Over the last decade, increasing attention has been focused on plasmids that harbour the antimicrobial resistance gene *bla*CMY-2, which encodes an AmpC-type beta-lactamase that hydrolyzes third-generation cephalosporins (Bauernfeind et al., 1996; Zhao et al., 2001; Carattoli et al., 2002; Wiesner et al., 2009; Wiesner et al., 2011). In *Salmonella enterica, bla*CMY-2 is frequently carried by IncA/C or IncI1 plasmids (Bauernfeind et al., 1996; Carattoli et al., 2002; Hopkins et al., 2006; Lindsey et al., 2009). In a recent study, Call et al. (2010) analyzed five *E. coli* and *Salmonella* Newport IncA/C plasmids carrying *bla*CMY-2, and showed that although they share a common ancestor with the *Yersinia* and *Photobacterium* plasmids, they are genetically distinct (Call et al., 2010). In a population study we found that IncA/C plasmids were associated to the Mexican Typhimurium ST213 genotype. We determined that the *bla*CMY-2 gene was carried in IncA/C plasmids, and genetic variability was observed using a plasmid typing scheme, targeting ten conserved regions in IncA/C plasmids (Wiesner et al., 2009; Wiesner et al., 2011). The Typhimurium *bla*CMY-2-bearing IncA/C plasmids possessed most of the accessory elements found in other *Salmonella* and *E. coli* plasmids (Call et al., 2010), but also more than half contained a class 1 integron (*dfrA12*-*orfF*-*aadA2*). The screening of the total Mexican Typhimurium population showed the presence of another IncA/C plasmid harboured by ST213 strains, yet lacking *bla*CMY-2. These plasmids also carried antibiotic resistance determinants, but they shared only three of the ten genetic markers used to study the IncA/C plasmids, and were smaller than the *bla*CMY-2-bearing IncA/C plasmids (100 vs. 150-160 kb). Nevertheless, the nucleotide sequences of the regions shared with the *bla*CMY-2 bearing IncA/C plasmids were identical, suggesting that the *bla*CMY-2-bearing plasmids could be the result of the insertion of DNA modules into this smaller precursor plasmid (Wiesner et al., 2009; Wiesner et al., 2011). The general agreement from the analysis of the genetic structure of the IncA/C group,is that plasmid evolution progresses faster through the insertion/deletion of DNA stretches rather than by point mutations (Welch et al., 2007; Kim et al., 2008; Fricke et al., 2009; Call et al., 2010; Wiesner et al., 2011).

Large resistance plasmids circulate among microbial populations in distinct environmental niches, even in the absence of antibiotic selective pressure. In other environments the target of the selective pressure could be, for example, mercury resistance carried by many transposons, such as Tn*21* (Liebert et al., 1999; McIntosh et al., 2008). Regardless of the primary selective agent, the complete battery of resistance determinants will be maintained, imposing a global health risk (Liebert et al., 1999; Frost et al., 2005; Welch et al., 2007; McIntosh et al., 2008; Pan et al., 2008; Davies and Davies 2010). Another worrisome situation is the emergence of *Salmonella* virulence-(antibiotic) resistance plasmids.  In some of the studies it was demonstrated that the hybrid plasmids were conjugative, which may lead to their spread to new recipients and allow the co-selection of the antibiotic and virulence genes, representing a hazard to human and animal health (Fluit 2005).

#### **5. Integrons**

234 Salmonella – A Diversified Superbug

correlate genomic content with the outcome of disease. They used a DNA microarray targeting 281 known virulence factors, and found that the presence or absence of the virulence plasmid did not correspond to disease symptoms (Litrup et al., 2010). On the other hand, Heithoff et al. (2008) found that all the Typhimurium strains isolated from animals or humans with bacteremia possessed the virulence plasmid, while 34% of the strains isolated from human gastroenteritis lacked the plasmid (Heithoff et al., 2008). These contrasting results highlight the complex nature of specific host-pathogen interactions, and call to avoid making generalizations since the diversity of environmental (biotic and abiotic), host, and

Large antimicrobial resistance plasmids are of public health concern. The global scene is that the therapeutic options for MDR microbes are reduced, periods of hospital care are more extended and costly and, in some cases, the strains have also acquired increased virulence and enhanced transmissibility. Realistically, antibiotic resistance can be considered a virulence factor (Davies and Davies 2010). Resistance mechanisms are pandemic and create an enormous clinical and financial burden on health care systems worldwide (Davies and Davies 2010). The resistance genes found in *Salmonella* are closely related to, or are indistinguishable from, those found in other bacteria, including not only members of the *Enterobacteriaceae* but also distantly related bacteria. It is most likely that *Salmonella* acquired these genes from other bacteria, and probably *Salmonella* strains also play a role in the further dissemination of these resistance genes to other bacteria (Michael et al., 2006). Frequencies of conjugative transmission in nature are probably several orders of magnitude higher that those observed under laboratory conditions, and occur readily in networks of multi-host interactions (Dionisio et al., 2002; Sorensen et al., 2005; Davies and Davies 2010). The IncA/C plasmids exemplify the problematic of resistance plasmids in *Salmonella*. They have attracted the attention of the research community due to their ability to acquire antimicrobial resistance traits and to mobilize across geographical and taxonomical borders (Fricke et al., 2009). Recent comparative studies have addressed the evolutionary relationships among the IncA/C plasmids from *Salmonella enterica*, *Escherichia coli*, *Yersinia pestis*, *Yersinia ruckeri*, *Vibrio cholera*, *Photobacterium damselae* and *Aeromonas salmonicida* (Welch et al., 2007; Kim et al., 2008; McIntosh et al., 2008; Pan et al., 2008; Fricke et al., 2009; Call et al., 2010). The genomic comparison of seven IncA/C plasmids showed that these plasmids share a common backbone, including the origin of replication and a conjugative plasmid transfer system (Welch et al., 2007; Fricke et al., 2009). Several loci containing antimicrobial resistance determinants are distributed along the plasmids, and are integrated at few sites within the conserved plasmid backbone; they are generally located as resistance gene arrays, composed of resistance genes and mobile genetic elements such as insertion sequences, transposons or integrons (Fricke et al., 2009). For example, in the IncA/C plasmids of *Yersinia pestis* and *Salmonella* Newport, a Tn*21* transposon is inserted in a similar location but some nucleotide divergence is evident and its orientation is reversed (Welch et al., 2007; Fricke et al., 2009). These studies suggest an evolutionary model in which each IncA/C plasmid diverged from a common ancestor, through processes of stepwise integration events of horizontally-acquired resistance genes

Over the last decade, increasing attention has been focused on plasmids that harbour the antimicrobial resistance gene *bla*CMY-2, which encodes an AmpC-type beta-lactamase that hydrolyzes third-generation cephalosporins (Bauernfeind et al., 1996; Zhao et al., 2001;

bacterial genetic conditions may produce different outcomes.

arrays (Welch et al., 2007; Fricke et al., 2009).

Integrons are assembly platforms that incorporate genes by site-specific recombination and convert them to functional genes by ensuring their correct expression. They are composed of three key elements: a gene encoding an integrase, a primary recombination site, and a promoter that directs the transcription of the captured genes. The integrase can recombine discrete units of circularized DNA known as "gene cassettes"; they are transcribed only

The Importance of Mobile Genetic Elements in the Evolution

across bacterial phyla (Wiesner et al., 2009).

**6. Bacteriophages**

of *Salmonella*: Pathogenesis, Antibiotic Resistance and Host Adaptation 237

Since the discovery of the importance of integrons in the dissemination of antibiotic resistance, many studies have addressed the presence of integrons in *Salmonella*. At the moment of writing there were more than 300 research papers regarding integrons in *Salmonella* (http://www.ncbi.nlm.nih.gov/pubmed). An overview of these studies shows that integrons have been detected in many different *Salmonella* serovars (i. e. Agona, Albany, Anatum, Braenderup, Branderburg, Bredeny, Brikama, Derby, Dublin, Emek, Enteritidis, Eppendorf, Goldcoast, Grumpensis, Hadar, Haifa, Heildelberg, Infantis, Javiana, Kedougou, Kentucky, Kingston, Krefekd, Mbandaka, Muenster, Newport, Panama, Paratyphi B, Rissen, Rough, Saintpaul, Schwarzengrund, Stanley, Senftenberg, Tees, Tshiongwe, Typhimurium, Virchow, Weltevreden, Wien and Worthington), that were isolated from diverse sources (i. e. animal feed, beef, chicken, camel, environment, feline, foodstuff, goat, horse, human, milk, pork and turkey), and from countries all around the world (i. e. Albania, Algeria, Brazil, China, Chile, England, Ethiopia, Germany, Great Britain, Iran, Ireland, Italy, Japan, Lithuania, Mexico, Portugal, Slovak Republic, Spain, Thailand, The Netherlands, United States and Vietnam). Among the most studied cases are the chromosomally-located integrons present in the SGI1. There is a great diversity of integron cassettes detected in *Salmonella*, most of them encoding antibiotic resistance genes, carried in a wide variety of cassette arrays. It is noteworthy that similar cassette arrays are found in different *Salmonella* serovars isolated from different sources and distant countries, and even in other bacterial species. For example, in the study of the integrons present in a Mexican Typhimurium population (Wiesner et al., 2009), we found that the two most abundant integrons (*dfrA12*-*orfF*-*aadA2* and *dfrA17*-*aadA5*) were reported for other *Salmonella* serovars (Anatum, Branderup, Brikama, Enteritidis, Mbandaka, Rissen and Saintpaul) and in other *Enterobacteriaceae* (Lindstedt et al., 2003; Antunes et al., 2006; Su et al., 2006; Molla et al., 2007; Zhao et al., 2007). More surprising was that these integrons were also found in different species of the Gram-positive genera *Staphylococcus* isolated in China (Xu et al., 2008), providing evidence of the successful spread of these integrons around the world and

Although bacteriophages carrying antibiotic resistance genes have rarely been identified (Davies and Davies 2010), their role in the dissemination of virulence factors has been widely documented (Boyd and Brussow 2002). Bacteriophage-encoded virulence factors can convert their bacterial host, in a process known as phage conversion, from a non-pathogenic strain to a virulent strain or a strain with increased virulence. The phage-encoded proteins involved in lysogenic conversion provide mechanisms to invade host tissues, avoid host immune defenses, and damage host cells (Boyd and Brussow 2002). The extra genes present in prophage genomes which do not have a phage function, but may act as fitness factors for the bacteria, are termed "morons". The moron-encoded genes are not required for the phage life cycle. Their expression is controlled by an autonomous promoter and, thus, can be expressed while the rest of the prophage genes remain silent (Hendrix et al., 2000). Morons enhance phage replication indirectly since moron-encoded functions enhance fitness of the lysogen. This hypothesis provides the theoretical framework for phage-mediated horizontal transfer of fitness factors between bacteria (Hendrix et al., 2000). The ecological success of a lysogenic bacterium contributes to the dissemination of phage genes, providing a case of coevolution of viruses and bacteria. It has been hypothesized that the driving force behind the emergence of new epidemic clones is the phage-mediated re-assortment of virulence and

when captured into an integron, since most of them lack a promoter. Integration occurs downstream of the resident promoter, at the primary recombination site, allowing the expression of the genes in the cassette (Figure 1). The integron inserted gene cassettes share specific structural characteristics and contain an imperfect inverted repeat at the 3' end, called the "59-base element". This site functions as a secondary recognition site for the sitespecific integrase, and can further integrate gene cassettes. The ability to capture disparate individual genes and to physically link them in arrays suitable for co-expression is a trait unique to integrons, and theoretically facilities the rapid evolution of new phenotypes (Stokes and Hall 1989; Recchia and Hall 1995; Fluit and Schmitz 1999; Holmes et al., 2003; Fluit and Schmitz 2004; Mazel 2006; Boucher et al., 2007; Joss et al., 2009).

