on the chromosome or on a plasmid, are flanked by repeat sequences, and tend to have a varied G/C composition compared with the surrounding genome. In the bacterial genome, the PI are typically associated with tRNA genes, are often mobile, and can move to different tRNA loci. Some PI contain other genetic structures such as transposons, integrons, or insertion sequences (Gal-Mor and Finlay, 2006). The T3SS structural genes located on SPI-1 include prgHIJK, spaMNOPQRS, and invABCEFGH, as well as multiple regulatory and effector genes (Lostroh and Lee, 2001).

The assembly of the SPI-1 T3SS appears to be built from the base up. An assembly model starts with the assembly of the inner ring structure, which spans the cell membrane and is assembled from PrgH and PrgK protein subunits (Galán and Wolf-Watz, 2006). Next, the cytoplasmic export machinery, which is composed of the InvA, InvC, SpaP, SpaQ, SpaR, and SpaS proteins, is assembled. Also, the outer ring structure, composed of InvG and InvH, is assembled in the outer membrane and connected to the inner ring structure and stabilized with the aid of the regulatory protein InvJ. The completed base structure allows for the assembly of the needle and inner rod structures, which are made up of PrgJ and PrgI subunits, respectively (Marlovits et al., 2004; Galán and Wolf-Watz, 2006).

The completed SPI-1 T3SS allows for effector proteins to be translocated from the bacterial cytoplasm to the host cell. In the bacterial cytoplasm, chaperone molecules bind to the effector proteins and accompany the molecule to the export machinery of the T3SS (Figure 2). The chaperone interacts with the ATPase allowing the effector protein to be released from the chaperone and enter the needle structure for translocation into the host cell. The needle structure cannot directly inject the proteins into the cytosol of the host cells. To facilitate transfer of proteins into the host cell cytosol, the needle structure interacts with a translocation complex embedded in the host cell membrane. The proteins that make up translocation complex are produced by the bacterium and are some of the initial effector molecules secreted by the T3SS. These proteins are likely inserted into the host cell membrane to form a channel for delivery of additional effector proteins into the cytoplasm. The needle complex, either directly or facilitated by accessory proteins at the needle tip, interacts with the translocation complex to allow for delivery of bacterial proteins into the host cytosol (Figure 2; Galán and Wolf-Watz, 2006).

One of the major clinical features of salmonellosis is diarrhea, which is caused by SPI-1 T3SS translocated proteins. The SopB protein appears to play an important role in the activation of secretory pathways, the attraction of neutrophils to the sites of infection (thereby increasing inflammation), and an alteration of ion balances within cells (Wallis and Galyov, 2000). Additionally, SopB is an inositol phosphate phosphatase, which likely influences the ion balance in cells through the antagonism of chloride channels in the infected cells. The alteration of ion balances within the cells can lead to fluid secretion into the intestinal tract and subsequent diarrhea (Norris et al., 1998). Other proteins such as SipA, SopA, SopD, and SopE2 may also play a role in Salmonella-associated gastroenteritis (Wallis and Galyov, 2000; Zhang et al., 2003). Table 2 shows some of the translocated proteins and their functions.

A number of additional proteins are translocated via the SPI-1 T3SS including SipA, SipC, and SopB, which interact with the actin cytoskeleton causing cytoskeletal rearrangements leading to membrane ruffling (Lostroh and Lee, 2001; McGhie et al., 2001). Membrane ruffling is characterized by a rearrangement of the cell membrane and cytosol such that the Salmonella bacterium is surrounded by the host cell and internalized (Figure 3; Jones et al., 1993; Goosney et al., 1999). Once the Salmonella organism is internalized, the microorganism resides in the membrane bound Salmonella-containing vacuole (SCV; Figure 3; Knodler and Steele-Mortimer, 2003). As the SCV matures, it migrates from the luminal border of the cell to the basal membrane where the Salmonella interact with and enter macrophages associated with Peyer’s patches in the submucosal space (Ohl and Miller, 2001; Pegues et al., 2005). The formation of SCV likely occurs separately from the normal endocytic processing pathways present in host cells. As the SCV matures, it acquires some of the endosomal markers involved in intracellular processing; however, it does not fuse with lysosomal compartments (Unsworth and Holden, 2000; Alonso and Garcia-del Portillo, 2004). Because of this separation, Salmonella avoid being killed by the normal phagolysosomal processing pathways (Holden, 2002). The SCV are important for Salmonella survival and transport in epithelial cells and play a key role in the survival of the bacterium within phagocytic cells such as macrophages during invasive infections. Therefore, the ability to survive and proliferate in the SCV is a major virulence factor for Salmonella (Tierrez and Garcia-del Portillo, 2005).

