clinical and environmental point of view (Baird-Parker, 1990; Baudart et al., 2000).

Transcription

1 II. REVIEW II. Review OF of LITERATURE literature Salmonella is a Gram negative, facultative anaerobic, rod shaped bacteria capable of causing disease in humans as well as in domestic animals. More than 2500 serovars of Salmonella have been identified but only a limited set of them has found importance from clinical and environmental point of view (Baird-Parker, 1990; Baudart et al., 2000). Salmonellosis is one of the common foodborne disease caused by Salmonella spp. It is a significant pathogen of food producing animals and these animals are the primary source of salmonellosis (Forshell and Wierup, 2006). Salmonella spp. has been constantly reported in environmental samples because of their excretion through the faeces of humans and animals. The incidence of foodborne infection due to Salmonella has been reported from several parts of the globe. In the US, the Center for Disease Control and Prevention (CDC), reported 39,027 cases of salmonellosis in 1996 (El-Begearmi, 1998). In 2004, the European Union (EU) alone showed 192,703 human cases of salmonellosis (EFSA, 2006). The 2012 outbreak investigation by CDC showed the association of different serovars of Salmonella from different food sources. The infection by Salmonella spp. is mainly through the consumption of contaminated food or water. The infection can result in gastroenteritis, enteric fever and septicemia. In humans, Salmonella spp. are the cause of two diseases called enteric fever which results from the invasion of bacteria into the blood stream and acute gastroenteritis, which results from the foodborne infection or intoxification. Page 6

2 2.1. History of Salmonella Typhoid fever was the first documented enteropathogenic disease in the latter part of the nineteenth century (Le Minor, 1981). Since then, contaminated shellfish have been associated with the transmission of a wide range of diseases including paratyphoid fever, cholera, viral hepatitis and many other gastroenteric conditions. Eberth was the first to observe typhoid bacillus in spleen sections and mesenteric lymph nodes from a typhoid patient in Subsequently, Robert Koch and Graffky succeeded in cultivating the bacterium in Serodiagnosis was made possible by 1896 due to the agglutination of typhoid bacterial cells with the serum obtained from an animal immunized with typhoid bacillus Nomenclature The genus Salmonella belongs to the family Enterobacteriaeceae with DNA base composition of mol % G+C (Marmur et al., 1963). Salmonella nomenclature has been complicated since the original taxonomy of the genus was not based on DNA relatedness; instead, the names were given according to clinical considerations and host specificity eg. Salmonella Typhi, Salmonella Choleraesuis etc. Later in 1941, Kauffman defined the species as a group of related fermentation and phage types (Kauffman, 1941, 1966, 1978) and considered each serovar as species. Since the host specificity suggested earlier did not exist, names derived from the geographical origin of the first isolated strain of the newly discovered serovars were next chosen, e.g., S. London, S. Panama, S. Stanleyville, S. Bareilly, etc. Page 7

3 According to the CDC, Salmonella genus contains only 2 species, S. enterica and S. bongori. It was found that all Salmonella serovars belong to a single hybridization group i.e., a single species composed of seven subspecies. Strains under Subspecies I represent more than 99.5 % of Salmonella isolated from humans and warm blooded animals. Subspecies II and III include organisms from cold blooded animals. Subspecies IV and V are found mainly in environment and have been rarely pathogenic to human beings (Le Minor, 1981). The role of a number of Salmonella serovars in pathogenesis of the disease is unknown. This is especially in the case of serovars from subspecies II to VI, where most of the serovars have been isolated rarely (some only once) during a systemic infection in cold blooded animals. Further, a new species of Salmonella (Salmonella subterranea) has been identified on March 18, 2005 that is adapted to live in soil with mild acidic ph (Shelobolina et al., 2004) Current Salmonella Nomenclature Genus: Species: Subspecies: Salmonella enterica, bongori, subterranea enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), obsolete (now designated S. bongori) (V), indica (VI), Serovars of subspecies I: Enteritidis, Typhi, Typhimurium, Paratyphi, Choleraesuis etc. The subspecies I to IV and VI belong to the species enterica and subspecies V belong to the species bongori. Page 8