Fig. 1. Diagrammatic representation of the basic features of an integron. Integrons consist of the gene for the integrase, the promoters for the expression of the integrase (Pint) and the gene cassettes (Pc), and the primary recombination site, where the cassettes are integrated. Gene cassettes consist of a single promoter-less gene and a recombination site known as a 59-base element (59-be), which functions as a secondary recognition site for the site-specific integrase, and can further integrate gene cassettes (Stokes and Hall 1989; Hall and Collis 1995; Levesque et al., 1995). An integron carrying an array of two inserted cassettes, and a free circularized gene cassette is shown.

Analyses on the diversity of gene cassettes in environmental samples have shown a great diversity of predicted genes, suggesting that essentially any DNA-encoded function may be contained within a gene cassette (Stokes et al., 2001; Holmes et al., 2003). However, the main part of the gene cassettes found in mobile integrons from cultured bacteria contain antibiotic-resistance determinants (Levesque et al., 1995; Fluit and Schmitz 1999, 2004). Class 1 integrons are found extensively in clinical isolates, and most of the known antibiotic resistance gene cassettes belong to this class (Fluit and Schmitz 2004). Although gene cassettes conferring resistance to nearly every major class of antibiotics have been identified, there are some antibiotic resistance determinants that are preferentially associated to integrons, such as streptomycin, trimethoprim, sulfafurazole, and the early aminoglycosides (White et al., 2001; Fluit and Schmitz 2004). Moreover, it has been reported that MDR is associated significantly with the presence of integrons (Leverstein-van Hall et al., 2003; Fluit 2005; Wiesner et al., 2009). Integrons themselves are not mobile although they can be part of transposons, which are capable of moving from one carrier replicon to another (Fluit and Schmitz 1999; Liebert et al., 1999). A well-known example are the integrons found in Tn*21*  and related transposons. These transposons generally are located on plasmids, which further enhances the spread of gene cassettes (Fluit and Schmitz 1999; Liebert et al., 1999). The association of integrons with mobile elements and resistance genes has led to their rapid dispersal among various bacteria found in environments exposed to antibiotics (Martinez-Freijo et al., 1999; White et al., 2001; Boucher et al., 2007).

Since the discovery of the importance of integrons in the dissemination of antibiotic resistance, many studies have addressed the presence of integrons in *Salmonella*. At the moment of writing there were more than 300 research papers regarding integrons in *Salmonella* (http://www.ncbi.nlm.nih.gov/pubmed). An overview of these studies shows that integrons have been detected in many different *Salmonella* serovars (i. e. Agona, Albany, Anatum, Braenderup, Branderburg, Bredeny, Brikama, Derby, Dublin, Emek, Enteritidis, Eppendorf, Goldcoast, Grumpensis, Hadar, Haifa, Heildelberg, Infantis, Javiana, Kedougou, Kentucky, Kingston, Krefekd, Mbandaka, Muenster, Newport, Panama, Paratyphi B, Rissen, Rough, Saintpaul, Schwarzengrund, Stanley, Senftenberg, Tees, Tshiongwe, Typhimurium, Virchow, Weltevreden, Wien and Worthington), that were isolated from diverse sources (i. e. animal feed, beef, chicken, camel, environment, feline, foodstuff, goat, horse, human, milk, pork and turkey), and from countries all around the world (i. e. Albania, Algeria, Brazil, China, Chile, England, Ethiopia, Germany, Great Britain, Iran, Ireland, Italy, Japan, Lithuania, Mexico, Portugal, Slovak Republic, Spain, Thailand, The Netherlands, United States and Vietnam). Among the most studied cases are the chromosomally-located integrons present in the SGI1. There is a great diversity of integron cassettes detected in *Salmonella*, most of them encoding antibiotic resistance genes, carried in a wide variety of cassette arrays. It is noteworthy that similar cassette arrays are found in different *Salmonella* serovars isolated from different sources and distant countries, and even in other bacterial species. For example, in the study of the integrons present in a Mexican Typhimurium population (Wiesner et al., 2009), we found that the two most abundant integrons (*dfrA12*-*orfF*-*aadA2* and *dfrA17*-*aadA5*) were reported for other *Salmonella* serovars (Anatum, Branderup, Brikama, Enteritidis, Mbandaka, Rissen and Saintpaul) and in other *Enterobacteriaceae* (Lindstedt et al., 2003; Antunes et al., 2006; Su et al., 2006; Molla et al., 2007; Zhao et al., 2007). More surprising was that these integrons were also found in different species of the Gram-positive genera *Staphylococcus* isolated in China (Xu et al., 2008), providing evidence of the successful spread of these integrons around the world and across bacterial phyla (Wiesner et al., 2009).

#### **6. Bacteriophages**

236 Salmonella – A Diversified Superbug

when captured into an integron, since most of them lack a promoter. Integration occurs downstream of the resident promoter, at the primary recombination site, allowing the expression of the genes in the cassette (Figure 1). The integron inserted gene cassettes share specific structural characteristics and contain an imperfect inverted repeat at the 3' end, called the "59-base element". This site functions as a secondary recognition site for the sitespecific integrase, and can further integrate gene cassettes. The ability to capture disparate individual genes and to physically link them in arrays suitable for co-expression is a trait unique to integrons, and theoretically facilities the rapid evolution of new phenotypes (Stokes and Hall 1989; Recchia and Hall 1995; Fluit and Schmitz 1999; Holmes et al., 2003;

Fluit and Schmitz 2004; Mazel 2006; Boucher et al., 2007; Joss et al., 2009).

Primary integration site

Pc 59-be 59-be

Gene cassette

59-be

Secondary integration sites

Fig. 1. Diagrammatic representation of the basic features of an integron. Integrons consist of the gene for the integrase, the promoters for the expression of the integrase (Pint) and the gene cassettes (Pc), and the primary recombination site, where the cassettes are integrated. Gene cassettes consist of a single promoter-less gene and a recombination site known as a 59-base element (59-be), which functions as a secondary recognition site for the site-specific integrase, and can further integrate gene cassettes (Stokes and Hall 1989; Hall and Collis 1995; Levesque et al., 1995). An integron carrying an array of two inserted cassettes, and a

Analyses on the diversity of gene cassettes in environmental samples have shown a great diversity of predicted genes, suggesting that essentially any DNA-encoded function may be contained within a gene cassette (Stokes et al., 2001; Holmes et al., 2003). However, the main part of the gene cassettes found in mobile integrons from cultured bacteria contain antibiotic-resistance determinants (Levesque et al., 1995; Fluit and Schmitz 1999, 2004). Class 1 integrons are found extensively in clinical isolates, and most of the known antibiotic resistance gene cassettes belong to this class (Fluit and Schmitz 2004). Although gene cassettes conferring resistance to nearly every major class of antibiotics have been identified, there are some antibiotic resistance determinants that are preferentially associated to integrons, such as streptomycin, trimethoprim, sulfafurazole, and the early aminoglycosides (White et al., 2001; Fluit and Schmitz 2004). Moreover, it has been reported that MDR is associated significantly with the presence of integrons (Leverstein-van Hall et al., 2003; Fluit 2005; Wiesner et al., 2009). Integrons themselves are not mobile although they can be part of transposons, which are capable of moving from one carrier replicon to another (Fluit and Schmitz 1999; Liebert et al., 1999). A well-known example are the integrons found in Tn*21*  and related transposons. These transposons generally are located on plasmids, which further enhances the spread of gene cassettes (Fluit and Schmitz 1999; Liebert et al., 1999). The association of integrons with mobile elements and resistance genes has led to their rapid dispersal among various bacteria found in environments exposed to antibiotics (Martinez-

*integrase orf orf*

Freijo et al., 1999; White et al., 2001; Boucher et al., 2007).

Pint

free circularized gene cassette is shown.

Although bacteriophages carrying antibiotic resistance genes have rarely been identified (Davies and Davies 2010), their role in the dissemination of virulence factors has been widely documented (Boyd and Brussow 2002). Bacteriophage-encoded virulence factors can convert their bacterial host, in a process known as phage conversion, from a non-pathogenic strain to a virulent strain or a strain with increased virulence. The phage-encoded proteins involved in lysogenic conversion provide mechanisms to invade host tissues, avoid host immune defenses, and damage host cells (Boyd and Brussow 2002). The extra genes present in prophage genomes which do not have a phage function, but may act as fitness factors for the bacteria, are termed "morons". The moron-encoded genes are not required for the phage life cycle. Their expression is controlled by an autonomous promoter and, thus, can be expressed while the rest of the prophage genes remain silent (Hendrix et al., 2000). Morons enhance phage replication indirectly since moron-encoded functions enhance fitness of the lysogen. This hypothesis provides the theoretical framework for phage-mediated horizontal transfer of fitness factors between bacteria (Hendrix et al., 2000). The ecological success of a lysogenic bacterium contributes to the dissemination of phage genes, providing a case of coevolution of viruses and bacteria. It has been hypothesized that the driving force behind the emergence of new epidemic clones is the phage-mediated re-assortment of virulence and

The Importance of Mobile Genetic Elements in the Evolution

**7. Host adaptation**

clones and the ability of *Salmonella* to adapt to a broad range of hosts.

of *Salmonella*: Pathogenesis, Antibiotic Resistance and Host Adaptation 239

transfer of the SopE cassette between lambda and P2-like phages families (Mirold et al., 2001). They proposed that the transfer of virulence factors between phages increases the flexibility of the re-assortment of effector protein repertories, by circumventing restrictions imposed by immunity functions or the occupancy of the attachment sites by resident prophages (Mirold et al., 2001). By this mean, phages would contribute a great deal to the evolution of bacterial pathogens, and might explain the rapid emergence of new epidemic

The development of genome-based methods such as microarrays, and the tools to compare complete genome sequences, has opened a new era in the study of *Salmonella* evolution. Several studies have addressed the importance of bacteriophages in the evolution of *Salmonella*, and the role of prophage-encoded virulence factors in pathogenicity.  The detection of prophage sequences has been recently developed and proposed as a tool for the subtyping of strains (Hermans et al., 2006; Cooke et al., 2007). Recent population studies are supporting the notion that the prophage content is one the most dynamic part of the genome, indicating that phage integration/excision are frequent events shaping *Salmonella* genome evolution (Hermans et al., 2006; Cooke et al., 2007; Drahovska et al., 2007; Matiasovicova et al., 2007; Cooke et al., 2008; Litrup et al., 2010). These observations are in agreement with the phage remnants found in the genomes of *Salmonella* (McClelland et al., 2001; Parkhill et al., 2001; Porwollik et al., 2002; Chan et al., 2003; Porwollik and McClelland 2003; Matiasovicova et al., 2007).