### Salmonella Pathogenicity Island-2 Type III Secretion System: Intracellular Survival and Systemic Infection

Invasive Salmonella infections are associated with T3SS encoded on SPI-2. The SPI-2 T3SS genes are only expressed inside the host cell SCV. Many of the genes encoded on SPI-2 encode for the structure of secretion system apparatus (ssaG through ssaU), secretion system effector (sseABCDEF), secretion system chaperones (sscAB), and secretion system regulatory (ssrAB) genes required for a functional T3SS (Hensel, 2000). The SPI-2 T3SS secretion apparatus expression is regulated through SsrA-SsrB 2-component regulatory system, which is subsequently regulated by a second 2-component regulatory system, OmpR-EnvZ (Lee et al., 2000; Garmendia et al., 2003). A number of environmental conditions have been associated with inducing the expression of the SPI-2 T3SS genes through the OmpR-EnvZ regulatory system, including low osmolarity, low levels of certain nutrients, and acidification of the SCV (Cirillo et al., 1998; Lee et al., 2000). The activated SPI-2 T3SS functions to transfer effector proteins from Salmonella across the SCV membrane to interact with targets in the host cells.

Many SPI-2 T3SS effector proteins including SifA, SseF, and SseG interact with microtubule bundles and their associated motor proteins and are involved in the formation of Salmonella-induced filaments (SIF) that extend from SCV (Table 3 and Figure 3; Waterman and Holden, 2003; Abrahams and Hensel, 2006). The formations of SIF are likely the result of the fusion of SCV with other vesicles in the cell (Abrahams and Hensel, 2006). The major functions of SIF are not fully understood; however, it is likely that SIF are important for pathogenesis and may play a role in intracellular replication of Salmonella because their formation often coincides with replication of the microorganisms (Knodler and Steele-Mortimer, 2003). An additional SPI-2 T3SS effector protein, SpiC, is translocated into the cytosol of host macrophages, where it interacts with the endomembrane system and likely interferes with the normal secretory pathways of the host (Uchiya et al., 1999). This disruption likely protects organisms from bactericidal compounds, including reactive oxygen and reactive nitrogen molecules that are able to kill many types of bacteria (Hensel, 2000).

Systemic infections are severe manifestations of salmonellosis. To facilitate systemic infection, intracellular Salmonella present in immune cells such as macrophages and dendritic cells (DC) may be carried from the intestinal tract to other areas of the body. Dendritic cells are important migratory phagocytes that are widely distributed throughout the body in lymphoid and nonlymphoid tissues (Sundquist et al., 2004). The ability of DC to migrate throughout the body potentially facilitates the spread of Salmonella to various parts of the body. While in the DC, the Salmonella do not appear to replicate but remain viable, possibly in a small colony variant state with reduced metabolic activity and increased persistence (Tierrez and Garcia-del Portillo, 2005). Genes encoded on SPI-2 T3SS appear to suppress antigen presentation by DC, which limits a robust immune response to the infected cell (Waterman and Holden, 2003). The combination of lowered metabolic activity and immunosuppression likely contributes to the persistence of Salmonella within host cells. When the macrophages or DC enter certain organ systems, the Salmonella can spread to adjacent cells and trigger apoptosis, which leads to increased pathology among the infected cells (Richter-Dahlfors et al., 1997; Sheppard et al., 2003; Tierrez and Garcia-del Portillo, 2005).