4 2.3. General features of Salmonella The Salmonella genus like all Enterobacteriaceae members Gram negative, oxidase negative, straight, rod shaped bacteria which are catalase positive and capable of fermenting carbohydrates. They are facultative anaerobes, non spore forming and usually motile with peritrichous flagella. However, some of the serovars like S. Pullorum and S. Gallinarum are usually nonmotile. Three major groups have been reported based on their host adaptation capability. Human adapted serovars like S. Typhi, S. Paratyphi, and S. Sendai usually causes conditions like septicemia typhoid syndrome or enteric fever which is non pathogenic to other creatures. Ubiquitous serovars like S. Typhimurium which is capable of causing gastrointestinal infections in humans as well as in certain animals. Some of the serovars S. Abortusovis and S. Gallinarum are highly adapted to animal hosts and infect sheep and poultry respectively, producing little or very mild symptoms in humans. One of the principal habitats of Salmonella spp. is the intestinal tract of humans and animals (Le Minor, 1981). It can be frequently found in a particular host, can be ubiquitous, or can have an unknown habitat. Human adapted serovars like S. Typhi and S. Paratyphi A, cause grave disease due to their ability to invade the blood stream. S. Gallinarum, S. Abortusovis, and S. Typhisuis are avian, ovine, and porcine adapted serovars which usually prefer certain growth factors, during culture in the minimal medium. Diverse clinical symptoms are very common in non-host adapted ubiquitous Salmonella serovars like S. Typhimurium where the symptoms could range from being Page 9

5 asymptomatic to serious typhoid like syndromes in infants or in certain highly susceptible animals (mice). In humans, these ubiquitous Salmonella are responsible for foodborne toxic infections Ecology of Salmonella Study of Salmonella ecology relates to the study of living organisms with which the Salmonella has an important relationship. Control of the cyclic phenomena of salmonellosis will be effective only when all sources of Salmonella are addressed simultaneously for which ecological issues must be studied. The natural habitat of Salmonella being the intestinal tract of animals (Pelczar, 1989; Holt et al., 1994), it could find its way to the natural environment through feces (Thong et al., 2002). The ecology of this organism was studied by examining the fishes recovered from sewage polluted streams that revealed a high percentage of Salmonella in the intestinal tract. An experimental study on the survival of Salmonella in catfish gut demonstrated its viability in the stomach and intestine for at least 29 days (Foltz, 1969) Salmonella spp. and food Outbreaks due to Salmonella spp. have occurred from variety of foods including poultry, meats, eggs, milk products, fruit juice, tomatoes, fish, shrimp, froglegs, yeast, coconut, etc. Salmonellosis in animal may leads to the contamination of meat which contributes to the contamination of muscles during evisceration, transportation of carcasses, washing, etc. (Le Minor, 1981). A variety of foods have been considered vehicles for the spread of salmonellosis in human as well as in animals which include fish, shellfish, fresh fruits, juices and vegetables (Gomez et al., 1997; Esaki et al., 2004). The incidence of Page 10

6 Salmonella spp. is much higher in raw agricultural products (e.g. raw eggs or uncooked poultry or meat) than in cooked or processed food products. However, Salmonella spp. can occur in other foods as a result of cross contamination with raw foods or contamination from humans, animals, birds, or reptiles. Further, due to its ability to survive in a wide range of environments, Salmonella spp. have been encountered in dry and dehydrated foods (e.g., cocoa, chocolate, dry milk, spices, and cereal products) and in acid food products (e.g., nonpasteurized orange juice) (Rushdy et al., 1998; Smith et al., 2004; Koch et al., 2005). Salmonellosis is one of the most common bacterial foodborne illnesses; however in most cases it could be prevented by proper food handling practices. Thus, preventative measures are extremely important during all food handling and processing steps Incidence of Salmonella spp. in seafood The majority of seafood associated illness is mainly due to the consumption of shellfish from faecally polluted water. Fish/shefish normally do not carry microorganism like Salmonella but acquire it from contaminated water from which it has been harvested (Shewan, 1962). Salmonella spp. being the most important contaminant of fish and fishery products has prompted the detention of imported food by Japan. In 2001, according to the FDA reports, of 6405 total violated foods, 28.6 % were found to be contaminated with Salmonella spp. (Doyle, 2006). More recently 2 of 47 black tiger prawn and two white tiger shrimp consignments that were imported were found to be contaminated with S. Weltevreden (Asai et al., 2008). These results indicate the possibility of foodborne infection through imported seafood like shrimp and prawns. Few studies showed that the overall incidence of Salmonella spp. was 7.2 % for import and 1.3 % for domestic seafood samples over a period of nine years with raw seafood Page 11