More than a decade ago, Baumler (1998) postulated that the genus *Salmonella* evolved in three phases (Baumler 1997; Baumler et al., 1998). The first phase involved acquisition of SPI1 by an ancestral lineage to all *Salmonella*, since it is present in all phylogenetic lineages of the genus *Salmonella* but absent from *E. coli* and other enterobacteria. In the second phase, the split of *S. enterica* from *S. bongori* involved the acquisition of SPI2, which is not present in *S. bongori* serovars (Ochman and Groisman 1996). Finally, the lineage of *S. enterica* branched into several phylogenetic groups. The formation of *S. enterica* subspecies *enterica* (I) involved a dramatic expansion in host range: while *S. bongori* and *S. enterica* subspecies II, IIIa, IIIb, IV, VI and VII are mainly associated with cold-blooded vertebrates, members of *S. enterica* subspecies I are most frequently isolated from avian and mammalian hosts. The host adaptation of *S. enterica* subspecies I to warm-blooded vertebrates, characterized the third phase in the evolution of virulence in the genus *Salmonella*. The immune system of higher vertebrates is more developed and organized than that of cold-blooded vertebrates. The common ancestor of subspecies I, II, IIIb and VI acquired mechanisms of flagellar antigen shifting (diphasic condition), which is thought to play a role in adaptation to warm-blooded hosts (Li et al., 1995; Baumler et al., 1998; Porwollik and McClelland 2003). The mechanism of phase shifting is amazing and involved the acquisition of the *fljBA* operon, which contains *hin*, encoding for a recombinase that catalyzes the reversible inversion of a segment of the chromosome containing the promoter for the *fljBA* operon. In one orientation, the promoter directs the transcription of the *fljA* (repressor of *fliC*) and *fljB* (phase 2 flagellin)

fitness factors, optimizing the *Salmonella*-host interaction (Mirold et al., 1999; Figueroa-Bossi et al., 2001; Brussow et al., 2004). The contribution of phages to *Salmonella* evolution is one of the best documented cases, and many phage-encoded virulence factors have been documented (Table 1). In particular, the functional biology of many phage-encoded genes has been studied in detail for Typhimurium strains.

Table 1. Bacteriophage-encoded virulence factors of *Salmonella enterica* (Figueroa-Bossi et al., 2001; Boyd and Brussow 2002; Porwollik and McClelland 2003; Ehrbar and Hardt 2005; Kropinski et al., 2007).

Prophages contribute significantly to the diversity among *Salmonella* strains (Boyd and Brussow 2002), and different Typhimurium strains harbor distinct sets of prophages (Figueroa-Bossi et al., 2001; Mirold et al., 2001; Mmolawa et al., 2002). Most of them belong to the P2 family (SopEΦ, Fels-1, and Fels-2) or the lambda family (GIFSY-1, GIFSY-2, GIFSY-3 and P22). Several of the Typhiumurium prophages encode the so-called type three effector proteins, which are injected by the bacterium into animal cells via a type three secretion system (TTSS) (Ehrbar and Hardt 2005). These effector proteins manipulate signal transduction pathways of the cells, which provoke a strong intestinal inflammation and diarrhea. The SopE effector, encoded by SopEΦ, is one of the better studied cases. It is injected into the intestinal cells by the TTSS encoded by SPI-1, and its expression is coregulated with other genes. The proper timing of SopE expression and delivery into the host cell depends on the regulatory circuits of SPI-1 (Mirold et al., 1999; Brussow et al., 2004; Ehrbar and Hardt 2005). Since the earlier studies, Mirold et al. (1999) demonstrated that SopEΦ is capable of infecting a range of Typhimurium strains (Mirold et al., 1999). In an experimental study, it was demonstrated that the lysogenic conversion of the laboratory Typhimurium strain ATCC14028 with SopEΦ provided increased enteropathogenicity compared with the wild-type strain (Zhang et al., 2002). Thus, it was shown that the horizontal transfer of phage-mediated genes may contribute to the emergence of more pathogenic epidemic clones. Moreover, Mirold et al. (2001) provided evidence for the transfer of the SopE cassette between lambda and P2-like phages families (Mirold et al., 2001). They proposed that the transfer of virulence factors between phages increases the flexibility of the re-assortment of effector protein repertories, by circumventing restrictions imposed by immunity functions or the occupancy of the attachment sites by resident prophages (Mirold et al., 2001). By this mean, phages would contribute a great deal to the evolution of bacterial pathogens, and might explain the rapid emergence of new epidemic clones and the ability of *Salmonella* to adapt to a broad range of hosts.

The development of genome-based methods such as microarrays, and the tools to compare complete genome sequences, has opened a new era in the study of *Salmonella* evolution. Several studies have addressed the importance of bacteriophages in the evolution of *Salmonella*, and the role of prophage-encoded virulence factors in pathogenicity.  The detection of prophage sequences has been recently developed and proposed as a tool for the subtyping of strains (Hermans et al., 2006; Cooke et al., 2007). Recent population studies are supporting the notion that the prophage content is one the most dynamic part of the genome, indicating that phage integration/excision are frequent events shaping *Salmonella* genome evolution (Hermans et al., 2006; Cooke et al., 2007; Drahovska et al., 2007; Matiasovicova et al., 2007; Cooke et al., 2008; Litrup et al., 2010). These observations are in agreement with the phage remnants found in the genomes of *Salmonella* (McClelland et al., 2001; Parkhill et al., 2001; Porwollik et al., 2002; Chan et al., 2003; Porwollik and McClelland 2003; Matiasovicova et al., 2007).

#### **7. Host adaptation**

238 Salmonella – A Diversified Superbug

fitness factors, optimizing the *Salmonella*-host interaction (Mirold et al., 1999; Figueroa-Bossi et al., 2001; Brussow et al., 2004). The contribution of phages to *Salmonella* evolution is one of the best documented cases, and many phage-encoded virulence factors have been documented (Table 1). In particular, the functional biology of many phage-encoded genes

**Phage Gene Protein Function in virulence**

Gifsy-2 *grvA* Antivirulence gene Decreases the pathogenicity in

Table 1. Bacteriophage-encoded virulence factors of *Salmonella enterica* (Figueroa-Bossi et al., 2001; Boyd and Brussow 2002; Porwollik and McClelland 2003; Ehrbar and Hardt 2005;

Prophages contribute significantly to the diversity among *Salmonella* strains (Boyd and Brussow 2002), and different Typhimurium strains harbor distinct sets of prophages (Figueroa-Bossi et al., 2001; Mirold et al., 2001; Mmolawa et al., 2002). Most of them belong to the P2 family (SopEΦ, Fels-1, and Fels-2) or the lambda family (GIFSY-1, GIFSY-2, GIFSY-3 and P22). Several of the Typhiumurium prophages encode the so-called type three effector proteins, which are injected by the bacterium into animal cells via a type three secretion system (TTSS) (Ehrbar and Hardt 2005). These effector proteins manipulate signal transduction pathways of the cells, which provoke a strong intestinal inflammation and diarrhea. The SopE effector, encoded by SopEΦ, is one of the better studied cases. It is injected into the intestinal cells by the TTSS encoded by SPI-1, and its expression is coregulated with other genes. The proper timing of SopE expression and delivery into the host cell depends on the regulatory circuits of SPI-1 (Mirold et al., 1999; Brussow et al., 2004; Ehrbar and Hardt 2005). Since the earlier studies, Mirold et al. (1999) demonstrated that SopEΦ is capable of infecting a range of Typhimurium strains (Mirold et al., 1999). In an experimental study, it was demonstrated that the lysogenic conversion of the laboratory Typhimurium strain ATCC14028 with SopEΦ provided increased enteropathogenicity compared with the wild-type strain (Zhang et al., 2002). Thus, it was shown that the horizontal transfer of phage-mediated genes may contribute to the emergence of more pathogenic epidemic clones. Moreover, Mirold et al. (2001) provided evidence for the

Involved in invasion Critical for survival in Peyer´s patches

Involved in invasion Intracellular survival Required for full virulence

Involved in invasion

O-antigen conversion O-antigen conversion

the host

Bacterial envelope for invasion

Fels-1 *sodC-III* Superoxide dismutase Intracellular survival Fels-1 *nanH* Neuraminidase Intracellular survival

Type III efector

Type III efector Superoxide dismutase Type III efector

Type III efector *phoPQ*-activated gene

Flippase

Glucosyl transferase

SopEΦ *sopE* Type III efector Involved in invasion ε34 *rfb* Glucosyl transferase Altering antigenicity

IS-like

has been studied in detail for Typhimurium strains.

Gifsy-1 *gogB*

Gifsy-3 *sspH1*

P22 *gtrB*

Kropinski et al., 2007).

Gifsy-2

Fels-1 and

*gipA*

*pagJ*

*gtrA*

*sseI (gtgB) sodC-I gtgE*

> More than a decade ago, Baumler (1998) postulated that the genus *Salmonella* evolved in three phases (Baumler 1997; Baumler et al., 1998). The first phase involved acquisition of SPI1 by an ancestral lineage to all *Salmonella*, since it is present in all phylogenetic lineages of the genus *Salmonella* but absent from *E. coli* and other enterobacteria. In the second phase, the split of *S. enterica* from *S. bongori* involved the acquisition of SPI2, which is not present in *S. bongori* serovars (Ochman and Groisman 1996). Finally, the lineage of *S. enterica* branched into several phylogenetic groups. The formation of *S. enterica* subspecies *enterica* (I) involved a dramatic expansion in host range: while *S. bongori* and *S. enterica* subspecies II, IIIa, IIIb, IV, VI and VII are mainly associated with cold-blooded vertebrates, members of *S. enterica* subspecies I are most frequently isolated from avian and mammalian hosts. The host adaptation of *S. enterica* subspecies I to warm-blooded vertebrates, characterized the third phase in the evolution of virulence in the genus *Salmonella*. The immune system of higher vertebrates is more developed and organized than that of cold-blooded vertebrates. The common ancestor of subspecies I, II, IIIb and VI acquired mechanisms of flagellar antigen shifting (diphasic condition), which is thought to play a role in adaptation to warm-blooded hosts (Li et al., 1995; Baumler et al., 1998; Porwollik and McClelland 2003). The mechanism of phase shifting is amazing and involved the acquisition of the *fljBA* operon, which contains *hin*, encoding for a recombinase that catalyzes the reversible inversion of a segment of the chromosome containing the promoter for the *fljBA* operon. In one orientation, the promoter directs the transcription of the *fljA* (repressor of *fliC*) and *fljB* (phase 2 flagellin)

The Importance of Mobile Genetic Elements in the Evolution

of *E. coli* and *Salmonella* (Retchless and Lawrence 2007).