### Plasmid-Encoded Virulence

In addition to the virulence factors associated with the SPI-1 and SPI-2 T3SS, some factors can be found on plasmids, which are extrachromosomal, circular DNA molecules that typically contain genes that impart selective advantage to the host, such as virulence or anti-microbial resistance (Richter-Dahlfors et al., 1997; Rotger and Casadesus, 1999; Sheppard et al., 2003). The plasmids that are known to carry virulence gene clusters are called virulence plasmids. Strains from many serovars lack virulence plasmids; however, some of the most important serovars for human health, including Typhimurium, Enteritidis, and Choleraesuis are known to harbor virulence plasmids (Lu et al., 1999; Chiu et al., 2005; Villa and Carattoli, 2005; Yu et al., 2006). These virulence plasmids have a genetic region called Salmonella plasmid virulence, which contains spvRABCD genes. The spv genes appear to be important for bacterial multiplication within the host cells during extraintestinal infections (Gulig et al., 1993; Guiney et al., 1995). Additional virulence genes located on virulence plasmids include those encoding fimbriae (pef-BACDI) and serum resistance (traT; Rotger and Casadesus, 1999). Although most virulence plasmids are not self-transmissible, some appear to contain a full concert of transfer (tra) genes that allow the plasmids to be transferred to additional strains by conjugation (discussed subsequently), potentially increasing the virulence of the recipients (Ahmer et al., 1999). Because of their conservation among members of a particular serovar, virulence plasmids likely provide a significant advantage to the strains harboring plasmids.

## ANTIMICROBIAL RESISTANCE

### Trends in Salmonella Resistance

Not only is Salmonella a public health concern due to the number of cases per year, but many strains are resistant to a number of antimicrobial agents. The National Antimicrobial Resistance Monitoring System (NARMS) is a collaborative program among the Food and Drug Administration (FDA), the USDA, and the CDC that tracks resistance of certain enteric bacteria to antimicrobial agents (Tollefson et al., 1998). The antibiotics tested by NARMS include amikacin, amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfasoxazole, tetracycline, and trimethoprim/sulfamethoxazole (FDA, 2006). In a recent summary of resistance trends, the NARMS Executive Report indicated that in 2003, 22.5% of non-Typhi Salmonella isolates from humans were resistant to at least 1 antimicrobial agent, which is a decrease from the 33.8% reported in 1996 (FDA, 2006). The most common multidrug resistance phenotype reported was to ampicillin, chlorampheniol, streptomycin, sulfonamides, and tetracyclines (ACSSuT), which was detected in 9.3% of isolates tested. The resistance trends for individual antibiotics are summarized in Figure 4. The antibiotics with the greatest percentage of resistant isolates include ampicillin, streptomycin, sulfonamides, and tetracyclines; however, the percentage of isolates resistant to these drugs has decreased or remained stable since 1996. Drugs that have an upward trend in resistance include amoxicillin/clavulanic acid, ceftriaxone, ceftiofur, and nalidixic acid, but there remains a relatively low prevalence of resistance among Salmonella isolated from humans (< 6%; FDA, 2006).

On the veterinary side, 44% of the Salmonella samples isolated from animal slaughter and veterinary diagnostic sources were resistant to at least 1 antimicrobial agent (FDA, 2006). The ACSSuT phenotype was also the most common multidrug resistance profile among veterinary isolates. In 2004, 4.8% of isolates displayed this phenotype, with most originating from diagnostic sources (USDA, 2006). Overall, when antimicrobial resistance data from the veterinary and human NARMS reports were compared, the trend appeared to indicate that the percentage of resistant isolates was greater in strains of veterinary origin (FDA, 2006). The trends for individual antibiotics are summarized in Figure 5. Veterinary-associated Salmonella isolates demonstrated the greatest percentage of resistance to ampicillin, streptomycin, sulfonamides, and tetracyclines. Resistance to each of the drugs was on an upward trend from 1997 to 2004, before a slight decrease in 2005. There have been sharp increases in resistance to amoxicillin/clavulanic acid, ceftriaxone, and ceftiofur among veterinary-associated Salmonella isolates. The percent-age of isolates resistant to amoxicillin/clavulanic acid and ceftiofur increased from <2% in 1997 to >15% in 2005, and resistance to ceftriaxone has increased from no resistance through 1998 to nearly 1% resistance in 2005. The increase in resistance among Salmonella to extended-spectrum cephalosporins (ceftiofur and ceftriaxone) has become a significant public health concern because ceftriaxone is an important drug for the treatment of severe salmonellosis in children (Rabsch et al., 2001). Ceftiofur is the only extended-spectrum cephalosporin approved for veterinary use in the United States (Bradford et al., 1999). Because ceftiofur-resistant organisms are cross resistant to ceftriaxone, the use of this antimicrobial agent in food animals has come under increasing scrutiny as a selective agent potentially responsible for the emergence and dissemination of ceftriaxone resistance in Salmonella and other enteric pathogens (Alcaine et al., 2005). Resistance rates to the remaining antibiotics tested have remained stable over the past decade (FDA, 2006; USDA, 2007).