7 demonstrating the highest contamination rates of 10 % for import and 2.8 % for domestic (Heinitz et al., 2000). Incidences of Salmonella spp. causing gastroenteritis have also been reported due to the consumption of sewage contaminated shellfish (Allen, 1899; Goh, 1981; Greenwood et al., 1998; Mead et al., 1999). The contamination of fish and fishery products by Salmonella spp. has also been reported by other countries like Thailand (Rattagool et al., 1990), Hong Kong (Yam et al., 1999), Spain (Martinez-Urtaza et al., 2004) and Singapore (Huang et al., 2012). More than 1.6 % of the shellfish sampled from open harvesting waters were found to be contaminated with S. Typhi in the UK (Rippey, 1994; Wright et al., 1996; Lipp and Rose, 1997). The work of Koonse et al. (2005) showed S. Weltevreden as the most frequent serotype in 21 % of the total serotypes isolated and reported from aquaculture shrimp farms in three different countries. Similar observation made by Ponce et al. (2008) confirmed S. Weltevreden as the most important serovar among the 64 different serovars isolated from seafood. S. Senftenberg was found to be the leading serovar isolated from live molluscan shellfish from the marine environments of Galicia region of Spain (Martinez-Urtaza et al., 2003). In Vietnam 24.5 % of the shrimp samples were found to be contaminated with S. Weltevreden and S. Tennessee (Phan et al., 2005). A much incidence of Salmonella spp. is found in tropical countries. In India, the incidence of the organism showed wide variation in levels found in seafood samples. Its prevalence in fish and shellfish obtained from Coimbatore market was 14.2 % and %, respectively (Hatha and Lakshmanaperumalsamy, 1997) which is in contrast to the prevalence of 7.8 % in seafood samples from Cochin market (Nambiar and Iyer, 1991). Fish and fishery Page 12

8 products in Mumbai recorded thirty four different Salmonella serovars (Iyer and Shrivastava, 1989). S. Weltevreden has been reported to be the predominant serovar in seafood in the Asian region (Boonmar et al., 1998; Phan et al., 2005; Shabarinath et al., 2007). A study conducted by Kumar et al. (2009) showed 23.2 % of the seafood samples to be positive for Salmonella and a total of 241 Salmonella isolates comprising 27 different serovars were isolated. S. Weltevreden, S. Rissen, S. Typhimurium and S. Derby were the most predominant serovars in seafood. This widespread occurrence of multiple clones of Salmonella spp. in seafood suggests the diverse routes of Salmonella contamination in seafood. S. Weltevreden was found to be the most common serovar causing human infections in India during early 1970 s (Basu et al., 1997). 30 % finfish, 20 % clams, and 1 % shrimp samples caught off Mangalore coast were reported to be contaminated with Salmonella spp. (Kumar et al., 2003) Virulence characteristics of Salmonella spp. In order to prevent the suffering among humans and economic losses caused by the presence of Salmonella in seafood, it is necessary to understand the mechanism of its survival inside the host body. The ability of Salmonella to cause disease relies on several determinants of virulence which include the genes involved in nutrient biosynthesis, stress response and repair of cell damage. These genes are together considered as housekeeping genes which are also present in other closely related bacteria such as E. coli (Baumler et al., 2000). The presence of true virulence determinants which is specific for the genus Salmonella, help them overcome the host defense mechanism. The genetic control of virulence in Salmonella is not fully known. However, it involves both plasmid and chromosomal genes. These virulence genes are mainly located on the pathogenicity islands of the chromosomes referred to as Page 13