of *Salmonella*: Pathogenesis, Antibiotic Resistance and Host Adaptation 241

deleted rather than merely silenced (Selander et al., 1996; Echeita et al., 2001). Likewise, in a study analyzing *E. coli* and *Salmonella* genomes, Retcheless and Lawrence (2007) found that their chromosomes diverged over a 70 million year period, and that the regions flanking SPI1 and SPI2 diverged more recently, suggesting that they did not promote the separation

The distribution of pathogenicity islands, fimbrial operons, and capsular biosynthesis genes among *S. enterica* suggests that during evolution, new combinations of virulence determinants arose through multiple horizontal transfer events, a process which may have driven the development of host adaptation. In addition, deletion events and sequence divergence by point mutations were likely among the events which contributed to changes in the host range of *S. enterica* serovars (Baumler et al., 1998; Porwollik and McClelland 2003). The *S. enterica* subspecies I serovars form a group of pathogens that differ widely in their spectrum of host range within mammals and birds. For classification purposes, they can be categorized into three different groups: broad-host range, host-adapted, and hostrestricted serovars. For example, serovars Typhi, Gallinarum, and Abortusovis are hostrestricted serovars that are associated with systemic disease in humans, fowl and ovine hosts, respectively. Serovars Dublin and Cholerasuis are host-adapted serovars that are often associated with systemic disease in cattle and pigs, respectively, but can cause disease in other animals. Typhimurium and Enteritidis are broad-host range serovars capable of causing systemic disease in a wide range of animals, but are usually associated with gastroenteritis in a broad range of phylogenetically unrelated host species (Baumler et al., 1998; Kingsley and Baumler 2000; Rabsch et al., 2002). However, even within a single serovar there are differences in host range. For example, Rabsch *et al*. (2002) showed that two Typhimurium variants (DT2 and DT99) were almost exclusively associated with pigeons during decades, over a wide geographic range, indicative of a narrow host range; while other Typhimurium variants, such as DT104, are truly broad-host-range, thus circulating in cattle, swine, poultry and humans. Therefore, it may be more accurate to describe serovar Typhimurium as a collection of variants that vary significantly in their host range and degree of host adaptation. One possible mechanism by which such variants arise is through phage-mediated transfer, of a small number of host-specific virulence factors (Rabsch et al., 2002; Porwollik and McClelland 2003; Brussow et al., 2004; Porwollik et al., 2004; Ehrbar and Hardt 2005; Vernikos et al., 2007). There is currently no genetic explanation for the phenotype of host adaptation; it is unlikely that a single locus will be found to be responsible for this complex biological trait. Instead, a combination of multiple genes is

likely to contribute to the overall virulence phenotype (Fierer and Guiney 2001).

In this chapter, we have provided extensive evidence on the importance of lateral transfer of genetic information in the evolution of *Salmonella*. Compiling evidence on the role of gene loss and acquisition in the origin of the genus *Salmonella* has been presented in several evolutionary studies (Groisman and Ochman 1997; Lawrence 1999; Porwollik et al., 2002; Lerat et al., 2005; Retchless and Lawrence 2007; Vernikos et al., 2007), exemplified by the acquisition of pathogenicity islands (Ochman and Groisman 1996; Porwollik and McClelland 2003; Hensel 2004). Likewise, the evolutionary processes shaping the genetic structure within serovars and host-adapted ecotypes involve in many cases lateral transfer events, such as prophage insertions (Porwollik and McClelland 2003; Thomson et al., 2004;

**8. How much lateral transfer occurs in natural populations?**

genes, inducing the repression of *fliC* (phase 1 flagellin). In the other orientation, *hin*, *fljB* and *fljA* are not expressed and *fliC* is expressed (Zieg et al., 1977). A schematic representation of *Salmonella* evolution is presented in Figure 2 (Silva and Wiesner 2009).

Fig. 2. Schematic representation of *Salmonella* evolution. The cladogram shown is a modified version of that proposed by Porwollik & McClelland (2003), and published by Silva & Wiesner (2009). The number of genes acquired at crucial steps in *Salmonella* evolution is indicated on the nodes along with prominent examples (Porwollik et al., 2002). During the divergence of *E. coli* and *Salmonella*, about 120 million years (myr) ago (Ochman and Wilson 1987), more than 500 genes were acquired, including *Salmonella* pathogenicity island 1 (SPI-1). In the divergence between *S. bongori* and *S. enterica* more than 100 genes were acquired in the *S. enterica* lineage, including those of the SPI-2 (Ochman and Groisman 1996). Along the diversification of the *S. enterica* lineage, about 100 genes were acquired by the common ancestor of subspecies IIIb, II, IV and I, among them were the phase shifting genes (*fljBA* and *hin*), required to confer the diphasic condition to *Salmonella*. During the evolution of subspecies I, which is the most diverse of the subspecies and adapted to warm-blooded vertebrates, more than 200 genes were acquired. Kidgell et al. (2002) estimated that the time of divergence among serovars was around 10 myr, and that the last common ancestor of serovar Typhi existed about 50,000 yr (Kidgell et al., 2002).

However, not everything is said about *Salmonella* evolution. The report of serovar Senftenberg human clinical isolates lacking SPI-1 (Hu et al., 2008) is an example of subsequent loss of genetic determinants during the diversification of *Salmonella*. Another example is the loss of the diphasic trait in some of the serovars of subspecies I (e. g. Typhi) and II. They have reverted to the monophasic condition, usually by loss of expression of phase 2 flagella. In Enteritidis and serovar 4,5,12:i:- the phase 2 flagellin gene (*flj*B) has been

genes, inducing the repression of *fliC* (phase 1 flagellin). In the other orientation, *hin*, *fljB* and *fljA* are not expressed and *fliC* is expressed (Zieg et al., 1977). A schematic representation of

Fig. 2. Schematic representation of *Salmonella* evolution. The cladogram shown is a modified version of that proposed by Porwollik & McClelland (2003), and published by Silva & Wiesner (2009). The number of genes acquired at crucial steps in *Salmonella* evolution is indicated on the nodes along with prominent examples (Porwollik et al., 2002). During the divergence of *E. coli* and *Salmonella*, about 120 million years (myr) ago (Ochman and Wilson 1987), more than 500 genes were acquired, including *Salmonella* pathogenicity island 1 (SPI-1). In the divergence between *S. bongori* and *S. enterica* more than 100 genes were acquired in the *S. enterica* lineage, including those of the SPI-2 (Ochman and Groisman 1996). Along the diversification of the *S. enterica* lineage, about 100 genes were acquired by the common ancestor of subspecies IIIb, II, IV and I, among them were the phase shifting genes (*fljBA* and *hin*), required to confer the diphasic condition to *Salmonella*. During the evolution of subspecies I, which is the most diverse of the subspecies and adapted to warm-blooded vertebrates, more than 200 genes were acquired. Kidgell et al. (2002) estimated that the time of divergence among serovars was around 10 myr, and that the last common ancestor of

However, not everything is said about *Salmonella* evolution. The report of serovar Senftenberg human clinical isolates lacking SPI-1 (Hu et al., 2008) is an example of subsequent loss of genetic determinants during the diversification of *Salmonella*. Another example is the loss of the diphasic trait in some of the serovars of subspecies I (e. g. Typhi) and II. They have reverted to the monophasic condition, usually by loss of expression of phase 2 flagella. In Enteritidis and serovar 4,5,12:i:- the phase 2 flagellin gene (*flj*B) has been

serovar Typhi existed about 50,000 yr (Kidgell et al., 2002).

*Salmonella* evolution is presented in Figure 2 (Silva and Wiesner 2009).

deleted rather than merely silenced (Selander et al., 1996; Echeita et al., 2001). Likewise, in a study analyzing *E. coli* and *Salmonella* genomes, Retcheless and Lawrence (2007) found that their chromosomes diverged over a 70 million year period, and that the regions flanking SPI1 and SPI2 diverged more recently, suggesting that they did not promote the separation of *E. coli* and *Salmonella* (Retchless and Lawrence 2007).

The distribution of pathogenicity islands, fimbrial operons, and capsular biosynthesis genes among *S. enterica* suggests that during evolution, new combinations of virulence determinants arose through multiple horizontal transfer events, a process which may have driven the development of host adaptation. In addition, deletion events and sequence divergence by point mutations were likely among the events which contributed to changes in the host range of *S. enterica* serovars (Baumler et al., 1998; Porwollik and McClelland 2003). The *S. enterica* subspecies I serovars form a group of pathogens that differ widely in their spectrum of host range within mammals and birds. For classification purposes, they can be categorized into three different groups: broad-host range, host-adapted, and hostrestricted serovars. For example, serovars Typhi, Gallinarum, and Abortusovis are hostrestricted serovars that are associated with systemic disease in humans, fowl and ovine hosts, respectively. Serovars Dublin and Cholerasuis are host-adapted serovars that are often associated with systemic disease in cattle and pigs, respectively, but can cause disease in other animals. Typhimurium and Enteritidis are broad-host range serovars capable of causing systemic disease in a wide range of animals, but are usually associated with gastroenteritis in a broad range of phylogenetically unrelated host species (Baumler et al., 1998; Kingsley and Baumler 2000; Rabsch et al., 2002). However, even within a single serovar there are differences in host range. For example, Rabsch *et al*. (2002) showed that two Typhimurium variants (DT2 and DT99) were almost exclusively associated with pigeons during decades, over a wide geographic range, indicative of a narrow host range; while other Typhimurium variants, such as DT104, are truly broad-host-range, thus circulating in cattle, swine, poultry and humans. Therefore, it may be more accurate to describe serovar Typhimurium as a collection of variants that vary significantly in their host range and degree of host adaptation. One possible mechanism by which such variants arise is through phage-mediated transfer, of a small number of host-specific virulence factors (Rabsch et al., 2002; Porwollik and McClelland 2003; Brussow et al., 2004; Porwollik et al., 2004; Ehrbar and Hardt 2005; Vernikos et al., 2007). There is currently no genetic explanation for the phenotype of host adaptation; it is unlikely that a single locus will be found to be responsible for this complex biological trait. Instead, a combination of multiple genes is likely to contribute to the overall virulence phenotype (Fierer and Guiney 2001).