### Mechanisms of Antimicrobial Resistance

The increased level of antimicrobial resistance observed in Salmonella has become a public health issue. The development of resistance in Salmonella toward antimicrobial agents is attributable to one of multiple mechanisms, including production of enzymes that inactivate antimicrobial agents through degradation or structural modification, reduction of bacterial cell permeability to antibiotics, activation of antimicrobial efflux pumps, and modification of the cellular target for drug (Sefton, 2002). Much of the observed resistance to cephalosporins and penicillins by Salmonella is attributable the acquired ability of the strains to produce β-lactamase enzymes that are able to degrade the chemical structure of the antimicrobial agents. The β-lactamases are a diverse group of enzymes, some with affinities for the structures of a limited number of antimicrobial agents, whereas others are extended- or broad-spectrum β-lactamases that are able to degrade a wide array of antibiotics (Bush, 2003). The production of β-lactamases by Salmonella has become an important and common mechanism for β-lactam resistance (Revathi et al., 1998; Bush, 2003). Among the more worrisome β-lactamases is the AmpC enzyme, which is encoded by blacmy and has been associated with resistance to a large number of β-lactam antibiotics including ampicillin, ceftiofur, and ceftriaxone (Aarestrup et al., 2004). In other cases, enzymes inactivate antimicrobial agents by modifying their structures. Much of the aminoglycoside resistance in Salmonella is associated with modifying enzymes that include the aminoglyco-side phosphotransferases, aminoglycoside acetyltransferases, and aminoglycoside adenyltransferases, which function by phosphorylating, acetylating, and adenylating certain aminoglycosides, respectively (Poole, 2005). Aminoglycoside phosphotransferase encoded by aphA is responsible for kanamycin resistance, aminoglyco-side acetyltransferase encoded by aacC can cause gentamicin resistance, and aminoglycoside adenyltransferases encoded by aadA and aadB are associated with streptomycin and gentamicin resistance, respectively (Randall et al., 2004; Welch et al., 2007).

In addition to inactivation of the drug itself, other resistance is associated with the modification of the drug binding target within the cell. Much of the resistance to the quinolone and fluoroquinolone drugs is associated with mutations in the genes encoding the topoisomerase enzymes required for bacterial DNA replication. The mutations prevent the antimicrobial agents from binding to their topoisomerase targets and carrying out their antimicrobial activity (Heisig, 1993). Much of the resistance to tetracycline and chloramphenicol is associated with the acquisition and expression efflux pumps that remove toxic levels of the drug from the bacterial cells. In Salmonella, most tetracycline resistance efflux pumps are encoded by the tet genes, whereas chloramphenicol efflux pumps are encoded by floR or cml (Chopra and Roberts, 2001; Butaye et al., 2003). In addition to efflux-mediated resistance, some chloramphenicol resistance in Salmonella is associated with drug target modification by chloramphenicol acetyltransferases encoded by the cat genes (Murray and Shaw, 1997). The drug trimethoprim and the sulfonamides function by competitively inhibiting different enzymes in the folic acid biosynthetic pathway in bacterial cells. Resistance to sulfonamides is often associated with the acquisition of either sulI or sulII, which encode altered dihydropteroate synthetase enzymes that have a reduced affinity for the sulfonamides, but function in folic acid biosynthesis (Huovinen et al., 1995). Likewise, with trimethoprim resistance, dhfr genes encode altered dihydrofolate reductases that have reduced affinity for the antimicrobial agent, allowing folic acid biosynthesis to occur in the presence of trimethoprim (Huovinen et al., 1995).