9 Salmonella pathogenicity islands (SPI). It is also believed that these genes have been acquired by Salmonella from other organisms through horizontal transfer (van Asten et al., 2005). The main function of these islands includes host cell invasion and intracellular pathogenesis (Fig.1). Though about 17 SPIs have been identified so far, their function in pathogenesis of Salmonella spp. and virulence of many genes within the SPI is not yet fully known (Hensel, 2004) Salmonella pathogenicity islands SPI-1 a 40 kb island having a G+C content of 42 % encoding type 3 secretion system that translocates effector protein molecules into the host cell (Fig.2). It forms syringe like organelles on the surface of Gram negative bacteria and enables the injection of effector proteins directly into the cytosol of eukaryotic cells (Galan et al., 2001; Waterman et al., 2003). These effectors ultimately manipulate the cellular functions of the infected host and inturn facilitates the progression of the infection. SPI-1 primarily promotes the invasion of non-phagocytic intestinal epithelial cells and the initiation of the inflammatory response in the intestines (Hapfelmeier et al., 2004; Coombes et al., 2005). It is also involved in the survival and persistence of Salmonella in the systemic compartment of the host (Steele-Mortimer et al., 2002; Brawn et al., 2007). Page 14

10 Lopez et al., 2012 Fig. 1. Schematic representation of Salmonella infection (A) Free living state of Salmonella (B) Entry into the human intestinal mucus layer through contaminated water and foods (C) After the bacteria encounter the intestinal epithelial cell, the infection is established. To facilitate adhesion, Salmonella induce the expression of fimbriae and adhesins that are encoded in six SPIs (1). SPI-1 primarily promotes the invasion of nonphagocytic intestinal epithelial cells and the initiation of the inflammatory response in the intestines (2). The bacteria entry to the cell results in an early SCV (Salmonella containing vacuole) (3), that maturates to a late SCV (4). Several hours after invasion, Salmonella initiate the replication (5). Finally, the bacteria induce the host cell apoptosis (6) Page 15

11 The regulation of SPI-1 is complex. It encodes for the five regulators; HilA, HilC, HilD, InvF, and SprB, the first four of which are involved in regulatory pathways that lead to the activation of SPI-1 genes and of genes encoding type 3 secretion system (T3SS) effectors located outside SPI-1. Ellermeier et al., 2007 Fig. 2. Schematic representation of Salmonella pathogenicity Island-1 genes. Arrows below the gene names represent transcripts SPI-2 a 40 kb island was first characterized for its role in promoting the Salmonella survival and multiplication in phagocytic cells that constitute the main reservoir for dissemination of the bacteria into systemic organs (Fig.3) (Waterman et al., 2003). SPI-2 encoding T3SS also plays an important role in the intestinal phase of Salmonella infection in mice (Coburn et al., 2005; Coombes et al., 2005; Hapfelmeier et al., 2005). Salmonella strains proliferate within macrophages and epithelial cells in vitro with the help of SPI-2 (Ochman et al., 1996; Cirillo et al., 1998; Hensel et al., 1998; Vazquez-Torres et al., 2000; Gallois et al., 2001). Further, SPI-2 is essential for Salmonella to cause systemic infection and has been associated with enteric diseases in animal models (Hensel et al., 1995; Shea et al., 1996; Page 16

12 Everest et al., 1999; Bispham et al., 2001). In contrast to SPI-1 the regulation of SPI-2 genes is simpler with the SsrAB two component system being the only transcriptional regulator encoded within SPI-2 that activates the expression of SPI-2 genes and of genes encoding T3SS effectors located outside SPI-2. In addition to this, genes in the SPI-2 are crucial for prolonged bacterial growth in intestinal epithelial cells in mouse model (Paesold et al., 2002). SPI-1 regulators can regulate SPI-2 genes which include HilA that bind and repress the promoter of ssah, and HilD that binds and activates the promoter of the ssrab operon (Bustamante et al., 2008). In contrast, SsrAB has never been shown to act on the expression of SPI-1 genes. Recent studies showed that SPI-1 contributes to colonization of both the cecum and spleen in the chicken whereas SPI-2 contributes to colonization of the spleen but not the cecum and in the absence of SPI-1, inhibits cecal colonization (Dieye et al., 2009). Slonczewski and Foster et al., 2010 Fig. 3. The Salmonella pathogenicity island SPI-2 was identified using signature tagged mutagenesis. ORFs are indicated by colored block arrows pointing in the direction of transcription. Gene designations are positioned below each ORF. The functional classes of SPI-2 genes are represented by different colors Page 17