#### **8. How much lateral transfer occurs in natural populations?**

In this chapter, we have provided extensive evidence on the importance of lateral transfer of genetic information in the evolution of *Salmonella*. Compiling evidence on the role of gene loss and acquisition in the origin of the genus *Salmonella* has been presented in several evolutionary studies (Groisman and Ochman 1997; Lawrence 1999; Porwollik et al., 2002; Lerat et al., 2005; Retchless and Lawrence 2007; Vernikos et al., 2007), exemplified by the acquisition of pathogenicity islands (Ochman and Groisman 1996; Porwollik and McClelland 2003; Hensel 2004). Likewise, the evolutionary processes shaping the genetic structure within serovars and host-adapted ecotypes involve in many cases lateral transfer events, such as prophage insertions (Porwollik and McClelland 2003; Thomson et al., 2004;

The Importance of Mobile Genetic Elements in the Evolution

**75 80 85 90 95 100**

*Xba*I

of *Salmonella*: Pathogenesis, Antibiotic Resistance and Host Adaptation 243

**yuhs03-8 yuhs03-1 mipus03-2 yupus03-3 -1 slpus03-2 -1 yuhs04-2 slrapus05-3 slres02-108 mihs02-1 yuhs02-7 yuhs03-58A yuhs03-8 yuhs05-2 yuhs04-3 yuhs05-7 slrares04-8 slrapus04-6 yuhs04-1 yuhs04-6 yuhs03-7 yuhs03-2 mires03-1 -2 slhs03-1 slhs03-9 slres03-5 -2 yupols03-3 sores04-4 slhs03-1 slpus03-2 yuhs05-7 sorapus04-2 sores05-2 sorapus05-2 mires04-4 mirapus04-1 yures03-7 yupus03-1 yuhs03-2 mipols03-7 mipols03-7 sorapus04-2 sorares04-2 sohs03-1 slpus03-4 mihs03-1 mihs03-2 -1 slpus02-8 sohs04-1 slhs05-7 yuhs00-204 yuhs00-228 mihs03-1 yuhs00-141 yuhs00-8 yuhs01-401 mipus03-2 yuhs01-400 sohs04-4 sohs04-3 sohs02-6 sohs04-4 sohs02-2 sohs03-5 slhs02-2 sohs02-2**

**A/C+cmy A/C A/C+cmy A/C+cmy A/C+cmy A/C A/C A/C+cmy A/C+cmy A/C+cmy A/C A/C+my A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C A/C+cmy A/C A/C A/C A/C+cmy A/C A/C A/C A/C A/C A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C+cmy A/C A/C STV STV STV STV STV STV STV STV STV STV**

Strain ST Plasmid Integron

**A/C+cmy**

*dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2*

*dfrA17***-***aadA5 dfrA17***-***aadA5 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2 dfrA12***-***orfF***-***aadA2*

*aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1 aadA2, pse-1*

*oxa2-orfD*

**STV STV STV STV STV STV**

*aadA12*

Fig. 3. Dedrogram depicting the genetic relationships between 65 representative Mexican Typhimurium strains, and the associations among core and accessory genes. The data were obtained from Wiesner et al. (2009) and Wiesner et al. (2011). The *Xba*I restriction fingerprints were clustered by the UPGMA algorithm using Dice coefficients. The first column contains the strain designation. The second column displays the multi-locus sequence type (ST) for each strain. The plasmid column indicates if the strains harbored the *Salmonella* virulence plasmid (STV), the *bla*CMY-2-bearing IncA/C plasmid (A/C+cmy) or the smaller *bla*CMY-2-lacking IncA/C plasmid (A/C). The last column shows the integrons carried by the strains. The horizontal dashed line separates the ST213 strains from the remaining STs. SGI1 indicates the cluster

containing the strains with the *Salmonella* genomic island.

SGI1

Hermans et al., 2006; Cooke et al., 2007; Vernikos et al., 2007). The selective pressure that antimicrobial drugs have imposed on the survival of *Salmonella* probably has increased the acquisition of resistance determinants, often carried by mobile genetic elements that are acquired by lateral transfer. These processes are the result of a long evolutionary history of adaptation to changing environments and hosts. However, we do not want to leave the misconception that the amount of lateral transfer is so rampant that there are no limits to genetic exchange in *Salmonella* populations.

Since more than two decades ago, the clonal nature of Salmonella species was documented by several studies based on multilocus enzyme electrophoresis analysis (Beltran et al., 1988; Reeves et al., 1989; Selander et al., 1990; Boyd et al., 1996; Spratt and Maiden 1999). Evidences for the clonal structure of *Salmonella* include the global distribution of certain genotypes, the congruent relationships between isolates derived from several housekeeping genes, and the robust subspecies structure (Reeves et al., 1989; Boyd et al., 1996; Selander et al., 1996; Falush et al., 2006). In the past decade, multi-locus sequence typing (MLST) studies, analyzing *Salmonella* populations for epidemiological purposes, showed concordant results with the studies based on enzyme electrophoresis, which support the view that *Salmonella* has a clonal population structure. Among these results are the almost strict association between multilocus genotype and serovar, the low genetic diversity within serovars, and the maintenance of old globally-distributed clones (Sukhnanand et al., 2005; Harbottle et al., 2006; Tankouo-Sandjong et al., 2007; Wiesner et al., 2009). The clonal nature of *Salmonella* populations was observed in our study based on a survey of more than 100 Mexican Typhimurium strains. MLST and macrorestriction fingerprints by pulsed-field gel electrophoresis were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. SGI1 was found in a defined subset (16%) of the strains. They were in a compact cluster conformed by strains belonging to the second most abundant genotype (ST19), and in most of the cases they also carried the *Salmonella* virulence plasmid (Figure 3). On the other hand, the strains with the most abundant genotype (ST213) lacked *Salmonella* virulence plasmid or SGI1, but in most of the cases carried a multiple-drug resistant (MDR) IncA/C plasmid. The ST19 isolates carrying the virulence plasmid were significantly associated with the human host, whereas ST213 isolates were more frequently isolated from animal sources, indicating that the distinct accessory genes carried by these genotypes are probably involved in the interaction with the host (Wiesner et al., 2009). No strain carrying both the *Salmonella* virulence plasmid and the MDR IncA/C plasmid, nor hybrid virulence-resistance plasmids, was detected. We concluded that, in the Mexican Typhimurium population, the association between distinct core and accessory genes creates a structure of genetic subgroups within the population, which could be due to the existence of barriers to genetic exchange among subgroups (Wiesner et al., 2009).

It is intriguing why if *Salmonella* evolution is marked by lateral transfer events and genome rearrangements, the genetic structure of populations seems to be extremely clonal. It is possible that in an evolutionary time scale (millions of years) there were several occasions where the mismatch repair system was impaired and large scale recombination events occurred and marked the genomes of diverse *Salmonella* lineages (Taddei et al., 1997; Matic et al., 2000; Didelot et al., 2007). However, in the ecological time scales (thousands of years to decades) the recombination events are rare.

Hermans et al., 2006; Cooke et al., 2007; Vernikos et al., 2007). The selective pressure that antimicrobial drugs have imposed on the survival of *Salmonella* probably has increased the acquisition of resistance determinants, often carried by mobile genetic elements that are acquired by lateral transfer. These processes are the result of a long evolutionary history of adaptation to changing environments and hosts. However, we do not want to leave the misconception that the amount of lateral transfer is so rampant that there are no limits to

Since more than two decades ago, the clonal nature of Salmonella species was documented by several studies based on multilocus enzyme electrophoresis analysis (Beltran et al., 1988; Reeves et al., 1989; Selander et al., 1990; Boyd et al., 1996; Spratt and Maiden 1999). Evidences for the clonal structure of *Salmonella* include the global distribution of certain genotypes, the congruent relationships between isolates derived from several housekeeping genes, and the robust subspecies structure (Reeves et al., 1989; Boyd et al., 1996; Selander et al., 1996; Falush et al., 2006). In the past decade, multi-locus sequence typing (MLST) studies, analyzing *Salmonella* populations for epidemiological purposes, showed concordant results with the studies based on enzyme electrophoresis, which support the view that *Salmonella* has a clonal population structure. Among these results are the almost strict association between multilocus genotype and serovar, the low genetic diversity within serovars, and the maintenance of old globally-distributed clones (Sukhnanand et al., 2005; Harbottle et al., 2006; Tankouo-Sandjong et al., 2007; Wiesner et al., 2009). The clonal nature of *Salmonella* populations was observed in our study based on a survey of more than 100 Mexican Typhimurium strains. MLST and macrorestriction fingerprints by pulsed-field gel electrophoresis were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. SGI1 was found in a defined subset (16%) of the strains. They were in a compact cluster conformed by strains belonging to the second most abundant genotype (ST19), and in most of the cases they also carried the *Salmonella* virulence plasmid (Figure 3). On the other hand, the strains with the most abundant genotype (ST213) lacked *Salmonella* virulence plasmid or SGI1, but in most of the cases carried a multiple-drug resistant (MDR) IncA/C plasmid. The ST19 isolates carrying the virulence plasmid were significantly associated with the human host, whereas ST213 isolates were more frequently isolated from animal sources, indicating that the distinct accessory genes carried by these genotypes are probably involved in the interaction with the host (Wiesner et al., 2009). No strain carrying both the *Salmonella* virulence plasmid and the MDR IncA/C plasmid, nor hybrid virulence-resistance plasmids, was detected. We concluded that, in the Mexican Typhimurium population, the association between distinct core and accessory genes creates a structure of genetic subgroups within the population, which could be due to the existence of barriers to genetic exchange among

It is intriguing why if *Salmonella* evolution is marked by lateral transfer events and genome rearrangements, the genetic structure of populations seems to be extremely clonal. It is possible that in an evolutionary time scale (millions of years) there were several occasions where the mismatch repair system was impaired and large scale recombination events occurred and marked the genomes of diverse *Salmonella* lineages (Taddei et al., 1997; Matic et al., 2000; Didelot et al., 2007). However, in the ecological time scales (thousands of years to

genetic exchange in *Salmonella* populations.

subgroups (Wiesner et al., 2009).

decades) the recombination events are rare.

Fig. 3. Dedrogram depicting the genetic relationships between 65 representative Mexican Typhimurium strains, and the associations among core and accessory genes. The data were obtained from Wiesner et al. (2009) and Wiesner et al. (2011). The *Xba*I restriction fingerprints were clustered by the UPGMA algorithm using Dice coefficients. The first column contains the strain designation. The second column displays the multi-locus sequence type (ST) for each strain. The plasmid column indicates if the strains harbored the *Salmonella* virulence plasmid (STV), the *bla*CMY-2-bearing IncA/C plasmid (A/C+cmy) or the smaller *bla*CMY-2-lacking IncA/C plasmid (A/C). The last column shows the integrons carried by the strains. The horizontal dashed line separates the ST213 strains from the remaining STs. SGI1 indicates the cluster containing the strains with the *Salmonella* genomic island.

The Importance of Mobile Genetic Elements in the Evolution

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Beltran, P., J. M. Musser, R. Helmuth, J. J. Farmer, 3rd, W. M. Frerichs, I. K. Wachsmuth, K.

Boucher, Y., M. Labbate, J. E. Koenig, and H. W. Stokes. 2007. Integrons: mobilizable platforms that promote genetic diversity in bacteria. *Trends Microbiol.* 15:301-309. Boyd, D., G. A. Peters, A. Cloeckaert, K. S. Boumedine, E. Chaslus-Dancla, H. Imberechts,

Boyd, D. A., X. Shi, Q. H. Hu, L. K. Ng, B. Doublet, A. Cloeckaert, and M. R. Mulvey. 2008.