### Mechanisms of Antimicrobial Resistance Dissemination

Many strains of Salmonella demonstrate a high level of multidrug resistance to agents such as the tetracyclines, sulfonamides, streptomycin, kanamycin, chloramphenicol, and some of the β-lactam antibiotics (penicillins and cephalosporins; Olsen et al., 1994; Gebreyes et al., 2000; White et al., 2001; Gebreyes and Altier, 2002). Many of the β-lactamases are carried on plasmids in different Salmonella strains (Kratz et al., 1983; Garbarg-Chenon et al., 1989; Morosini et al., 1996; Llanes et al., 1999; AitMhand et al., 2002). In addition to β-lactamases, many resistance plasmids contain integrons, which may carry the genes needed for resistance to agents such as chloramphenicol, sulfonamides, tetracycline, and streptomycin (Recchia and Hall, 1997; Bennett, 1999; Briggs and Fratamico, 1999; Fluit and Schmitz, 1999; Rankin et al., 2002). Integrons are important in the dissemination of resistance genes because they are mobile genetic elements that can be located on plasmids and(or) integrated into the bacterial chromosome (Bennett, 1999). Integrons contain the genes needed for insertion and excision of genetic material from plasmids, transposons, and chromosomes and contain a complement of resistance genes and the factors needed for the expression of those genes (Recchia and Hall, 1997). Class 1 integrons are the most common antimicrobial resistance integrons and are found in a variety of species (Fluit and Schmitz, 2004). A schematic of a class 1 integron is shown in Figure 6. Class 1 integrons include a gene encoding an integrase (intI), a recombination site (attI), and a gene cassette (Fluit and Schmitz, 2004; Mazel, 2006). The gene cassette usually contains antimicrobial resistance genes and a short 59-bp element (attC) that functions as a recognition sequence for the specific recombination site (Bennett, 1999). The integrases can recombine the gene cassette into a specific recombination site (Mazel, 2006). Immediately downstream of the gene cassette are the genes qacEΔ and sul1, which encode resistance to quaternary ammonium compounds and sulfonamides, respectively (Fluit and Schmitz, 2004). Integron transfer of resistance genes has been shown to spread across serotype and even species barriers, raising concern for potential resistance gene transfers from multiple sources to and among Salmonella strains (Bennett, 1999).

Other mechanisms that are important in the transfer of resistance genes include transformation, transduction, and conjugation (Figure 7; Marlovits et al., 2004; Frost et al., 2005). Natural transformation is characterized by the uptake, integration, and expression of free DNA molecules. The DNA molecules are released into the environment from decomposing cells, damaged cells, or viruses. If the free DNA released into the environment contains resistance genes, there is potential for the recipient cell to acquire the corresponding resistance. In order for the recipient to acquire the DNA, it must be in a physiological state known as competence. Once the DNA has entered the cytoplasm, it is integrated into the genome through a recombination or additive integration (Marlovits et al., 2004). If a resistance gene is incorporated into the recipient genome, the strain may develop resistance to the particular antimicrobial agent. In transduction, DNA is transferred by bacterial viruses (i.e., phages). Phages that have infected a bacterial cell can accidentally package small segments of host DNA during the viral reproductive process and inject it into a bacterial recipient during the normal infectious process. This new DNA can integrate into the recipient genome and be expressed (Davidson, 1999). As with transformation, if the genetic material transferred from the donor host to recipient contains a resistance gene, the resistance could be expressed in the recipient. With conjugation, transfer of plasmids is mediated by a cell-to-cell bridge (pilus) that is encoded by the plasmid. During conjugation, the donor cell produces a pilus that contacts a recipient cell and forms a mating pair. The plasmid begins replication and one strand of DNA is transferred to the recipient. Both strands finish replication in their respective cells resulting with the donor cell and a transconjugant (recipient cell with newly acquired plasmid; Frost et al., 2005).

Plasmid-mediated spread of antibiotic resistance genes is likely an important means for Salmonella to acquire resistance. A number of studies have shown experimental conjugal transfer of antibiotic resistance among Salmonella and related organisms (Simmons et al., 1988; Carattoli et al., 2002; Gebreyes and Altier, 2002; Yan et al., 2003; Zhao et al., 2003; Aarestrup et al., 2004; Fakhr et al., 2006). In a study by Zhao et al. (2003), plasmids from multidrug-resistant S. Newport isolates were transferred to a susceptible Escherichia coli strain. The transconjugants acquired the phenotypic resistance profiles of the S. Newport donors. The transfer of the blacmy gene and a class 1 integron encoding streptomycin resistance was confirmed by PCR and sequence analysis.

Aarestrup et al. (2004) also indicated that multidrug resistance could be transferred among strains of Salmonella. In this study, a multidrug-resistant strain of S. Heidelberg was subjected to conjugation experiments and it was discovered that many of the resistance determinants were located on a conjugative plasmid and could be transferred into highly susceptible strains of S. Heidelberg, Typhimurium, and Dublin. Of special concern is the ability of the resistance genes to be transferred to S. Dublin strains, which, although not commonly associated with human infections, are known to be highly invasive and have a relatively high mortality rate when infections do occur (Helms et al., 2003). Thus, the natural transfer of this multidrug resistance from S. Heidelberg strains to highly invasive strains like those of S. Dublin would be problematic, because these invasive infections are most likely to require antimicrobial therapy to treat the infection.