13 SPI-3 a 17 kb pathogenicity island located at the selc trna locus of S. Typhimurium with 47.1 % of G+C content which differs from that of the Salmonella genome (52 %) (Fig.4). The island harbours mgtc, a Salmonella specific gene that is required for intramacrophage survival and virulence in mice (Blanc-Potard et al., 1997). Recently, it has been described that SPI-3 harbors 10 open reading frames organized in six transcriptional units, which include the previously described mgtcb operon encoding the macrophage survival protein MgtC and the Mg 2+ transporter protien MgtB. Among the newly identified open reading frames, one exhibits sequence similarity to the ToxR regulatory protein of Vibrio cholerae and one is similar to the AIDA-I adhesin of enteropathogenic E. coli (Blanc-Potard et al., 1999). Fig. 4. Genetic map of Salmonella pathogenicity island 3 (Blanc-Potard et al., 1999) SPI-4 a 24 kb pathogenicity island containing six open reading frames siia to siif as shown in Figure 5. The G+C content of SPI-4 is 37 to 44 % compared to 52 % for the rest of the S. Typhimurium genome (Wong et al., 1998). Recent studies have demonstrated the role of siie during the intestinal phase of infection. SPI-4 also has eighteen putative ORFs, encoding type I secretion system that mediates toxin secretion, much like E. coli haemolysin secretion. Page 18

14 Gerlach et al., 2007 Fig. 5. Genetic map of Salmonella pathogenicity island 4 SPI-5 a small locus of 7.6 kb, inserted adjacent to trna sert locus. The overall G+C content is of SPI-5 is approximately 43.6 % (Fig.6). It encodes the effector proteins for both T3SS encoded by SPI-1 and SPI-2. SopB of SPI-5 is secreted by T3SS of SPI-I and expression regulated by hila. The pipb of SPI-5 is translocated by the T3SS of SPI-2 to the Salmonella containing vacuole and Salmonella induced filaments. Fig. 6. Genetic map of Salmonella pathogenicity island 5 ( virulenec factors of pathogenic bacteria) SPI-6 a 59 kb genome sequence inserted adjacent to the aspv trna gene and contains the saf gene cluster for fimbriae, pagn encoding an invasin and several genes of unknown function. Reduced invasion of cultured cells was observed upon deletion of entire chromosomal island in S. Typhimurium (Folkesson et al., 2002). SPI-7 a large, mosaic, genetic island, found in several serovars of Salmonella enterica subsp. enterica associated with systemic disease. This 134 kb island was first discovered as a Page 19

15 large insertion in the genome of the pathogen S. Typhi relative to that of serovar Typhimurium (Liu and Sanderson, 1995). This island carries genes for potential virulence factors such as Vi antigen, SopE effector and type IVB pili. The stability of SPI-7 is of interest with respect to typhoid fever and related vaccines (Helena and Seth-Smith, 2008). SPI-8 a 6.8 kb region located adjacent to the phev trna gene sequence of S. Typhi (Parkhill et al., 2001). SPI-8 is widely distributed among Salmonella serovars. Bacteriocin production was found to be the virulence factor of this island but its exact role and distribution needs to be studied further to understand the specificity and invasiveness of Salmonella serovars. SPI-9 of Salmonella is a 16 kb region located adjacent to a lysogenic bacteriophage in the chromosome of S. Typhi (Parkhill et al., 2001). RTX like protein and type I secretion system are the major virulence factors encoded by SPI-9. SPI-10 of Salmonella is a 32.8 kb large insertion located at trna leux. There is a cryptic bacteriophage present within SPI-10 (Parkhill et al., 2001). The role of this bacteriophage in distribution of SPI-10 has not been elucidated so far. The major known virulence factors encoded by SPI-10 are the Sef fimbriae. SPI-11 was named following the sequencing of the S. Choleraesuis genome (Chiu et al., 2005). This island is a 14 kb region located next to the Gifsy-1 prophage. Many genes on this island have been characterized for their role in virulence of S. Typhimurium. The pagc and pagd genes are regulated by SlyA and the phop/phoq two component system which is a regulator of a large number of virulence genes (Gunn et al., 1995; Navarre et al., 2005). pagc contributes to intramacrophage survival and systemic infection in mice (Miller et al., 1989). Page 20