Boyd, E. F., and H. Brussow. 2002. Common themes among bacteriophage-encoded

Boyd, E. F., F. S. Wang, T. S. Whittam, and R. K. Selander. 1996. Molecular genetic relationships of the salmonellae. *Appl. Environ. Microbiol.* 62:804-808. Brussow, H., C. Canchaya, and W. D. Hardt. 2004. Phages and the evolution of bacterial

Call, D. R., R. S. Singer, D. Meng, S. L. Broschat, L. H. Orfe, J. M. Anderson, D. R. Herndon,

larger lineage of plasmids. *Antimicrob. Agents Chemother.* 54:590-596. Carattoli, A., F. Tosini, W. P. Giles, M. E. Rupp, S. H. Hinrichs, F. J. Angulo, T. J. Barrett, and

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Cornelis, G. R. 2006. The type III secretion injectisome. *Nat. Rev. Microbiol.* 4:811-825.

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and M. R. Mulvey. 2001. 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

*Salmonella* genomic island 1 (SGI1), variant SGI1-I, and new variant SGI1-O in *Proteus mirabilis* clinical and food isolates from China. *Antimicrob. Agents Chemother.*

virulence factors and diversity among the bacteriophages involved. *Trends*

pathogens: from genomic rearrangements to lysogenic conversion. *Microbiol. Mol.*

L. S. Kappmeyer, J. B. Daniels, and T. E. Besser. 2010. blaCMY-2-positive IncA/C plasmids from *Escherichia coli* and *Salmonella enterica* are a distinct component of a

P. D. Fey. 2002. Characterization of plasmids carrying CMY-2 from expandedspectrum cephalosporin-resistant *Salmonella* strains isolated in the United States

Kingsley, N. R. Thomson, and G. Dougan. 2008. Characterization of the genomes of a diverse collection of *Salmonella enterica* serovar Typhimurium definitive phage

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#### **9. Concluding remarks**

Mobile genetic elements are key to understanding *Salmonella* evolution and ecology. Actually there is a wealth of information regarding the record of horizontal gene transfer in *Salmonella*; for a revision we recommend the review of Porwollik & McClelland (2003) and the paper of Vernikos *et al*. (2007). In *Salmonella* many of the virulence factors are present as part of pathogenicity islands on the chromosome, yet some virulence factors are encoded on plasmids or bacteriophages. Their composition, presence or absence determines at large differences in pathogenicity and host range between serovars and strains (Fluit 2005). We certainly need to expand our vision, in order to integrate the knowledge about the great variety of genetic virulence determinants in *Salmonella* and the antibiotic-resistance "tool kit". A vision is emerging regarding different molecular routes that determine the plasticity of the accessory genome, which are subject to intricate interactions that we still do not completely understand. Complex interactions among pathogenicity islands, bacteriophages, plasmids and other mobile elements, such as transposons and integrons, are increasingly being evidenced. For example, the transfer of the resistance phenotypes associated with Typhimurium DT104 SGI1, by phage-transduction experiments, suggest the involvement of phages in the mobilization of SGI1 (Lawson et al., 2002b; Fluit 2005). In another example, derivatives of the original Typhimurium DT104 clone have emerged over the more than 20 years after the first report. Different phage-types, such as DT12 or DT120, are indistinguishable from the DT104 clone by genotyping, suggesting that they arose from DT104 through changes that led to different phage susceptibility patterns (Fluit 2005). These shifts in phage-type are probably due to the gain or loss of prophages. More than three decades ago, Threlfall et al. (1978) showed that changes in phage-types can be the result of the loss or acquisition of plasmids (Threlfall et al., 1978). Likewise, in another study related to Typhimurium DT104 and SGI1, some strains showed to be phage-untypeable or DT104B, and it was found that these strains carried a larger plasmid (140 kb) resulting from the recombination between a resistance and virulence plasmid (Guerra et al., 2002). In these latter cases, the changes in the plasmid content could be associated with the loss of determinants required by phages (Fluit 2005), such as surface lipopolysaccharides (Lawson et al., 2002a). Of particular concern is the increasing number of reports of co-integrates of resistance and virulence plasmids, envisioning that new *Salmonella* strains will arise posing a threat to public health (Fluit 2005).

#### **10. References**

Mobile genetic elements are key to understanding *Salmonella* evolution and ecology. Actually there is a wealth of information regarding the record of horizontal gene transfer in *Salmonella*; for a revision we recommend the review of Porwollik & McClelland (2003) and the paper of Vernikos *et al*. (2007). In *Salmonella* many of the virulence factors are present as part of pathogenicity islands on the chromosome, yet some virulence factors are encoded on plasmids or bacteriophages. Their composition, presence or absence determines at large differences in pathogenicity and host range between serovars and strains (Fluit 2005). We certainly need to expand our vision, in order to integrate the knowledge about the great variety of genetic virulence determinants in *Salmonella* and the antibiotic-resistance "tool kit". A vision is emerging regarding different molecular routes that determine the plasticity of the accessory genome, which are subject to intricate interactions that we still do not completely understand. Complex interactions among pathogenicity islands, bacteriophages, plasmids and other mobile elements, such as transposons and integrons, are increasingly being evidenced. For example, the transfer of the resistance phenotypes associated with Typhimurium DT104 SGI1, by phage-transduction experiments, suggest the involvement of phages in the mobilization of SGI1 (Lawson et al., 2002b; Fluit 2005). In another example, derivatives of the original Typhimurium DT104 clone have emerged over the more than 20 years after the first report. Different phage-types, such as DT12 or DT120, are indistinguishable from the DT104 clone by genotyping, suggesting that they arose from DT104 through changes that led to different phage susceptibility patterns (Fluit 2005). These shifts in phage-type are probably due to the gain or loss of prophages. More than three decades ago, Threlfall et al. (1978) showed that changes in phage-types can be the result of the loss or acquisition of plasmids (Threlfall et al., 1978). Likewise, in another study related to Typhimurium DT104 and SGI1, some strains showed to be phage-untypeable or DT104B, and it was found that these strains carried a larger plasmid (140 kb) resulting from the recombination between a resistance and virulence plasmid (Guerra et al., 2002). In these latter cases, the changes in the plasmid content could be associated with the loss of determinants required by phages (Fluit 2005), such as surface lipopolysaccharides (Lawson et al., 2002a). Of particular concern is the increasing number of reports of co-integrates of resistance and virulence plasmids, envisioning that new *Salmonella* strains will arise posing

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**14**

*Turkey*

*Salmonella* **as a Unique Tool**

*Biology Department of Atatürk University, Erzurum*

Mehmet Karaday, Özlem Barş and Medine Güllüce

In molecular biology and genetics, mutations are described as sudden and spontaneous or induced changes in a genomic sequence (Brown, 2007). They have wide effects on all living organisms from bacteria with a single prokaryotic cell construction to multicellular and eukaryotic organisms including human being with high-level cellular differentiation. Mutations occur also in the genomic materials (DNA or RNA) of viruses and affect their functionality (Hartl & Jones, 1998; Lewin, 2004). When a mutation happens, it can basically result in several different types of change in DNA (or RNA for some viruses) sequences; these can have no effect, alter the product of gene, and prevent the gene from functioning properly or completely. Alterations in the product of gene and partial or total loss of gene function generally result in a disadvantageous situation for the organism, which cause various symptoms and ailments affect the maintenance of life (Brown, 2007). Previous studies made to understand the relations between mutations and their negative effects on human being clearly showed that some diseases, such as most forms of cancer, heart disease and mental disorders, have a partly or completely genetic basis closely related to mutagenesis (Bertram, 2000; Alberts et al., 2002; Lodish et al., 2007).  Thus, the identification of substances capable of inducing mutations has become an important procedure in safety assessment. In the research studies, mutations can be divided in two main groups to get more comprehensive results according to their size. First group is described as gene mutations, where only single base is modified, or one or a relatively few bases are inserted or deleted (Brown, 2007). Other one consists of chromosome mutations, which are including chromosome breaks, large deletions, rearrangements, or gain or loss of whole chromosome (Hartl & Jones, 1998; Lewin, 2004).

Mutation test systems also divide in long-term and short-term systems according to obtaining of the results (Wickramasnghe, 1979; Mortelmans & Zeiger, 2000; Zeiger et al., 2005). The long-term mutagenicity tests, which use *in vivo* researches with various experimental animals, give more reliable results than short-term mutagenicity test systems. However, they are not preferred as beginning test systems due to their high cost and long time requirements, where mutagenic potential of many synthetic and natural chemicals are checked (Wickramasnghe, 1979; Gulluce et al., 2010). In these studies, the short-term test

**1. Introduction**

**for Genetic Toxicology**

### *Salmonella* **as a Unique Tool for Genetic Toxicology**

Mehmet Karaday, Özlem Barş and Medine Güllüce *Biology Department of Atatürk University, Erzurum Turkey*

#### **1. Introduction**

254 Salmonella – A Diversified Superbug

Zhao, S., P. F. McDermott, D. G. White, S. Qaiyumi, S. L. Friedman, J. W. Abbott, A. Glenn,

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animals and ground meat. *Antimicrob. Agents Chemother.* 45:3647-3650. Zieg, J., M. Silverman, M. Hilmen, and M. Simon. 1977. Recombinational switch for gene

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S. L. Ayers, K. W. Post, W. H. Fales, R. B. Wilson, C. Reggiardo, and R. D. Walker. 2007. Characterization of multidrug resistant *Salmonella* recovered from diseased

Maurer, R. Holland, and R. D. Walker. 2001. Identification and expression of cephamycinase *bla*(CMY) genes in *Escherichia coli* and *Salmonella* isolates from food

> In molecular biology and genetics, mutations are described as sudden and spontaneous or induced changes in a genomic sequence (Brown, 2007). They have wide effects on all living organisms from bacteria with a single prokaryotic cell construction to multicellular and eukaryotic organisms including human being with high-level cellular differentiation. Mutations occur also in the genomic materials (DNA or RNA) of viruses and affect their functionality (Hartl & Jones, 1998; Lewin, 2004). When a mutation happens, it can basically result in several different types of change in DNA (or RNA for some viruses) sequences; these can have no effect, alter the product of gene, and prevent the gene from functioning properly or completely. Alterations in the product of gene and partial or total loss of gene function generally result in a disadvantageous situation for the organism, which cause various symptoms and ailments affect the maintenance of life (Brown, 2007). Previous studies made to understand the relations between mutations and their negative effects on human being clearly showed that some diseases, such as most forms of cancer, heart disease and mental disorders, have a partly or completely genetic basis closely related to mutagenesis (Bertram, 2000; Alberts et al., 2002; Lodish et al., 2007).  Thus, the identification of substances capable of inducing mutations has become an important procedure in safety assessment. In the research studies, mutations can be divided in two main groups to get more comprehensive results according to their size. First group is described as gene mutations, where only single base is modified, or one or a relatively few bases are inserted or deleted (Brown, 2007). Other one consists of chromosome mutations, which are including chromosome breaks, large deletions, rearrangements, or gain or loss of whole chromosome (Hartl & Jones, 1998; Lewin, 2004).