Welch et al. (2007) recently reported on the complete sequence of a large plasmid from a multidrug-resistant S. Newport isolate. The plasmid backbone contained genes responsible for replication and maintenance, as well as a functional transfer region allowing for conjugal transfer of the plasmid. The plasmid had multiple genes encoding resistance to different antimicrobial agents. The resistance plasmid specifically contained 13 genes encoding resistance to sulfonamides, phenicols, tetracyclines, aminoglycosides, quaternary ammonium compounds, β-lactams, and mercury. Many of the resistance genes are found in transposon Tn21, a common mobile genetic element. The plasmid also contained a class 1 integron encoding gentamicin and streptomycin resistance. Similarly, Chiu et al. (2005) reported on the complete sequence of a resistance plasmid from an S. Choleraesuis isolate. The plasmid contained genes encoding resistance to trimethoprim, sulfonamides, chloramphenicol, ampicillin, streptomycin, tetraclycline, kanamycin, streptothricin, macrolides, mercury, ethidium bromide, and quaternary ammonium compounds. The S. Choleraesuis resistance plasmid was nonconjugative because it contained a defective transfer region (Chiu et al., 2005).

In addition to distinct resistance or virulence plasmids, plasmids containing both types of genes have been identified. Guerra et al. (2002) found a conjugal plasmid from S. Typhmurium carrying both virulence and resistance genes. The plasmid is about 140 kbp and carries the virulence genes spvA, spvB, spvC, and rck as well as genes for ampicillin, streptomycin, mercury, chloramphenicol, and tetracycline resistance (Guerra et al., 2002). The genes for ampicillin and streptomycin resistance were located in a class 1 integron. The authors hypothesized that an S. Typhimurium isolate containing a virulence plasmid acquired 1 or more resistance plasmids that were integrated into the virulence plasmid creating a hybrid plasmid. This is a potential public health concern because the transfer of this plasmid to susceptible isolates could render them more virulent as well as resistant to multiple antimicrobial agents.

In conclusion, following a progressive increase in the number of reported cases of Salmonella since the end of World War II through the late 1980s, the number of reported cases of salmonellosis has remained steady over the past decade. However, during the last decade, there have been changes in the relative contributions of different serovars to the overall Salmonella burden on human health. The numbers of cases of S. Typhimurium and S. Enteritidis have decreased significantly, including an approximately 30% decline in the number of S. Enteritidis cases from 1995 to 2005. The percentage decrease would be even greater had there not been a 40% increase from 2004 to 2005. Conversely, serovars such as I 4,[5],12:i:-, Javiana, and Mississippi have increased significantly over the past decade.

To become significant human pathogens, Salmonella have developed mechanisms to survive the acidic environment of the stomach and enter the intestinal tract. There, they are able to colonize the intestinal epithelium through fimbriae-mediated attachment and invade the intestinal epithelium through the use of the SPI-1 T3SS and translocated effectors. A number of the SPI-1 T3SS effector molecules are associated with increased inflammation and an alteration of the ion balance within cells that facilitate the release of fluid into the intestinal tract leading to the development of watery diarrhea. A number of Salmonella are subsequently taken up by phagocytes such as macrophages and dendritic cells, where they are capable of survival and spread to different areas of the body to cause severe invasive infections.

Severe Salmonella infections often require antimicrobial therapy to aid in the elimination of the infection. A potential problem that has been developing for many decades is the development of antimicrobial resistance. A positive trend is that for many drugs there has been a reduction in resistance over the past 5 yr, as seen recently through the NARMS program. However, this trend has not occurred for all antimicrobial agents. A special concern has been the emergence of resistance to extended-spectrum cephalosporins such as ceftiofur and ceftriaxone. Resistance to ceftriaxone is especially troublesome, because it is the primary drug of choice for treating severe salmonellosis in children under 16 yr. With over a quarter of reported Salmonella infections occurring in children under the age of 10, this resistance is significant. Salmonella continues to remain an important pathogen of humans and other animal species. As we gain better understanding of the mechanisms of pathogenicity and factors that lead to the development of antimicrobial resistance, there is increased potential to limit the burden of these pathogens.

Key Words