16 The enve and envf genes are predicted to encode envelope proteins but are not required for virulence in mice (Gunn et al., 1995). In contrast to SPI-11, SPI-12 is present in genomes of S. Typhi and S. Typhimurium. This island is inserted next to the prol trna gene and is 6.3 kb in length in S. Typhi and S. Choleraesuis. An additional 9.5 kb carrying phage genes is present in S. Typhimurium (Hansen-wester and Hensel, 2002). The only virulence factor present in SPI-12 is SspH2. This is a secreted effector protein of T2SS that influences the rate of actin polymerisation inside the infected cells (Miao et al., 2003). SPI-13 is a 19.5 kb region located next to the phev trna gene. Of the 18 genes on this island only three have been implicated in virulence viz., gacd, gtra and gtrb. These are required for the virulence of S. Gallinarum in one day old chick infection model (Shah et al., 2005). SPI-14 carries two genes viz., gpiab, which is required for the virulence of S. Gallinarum in one day old chicks (Shah et al., 2005). The island is present in S. Typhimurium and S. Choleraesuis but absent in S. Typhi and S. Paratyphi A. More recently, the least characterized SPI-16 described by Sabbagh et al. (2010) contains three open reading frames which could be involved in mediating O-antigen glycosylation and cell surface variation. Apart from this Blondel et al. (2009) discovered three pathogenicity islands (SPI-19, 20 and 21) in Salmonella. SPI-19 is a locus of 45 kb, present in subset of serotypes belonging to S. Dublin, Weltevreden, Agona, Gallinarum and Enteritidis. It encodes two ORFs for Hcp-1 and Hcp-2 proteins. Hcp-1 is a putative operon that includes most of the T6SS function and Hcp-2 is found upstream of a VgrG homologue. SPI-20 is a Page 21

17 locus of~34 kb located adjacent to aspv trna gene having 28 ORFs with 17 of them involved in T6SS function. SPI-21 locus is located adjacent to thrw trna gene and encodes a T6SS in the genome of serotype IIIa 62:z4, z 23. It has 57 ORFs, 20 of which encode T6SS function. In addition SPI-21 includes four ORFs encoding putative colicin/pyocin immunity proteins Salmonella virulence plasmid Many serovars of Salmonella harbor virulence plasmids which are important for systemic infection of experimental animals. They vary in their size depending on the serovars e.g. 95 kb for S. Typhimurium; 60 kb for S. Enteritidis; 80 kb for S. Dublin; and ranging from 50 to 110 kb for S. Choleraesuis (Chu et al., 1999). A highly conserved 8 kb operon consisting of five genes, designated spvrabcd is present in all Salmonella serotypes examined (Gulig et al., 1993). The gene spvb encodes an ADP-ribosylating toxin that modifies actin directly and totally disrupts the cytoskeleton of the cell (Slayers and Whitt, 2002) Drug resistance of Salmonella spp. The introduction of antibiotics for treating and preventing infections in humans is the most fascinating development in medicine. However, their indiscriminate use has led to the rapid evolution and spread of bacterial resistance, which poses a major threat for successful treatment of infectious diseases. Antimicrobial resistance is one of the growing concerns in many regions of the world. Unless a rational policy for its use is in place, there is a threat that infectious diseases will become gradually untreatable. Salmonella within the subspecies 1 are amongst the most resistant pathogenic bacteria and are wide spread in both developed and developing countries. Since the early 1990s, the multiresistant strains of S. Typhimurium definitive phage type 104, displayed resistance to up to six commonly used antibiotics. The Page 22