> Mutation test systems also divide in long-term and short-term systems according to obtaining of the results (Wickramasnghe, 1979; Mortelmans & Zeiger, 2000; Zeiger et al., 2005). The long-term mutagenicity tests, which use *in vivo* researches with various experimental animals, give more reliable results than short-term mutagenicity test systems. However, they are not preferred as beginning test systems due to their high cost and long time requirements, where mutagenic potential of many synthetic and natural chemicals are checked (Wickramasnghe, 1979; Gulluce et al., 2010). In these studies, the short-term test

*Salmonella* as a Unique Tool for Genetic Toxicology 257

Another disorder is Turner syndrome, an example of a monosomy where the individual is

Structural anomalies of the large-scale mutations include deletions, duplications, inversions and translocations. A deletion is a loss of one or more pieces from a chromosome after DNA breaks induced by various physical or chemical agents (Klug et al., 2005). Genetic information loss together with deletions causes serious disorders in humans, for example, Wolf-Hirschhorn syndrome, also known as deletion 4p syndrome, and Jacobsen syndrome, also known as deletion 11q syndrome (Hirschhorn et al., 1965; Jacobsen et al., 1973). Duplication is described as a phenomenon that a chromosome has extra copies of a chromosomal region, which may affect phenotype by altering gene function and transcriptional dosage (Zhang, 2003; Mao & Pevsner, 2005). Because most embryonic processes requires sensitively balanced protein levels, many duplications lead to developmental defects such as *Bar* eye mutation in *Drosophila* and Charcot-Marie-Tooth disease in humans (Sutton, 1943; Latour et al., 1997). An inversion type chromosomal mutation occurs when a portion of the chromosome breaks off, 180° rotates and reattaches, resulting in an inverted genetic material. There is little knowledge about the linkage between inversions and disease formation, and it is believed that many affect mechanisms of inversions directly associated with deletions. Juvenile Polyposis of Infancy, a rare genetic disorder, is a good example for a disease evolved by the cumulative effects of inversions (a paracentric inversion in 10q) and deletions (a deletion in 10p) (Gimelli et al., 2003; Antonacci et al., 2009; Vargas-Gonzales et al., 2010). The last group of the structural anomalies is translocations, defined as an exchange of segments among the non-homologues chromosomes. Several forms of cancer, leukemia and lymphoma are the best known disorders related to translocations (Li et al., 1999; Kurzrock et al., 2003; Anton et al., 2004). Figure 1 illustrates

Small-scale mutations, also known as gene mutations, include three main groups: point mutations, which are the most common type of the gene mutations and replace one nucleotide with another, insertions, which add one or a few extra nucleotides into the DNA,

and deletions, which remove one or a few nucleotides from the DNA (Brown, 2007).

born with only one X chromosome (Klug et al., 2005).

structural chromosome mutations.

Fig. 1. Structural chromosome mutations

systems, which eliminate disadvantages of the long-term test systems, are more suitable and acceptable. Many short-term studies result in gaining reliable and alternative data under controlled *in vitro* conditions. Another important advantage is that short-term mutation test systems are not only correlated with other short-term test systems, but also long-term systems (Mortelmans & Zeiger, 2000). Thus, the combinations of the mutagenicity test systems play a key role to get more meaningful results.

The bacterial mutation assays are known as most important short-term systems in order to determine mutagenic and antimutagenic potential of natural or synthetic chemicals related to gene mutations (Ames et al., 1973a, 1973b; Maron & Ames, 1983; Mortelmans & Riccio, 2000; Mortelmans & Zeiger, 2000). The *Salmonella* bacterial reverse mutation assay is one of the simplest, the most meaningful and acceptable short-term mutagenicity and antimutagenicity test systems. The test was initially developed by Ames in 1971. Therefore, it is also called as the Ames mutagenicity assay or the Ames/*Salmonella* mutagenicity assay (Ames et al., 1973a, 1973b; Maron & Ames, 1983; Zeiger, 2004, 2010). The main advantages of the assay, which employs mutant *Salmonella typhimurium* tester strains as model prokaryotic organisms, can be ordered as inexpensive applications enable studying a large number of test materials, quickly resulting (approximately 48 hours) allows making replicates in a short time, divers tester strains with several gene mutations allow to research the molecular effect mechanism of test materials, additional mutations in each strain result in more sensitivity such as *rfa* or *uvrB* and mesophile character of *Salmonella* allows to study several test materials affective at human body temperature. Furthermore, combination of the cytochrome-based P450 metabolic oxidation system, which usually consists of a 9000×g supernatant fraction of a rat liver homogenate (S-9 microsomal fraction), with the *Salmonella* mutagenicity test allows to determine some mutagenic agents, which are biologically inactive unless they are metabolized to active forms (Ames et al., 1973b; Mortelmans & Zeiger, 2000; Zeiger et al., 2005). Thus, the assay is used world-wide in genetic toxicology laboratories as a beginning mutation test to determine mutagenic and antimutagenic potentials of various chemicals.

The present study includes an introduction to use of *Salmonella* strains in genetic toxicology, principles of *Salmonella* bacterial reverse mutation assay, the most popular assay procedures with explanatory figures and clues for experimental design.

#### **2. Scientific background**

#### **2.1 Mutations and their effects on living organisms**

Genetic materials of all living organisms are dynamic structures that change and rearrange many times as a result of cumulative effects of mutations. Mutations, described as sudden and spontaneous or induced changes in a genomic sequence, are classified in two main groups depend on their physical effect sizes on the genome (Hartl & Jones, 1998; Lewin, 2004; Klug et al., 2005; Brown, 2007). First group is large-scale mutations in chromosomal level, including numerical and structural anomalies. Numerical anomalies are also called as aneuploidy, means an abnormal number of chromosomes. The most known examples for aneuploidy are monosomy (missing a chromosome from a pair), nullisomy (missing a pair of chromosomes), trisomy and polysomy (having one or more than two chromosomes of a pair). Down and Turner syndromes are important examples related to chromosomal anomalies in humans. An individual with Down syndrome has a developmental disorder caused by having three copies of chromosome 21. Therefore, it is also called as Trisomy 21.

systems, which eliminate disadvantages of the long-term test systems, are more suitable and acceptable. Many short-term studies result in gaining reliable and alternative data under controlled *in vitro* conditions. Another important advantage is that short-term mutation test systems are not only correlated with other short-term test systems, but also long-term systems (Mortelmans & Zeiger, 2000). Thus, the combinations of the mutagenicity test

The bacterial mutation assays are known as most important short-term systems in order to determine mutagenic and antimutagenic potential of natural or synthetic chemicals related to gene mutations (Ames et al., 1973a, 1973b; Maron & Ames, 1983; Mortelmans & Riccio, 2000; Mortelmans & Zeiger, 2000). The *Salmonella* bacterial reverse mutation assay is one of the simplest, the most meaningful and acceptable short-term mutagenicity and antimutagenicity test systems. The test was initially developed by Ames in 1971. Therefore, it is also called as the Ames mutagenicity assay or the Ames/*Salmonella* mutagenicity assay (Ames et al., 1973a, 1973b; Maron & Ames, 1983; Zeiger, 2004, 2010). The main advantages of the assay, which employs mutant *Salmonella typhimurium* tester strains as model prokaryotic organisms, can be ordered as inexpensive applications enable studying a large number of test materials, quickly resulting (approximately 48 hours) allows making replicates in a short time, divers tester strains with several gene mutations allow to research the molecular effect mechanism of test materials, additional mutations in each strain result in more sensitivity such as *rfa* or *uvrB* and mesophile character of *Salmonella* allows to study several test materials affective at human body temperature. Furthermore, combination of the cytochrome-based P450 metabolic oxidation system, which usually consists of a 9000×g supernatant fraction of a rat liver homogenate (S-9 microsomal fraction), with the *Salmonella* mutagenicity test allows to determine some mutagenic agents, which are biologically inactive unless they are metabolized to active forms (Ames et al., 1973b; Mortelmans & Zeiger, 2000; Zeiger et al., 2005). Thus, the assay is used world-wide in genetic toxicology laboratories as a beginning mutation test to

systems play a key role to get more meaningful results.

determine mutagenic and antimutagenic potentials of various chemicals.

with explanatory figures and clues for experimental design.

**2.1 Mutations and their effects on living organisms**

**2. Scientific background**

The present study includes an introduction to use of *Salmonella* strains in genetic toxicology, principles of *Salmonella* bacterial reverse mutation assay, the most popular assay procedures

Genetic materials of all living organisms are dynamic structures that change and rearrange many times as a result of cumulative effects of mutations. Mutations, described as sudden and spontaneous or induced changes in a genomic sequence, are classified in two main groups depend on their physical effect sizes on the genome (Hartl & Jones, 1998; Lewin, 2004; Klug et al., 2005; Brown, 2007). First group is large-scale mutations in chromosomal level, including numerical and structural anomalies. Numerical anomalies are also called as aneuploidy, means an abnormal number of chromosomes. The most known examples for aneuploidy are monosomy (missing a chromosome from a pair), nullisomy (missing a pair of chromosomes), trisomy and polysomy (having one or more than two chromosomes of a pair). Down and Turner syndromes are important examples related to chromosomal anomalies in humans. An individual with Down syndrome has a developmental disorder caused by having three copies of chromosome 21. Therefore, it is also called as Trisomy 21. Another disorder is Turner syndrome, an example of a monosomy where the individual is born with only one X chromosome (Klug et al., 2005).

Structural anomalies of the large-scale mutations include deletions, duplications, inversions and translocations. A deletion is a loss of one or more pieces from a chromosome after DNA breaks induced by various physical or chemical agents (Klug et al., 2005). Genetic information loss together with deletions causes serious disorders in humans, for example, Wolf-Hirschhorn syndrome, also known as deletion 4p syndrome, and Jacobsen syndrome, also known as deletion 11q syndrome (Hirschhorn et al., 1965; Jacobsen et al., 1973). Duplication is described as a phenomenon that a chromosome has extra copies of a chromosomal region, which may affect phenotype by altering gene function and transcriptional dosage (Zhang, 2003; Mao & Pevsner, 2005). Because most embryonic processes requires sensitively balanced protein levels, many duplications lead to developmental defects such as *Bar* eye mutation in *Drosophila* and Charcot-Marie-Tooth disease in humans (Sutton, 1943; Latour et al., 1997). An inversion type chromosomal mutation occurs when a portion of the chromosome breaks off, 180° rotates and reattaches, resulting in an inverted genetic material. There is little knowledge about the linkage between inversions and disease formation, and it is believed that many affect mechanisms of inversions directly associated with deletions. Juvenile Polyposis of Infancy, a rare genetic disorder, is a good example for a disease evolved by the cumulative effects of inversions (a paracentric inversion in 10q) and deletions (a deletion in 10p) (Gimelli et al., 2003; Antonacci et al., 2009; Vargas-Gonzales et al., 2010). The last group of the structural anomalies is translocations, defined as an exchange of segments among the non-homologues chromosomes. Several forms of cancer, leukemia and lymphoma are the best known disorders related to translocations (Li et al., 1999; Kurzrock et al., 2003; Anton et al., 2004). Figure 1 illustrates structural chromosome mutations.

Fig. 1. Structural chromosome mutations

Small-scale mutations, also known as gene mutations, include three main groups: point mutations, which are the most common type of the gene mutations and replace one nucleotide with another, insertions, which add one or a few extra nucleotides into the DNA, and deletions, which remove one or a few nucleotides from the DNA (Brown, 2007).

*Salmonella* as a Unique Tool for Genetic Toxicology 259

Fig. 3. Two possible effect mechanisms of the insertion or deletion type mutations on the

composed of tandem repetitions of relatively short motifs (Kashi & King, 2006).

Mutations are divided into spontaneous and induced alterations according to their formation sources. Spontaneous mutations arise from replication errors due to defective replication enzymes and alternative tautomer forms of nucleotide bases. These are rare types of mutations. On the other hand, induced mutations, common types of mutations, are caused by various mutagens. In the molecular mechanism of induced mutations, a physical or chemical mutagen reacts with the DNA strand, causing a structural change that affects the base-pairing capability of the altered nucleotide. The most important types of physical

Phenotypic results of mutations can be deleterious or advantageous for the affected organism. Many hereditable disorders are either directly caused by the mutations or indirectly associated with the mutagenesis. Cancer formation can be given as a satisfactory example to demonstrate deleterious effect of mutations. Recent studies clearly showed that there is a strict connection between mutagenesis and the formation of the several cancer types (Davidson et al., 2002). In this manner, mutations provide a unique resource for all tumors that show genomic instability with few exceptions. On the other hand, mutated organisms can gain various advantages as a result of mutagenesis. Gain of antibiotic resistance in microorganisms and HIV/AIDS protective mutations on the SDF1, CCR5 and CCR2 genes in the human genome are well examples for the advantageous mutations (Stephan et al., 1998; Galvani & Slatkin, 2003; Apostolakis et al., 2005). These are also very important for evaluation of the organisms. Therefore, mutagenesis can be considered as one of the most important evolutionary sources. For example; simple sequence repeats (SSRs, also called microsatellites and minisatellites) are defined as advantageous mutators in adaptive evolution. Recent studies showed that temperature compensation of circadian rhythm in *Drosophila*, adaptive divergence among barley and wheat populations, social behavior in voles, skeletal morphology in domestic dogs and sporulation efficiency and cell adhesion in yeast are closely related to SSRs, which are mutation-prone DNA tracts

coding region of a gene (Brown, 2007).

**2.2 The causes of mutations**

Point mutations are also divided into two categories as transitions and transversions. Transitions, which are purine-to-purine or pyrimidine-to-pyrimidine changes (A↔G or C↔T), are more common type of the point mutations than transversions, which are purineto-pyrimidine or pyrimidine-to-purine changes (A↔C, A↔T, G↔C or G↔T) (Brown, 2007).

Contrary to "small-scale" word in their names, these mutations can cause wide-range significant changes in genomes and phenotypes of living organisms with mutated genetic materials. For example, a point mutation may result in a synonymous change that causes forming a new codon specifying the same amino acid as the unmutated codon, a nonsynonymous change that causes a missense mutation where a new codon specifies a different amino acid from the unmutated codon, a nonsense mutation where the change converts an amino acid specifying codon into a termination codon, or a readthrough mutation where the change converts a termination codon into an amino acid specifying codon. Except synonymous changes, also called as silent mutations because the mutated gene codes for exactly the same protein as the unmutated gene, the other three types of point mutations have significant impacts on the genome and related phenotypes by effecting amino acid sequence of the coding protein (Hartl & Jones, 1998; Alberts et al., 2002; Lewin, 2004; Brown, 2007; Lodish et al., 2007). The effects of point mutations on the coding region of a gene are shown in Figure 2.

Fig. 2. The effects of point mutations on the coding region of a gene (Brown, 2007).

Insertion and deletion types of small-scale mutations affect the coding capabilities of the gene in a different way. It is defined as a frameshift mutation, caused by addition or deletion of a number of nucleotides that is not evenly divisible by three from a DNA sequence. Because codons consist of three nucleotides, an insertion or deletion type mutation can disrupt the reading frame, resulting in a completely different translation from the unmutated gene. Thus, insertion or deletion mutations generally have more significant effects on the protein function than the point mutations because the translated protein have completely different sequence from the mutated point to the end. An exception occurs that the number of inserted or deleted nucleotides is three or a multiple of three, which results in addition or deletion of one or more codons (Alberts et al., 2002; Lewin, 2004; Brown, 2007; Lodish et al., 2007). Figure 3 illustrates two possible effect mechanisms of the insertion or deletion type mutations on the coding region of a gene.

Point mutations are also divided into two categories as transitions and transversions. Transitions, which are purine-to-purine or pyrimidine-to-pyrimidine changes (A↔G or C↔T), are more common type of the point mutations than transversions, which are purineto-pyrimidine or pyrimidine-to-purine changes (A↔C, A↔T, G↔C or G↔T) (Brown, 2007). Contrary to "small-scale" word in their names, these mutations can cause wide-range significant changes in genomes and phenotypes of living organisms with mutated genetic materials. For example, a point mutation may result in a synonymous change that causes forming a new codon specifying the same amino acid as the unmutated codon, a nonsynonymous change that causes a missense mutation where a new codon specifies a different amino acid from the unmutated codon, a nonsense mutation where the change converts an amino acid specifying codon into a termination codon, or a readthrough mutation where the change converts a termination codon into an amino acid specifying codon. Except synonymous changes, also called as silent mutations because the mutated gene codes for exactly the same protein as the unmutated gene, the other three types of point mutations have significant impacts on the genome and related phenotypes by effecting amino acid sequence of the coding protein (Hartl & Jones, 1998; Alberts et al., 2002; Lewin, 2004; Brown, 2007; Lodish et al., 2007).

The effects of point mutations on the coding region of a gene are shown in Figure 2.

Fig. 2. The effects of point mutations on the coding region of a gene (Brown, 2007).

deletion type mutations on the coding region of a gene.

Insertion and deletion types of small-scale mutations affect the coding capabilities of the gene in a different way. It is defined as a frameshift mutation, caused by addition or deletion of a number of nucleotides that is not evenly divisible by three from a DNA sequence. Because codons consist of three nucleotides, an insertion or deletion type mutation can disrupt the reading frame, resulting in a completely different translation from the unmutated gene. Thus, insertion or deletion mutations generally have more significant effects on the protein function than the point mutations because the translated protein have completely different sequence from the mutated point to the end. An exception occurs that the number of inserted or deleted nucleotides is three or a multiple of three, which results in addition or deletion of one or more codons (Alberts et al., 2002; Lewin, 2004; Brown, 2007; Lodish et al., 2007). Figure 3 illustrates two possible effect mechanisms of the insertion or

Fig. 3. Two possible effect mechanisms of the insertion or deletion type mutations on the coding region of a gene (Brown, 2007).

Phenotypic results of mutations can be deleterious or advantageous for the affected organism. Many hereditable disorders are either directly caused by the mutations or indirectly associated with the mutagenesis. Cancer formation can be given as a satisfactory example to demonstrate deleterious effect of mutations. Recent studies clearly showed that there is a strict connection between mutagenesis and the formation of the several cancer types (Davidson et al., 2002). In this manner, mutations provide a unique resource for all tumors that show genomic instability with few exceptions. On the other hand, mutated organisms can gain various advantages as a result of mutagenesis. Gain of antibiotic resistance in microorganisms and HIV/AIDS protective mutations on the SDF1, CCR5 and CCR2 genes in the human genome are well examples for the advantageous mutations (Stephan et al., 1998; Galvani & Slatkin, 2003; Apostolakis et al., 2005). These are also very important for evaluation of the organisms. Therefore, mutagenesis can be considered as one of the most important evolutionary sources. For example; simple sequence repeats (SSRs, also called microsatellites and minisatellites) are defined as advantageous mutators in adaptive evolution. Recent studies showed that temperature compensation of circadian rhythm in *Drosophila*, adaptive divergence among barley and wheat populations, social behavior in voles, skeletal morphology in domestic dogs and sporulation efficiency and cell adhesion in yeast are closely related to SSRs, which are mutation-prone DNA tracts composed of tandem repetitions of relatively short motifs (Kashi & King, 2006).

#### **2.2 The causes of mutations**

Mutations are divided into spontaneous and induced alterations according to their formation sources. Spontaneous mutations arise from replication errors due to defective replication enzymes and alternative tautomer forms of nucleotide bases. These are rare types of mutations. On the other hand, induced mutations, common types of mutations, are caused by various mutagens. In the molecular mechanism of induced mutations, a physical or chemical mutagen reacts with the DNA strand, causing a structural change that affects the base-pairing capability of the altered nucleotide. The most important types of physical

*Salmonella* as a Unique Tool for Genetic Toxicology 261

After its introduction to the scientific world, Ames test has been widely accepted as a shortterm bacterial test system for determining chemicals that can cause gene mutations. The test has many advantages for identification of the chemicals that cause gene mutations. These

Short-term resulting: allows making replicates and obtaining more reliable results in a

Various tester strains with several gene mutations: enable to research the molecular

Additional mutations and genetic alterations: allow gaining more sensitivity for various

Mesophile character of *Salmonella* strains: enables to study mutagenic potential of the

Apart from all the maintained advantages, Ames/*Salmonella* test system is very versatile, and many modifications has been developed to determine mutagenic potencies of various materials such as environmental chemicals, environmental mixtures, body fluids, foods, drugs and physical agents. The most common assay procedures are the spot test: a primal method for determination of chemical mutagens, the standard plate incorporation method: an easily resulting and more comprehensive method than the spot test, the pre-incubation method: developed for performing more effective studies with lower volumes of test materials, the desiccator assay modifications: developed to study volatile materials and gases, and the modified *Salmonella* microsuspension assay (Kado): a highly sensitive method for testing the materials that are available only in small amounts (Kado et al., 1983; Hughes et al., 1987; Zeiger et al., 1992; Araki et al., 1994; Mortelmans & Zeiger, 2000; Tijs, 2008).

Although *Salmonella* has prokaryotic cell structure, combination of the cytochrome-based P450 metabolic oxidation system with the Ames/*Salmonella* test system allows determining some mutagenic agents, which are biologically inactive unless they are metabolized to active forms (Ames et al., 1973b; Mortelmans & Zeiger, 2000). Moreover, all the procedures of the test system can be altered to identify antimutagenic agents, inhibit mutagenesis and protect the organisms against deleterious effects of the mutagens, with some modifications (Nagabhushan et al., 1987; Bala & Grover, 1989; Edenharder et al., 1999; Edenharder &

The following items are required for performing the Ames/*Salmonella* mutagenicity and

Grünhage, 2003; Ozbek et al., 2008a, 2008b; Gulluce et al., 2010) (see 3.8).

Low-cost: allows studying a large number of test materials inexpensively.

advantages can be listed in:

chemicals.

**3. Material and methods 3.1 Supplies and equipment**

antimutagenicity protocols.

Sterile microbiological loops

Sterilizing membrane filters (0.2 µm)

Sterile glass test tubes (100×16 mm) and racks

**3.1.1 Supplies**

Magnetic stir bars

short duration. It takes only about 48 hours.

effect mechanism of test materials

chemicals at human body temperature.

mutagens are ultraviolet radiation of wavelength 260 nm, ionizing radiation and heat shock.