18 development of resistance to key antimicrobials such as fluoroquinolones, particularly in Asian countries, and also extended spectrum cephalosporins worldwide is a cause of concern (Miriagou, 2004). Since, Salmonella is an intracellular pathogen; the effective way to eradicate this organism is to use antibiotics that have intracellular activity. The earliest groups of drugs used in the treatment of Salmonella infections, were neomycin and colistin. This was followed by use of absorbable drugs such as ampicillin, amoxicillin chloramphenicol, tetracycline, and co-trimoxazole which unfortunately do not have substantial intracellular activity. The new era of drugs began with the use of intracellular drugs with potent activity such as 5-fluoroquinolone compounds (norfloxacin, ofloxacin, fleroxacin, ciprofloxacin). A word of caution needs to be kept in mind since frequent use of these antibiotics has been found to lead to resistance in Salmonella Development of antibiotic resistance Antibacterial agents used for the treatment of bacterial infections have four modes of action, namely (1) inhibition of cell wall synthesis by interfering with the enzymes required for the synthesis of the peptidoglycan layer (McManus, 1997; Martone, 1998); (2) inhibition of protein synthesis wherein the drug binds the 50S and 30S ribosomal subunit and inhibit the protein synthesis; (3) interference with nucleic acid synthesis by inhibiting DNA and RNA synthetase; and (4) inhibition of a metabolic pathway by targeting a metabolic enzyme. Susceptible bacteria can acquire resistance to new antimicrobial agent through many ways of which mutations in the genetic structure is one of the causes. Such mutations may cause resistance by (1) altering the target protein to which the antibacterial agent binds by modifying or eliminating the binding site; (2) up regulating the production of enzymes that inactivate the Page 23

19 antimicrobial agent; (3) down regulating or altering an outer membrane protein channel that the drug requires for cell entry; and (4) up regulating pumps that expel the drug from the cell (Fig. 7). In all these cases, the bacterial strains acquire resistance to new antibiotics owing to chromosomal mutation and such selection is termed as vertical evolution. Bacteria also develop resistance through the acquisition of new genetic material from other resistant organisms by several methods which include conjugation (via plasmids and conjugative transposons), transduction (via bacteriophages), and transformation (via uptake of chromosomal DNA, plasmids, and other DNAs of dying and dead organisms) (Levy and Marshall, 2004) and these modes of transfer result in horizontal evolution. Other mechanisms of transferring resistance include virulence plasmids, transposons, integrons etc Antibiotic resistance pattern in Salmonella spp. Antibiotic resistance is an emerging trend and seen frequently in multiresistant strains of S. Typhimurium isolated from animals (Martel et al., 1996) and humans (Breuil et al., 1996). Strains of Salmonella resistant to several antimicrobial agents have been reported worldwide (Angulo and Griffin, 2000; Breuil et al., 2000; Van Duijkeren et al., 2003). In 1964, genes conferring resistance to multiple antibiotics were first identified in UK in S. Typhimurium and could be transferred between unrelated bacteria such as Bacteroides and also between related members of Enterobacteriaceae such as Salmonella and E. coli (O Brien, 2002). Several studies have reported the prevalence of multiresistance genes in different serotypes of Salmonella (Aarts et al., 2001). Page 24

20 Levy and Marshall, 2004 Fig.7. Represents the mechanism of antibiotic resistance in bacteria The recent reports of NARMS (National Antimicrobial Resistance Monitoring System) indicated that in 2007, 53.9 %, 72 % and 43.1 % of nontyphoidal Salmonella isolates from chickens, cattle and swine, respectively were resistant to at least one antimicrobial agent, which had been similar to those reported in 1996 (FDA, 2010). The most common MDR phenotype ACSSuT, was detected in 1.5 %, 4.8 %, 16.2 % and 10.9 % of the isolates tested from chickens, turkeys, cattle and swine, respectively in 2007 (FDA, 2010). The extensive and indiscriminate use or misuse of antimicrobial agents, in human and veterinary treatment and also in growth promoting substances in livestock production, has greatly promoted the Page 25

21 appearance of antimicrobial resistant bacteria (Araque, 2009; Hur et al., 2011; Singh et al., 2010; Gousia et al., 2011) Transmission of antibiotic resistance in Salmonella spp. The mobile genetic elements such as plasmids, transposons and integrons, which disseminate antibiotic resistance genes by horizontal or vertical transfer, play an important role in the evolution and dissemination of multidrug resistance (Gomez et al., 1997; Boyd et al., 2002) Bacterial plasmids Plasmid mediated resistance is the transfer of antimicrobial resistant genes carried on plasmids and can be transferred between prokaryotes through horizontal gene transfer. They are important in bacterial evolution because they affect replication, metabolism, fertility as well as resistance to antibiotics, toxins (bacteriocins) and bacteriophages. The spread of multiple antimicrobial resistance has been enhanced by selective pressure from human and veterinary medicine (Carattoli, 2003) and plasmids often contain the information for resistance to antibiotics (Levy, 2002). A single R-plasmid can code for resistance of up to 10 different antibiotics simultaneously. Plasmids are able to replicate within a cell and are subject to mutations involving either the loss or gain of genes. Furthermore, they are capable of combining with other plasmids, thus conferring resistance to several antibiotics that can reside on one plasmid. Most importantly, bacteria are capable of transferring plasmids from one cell to another through a process termed conjugation, which is a mechanism of horizontal gene transfer and allows the transfer of plasmids coding for antibiotic resistance among an entire colony of bacterial cells (Levy, 2002). Many serovars of Salmonella harbor virulence plasmids Page 26

22 which are important for systemic infections. They vary in their size depending on the serovars, e.g. 95 kb for S. Typhimurium, 60 kb for S. Enteritidis, 80 kb for S. Dublin, and ranging from 50 to 110 kb for S. Choleraesuis (Chu et al., 1999). A highly conserved 8 kb operon of Salmonella virulence plasmid (spv) consisting of five genes, designated spvrabcd is present in all Salmonella serotypes and is found to contribute to colonization and resistance to complement killing by rck gene (Gulig et al., 1993). The gene spvb encodes an ADPribosylating toxin that modifies actin directly and totally disrupts the cytoskeleton of the cell (Slayers and Whitt, 2002) Bacteriophages Bacteriophages are bacterial viruses discovered independently by Twort in 1915 in England and by d Herelle at the Pasteur Institute in Paris in Like viruses, bacteriophages are incapable of independent growth in artificial media and are obligate intracellular parasites. They increase several fold by a process of multiplication in which many copies are made of each viral component and then assembled to produce many phage particles and involves the steps of adsorption, penetration, transcription, assembly and finally release by burst. Bacteriophages in the aquatic environment are very diverse and most of them have been studied elaborately and include the viruses that infect the enteric group such as E. coli and S. Typhimurium. Those most commonly found in nature have ds DNA genome though there are others that have ss RNA, ds RNA and ss DNA genomes. Two types of viral life cycles, namely virulent and temperate, exist. Virulent, also known as lytic phages, which lyse or kill the host after infection while the temperate or lysogenic as they are otherwise referred to, achieve a state where they get integrated into the genome and replicate along with the host Page 27

23 genome without killing them. They provide a mechanism to transfer antibiotic resistance genes through lysogenic cycle, the process being referred to as phage mediated transduction. When these viruses enter new hosts, they are able to integrate their DNA as well as the antibiotic resistance genes picked up from the previous host into the chromosome of new host (Levy, 2002). Zhang and Le Jeune (2008) showed that wild type phage mediated transduction also plays an important role in the dissemination of antimicrobial resistance genes. According to Porwollik and McClelland (2003), phage genomes are commonly encountered in the genome of most Salmonella. To date about 95 % of strains of S. Typhimurium examined known to contain complete inducible prophage genomes and of them 99 % were shown to be capable of generalized transduction of chromosomal host markers and plasmids (Schicklmaier et al., 1998). All these studies elucidate the importance of wild type phages in transmitting antibiotic resistance among Salmonella serovars Transposons Besides plasmid mediated mechanism, there are several other mechanisms of gene transfer. It is possible for resistance genes to reside on small pieces of DNA called transposons. In general, these pieces of DNA contain terminal regions that participate in recombination and express a protein(s) (e.g. transposase or recombinase) that facilitates incorporation into and movement from specific genomic regions. These pieces of DNA have the capability to jump from one region of the chromosome, to another and vice versa. This way, a resistant gene can be directly incorporated into host chromosomal DNA and not be dependent on plasmid transfer for spread (Levy, 2002). Pezzella et al. (2004) showed that most of the teta genes were localized within a deleted Tn1721 transposon variant. Conjugative Page 28

24 transposons are unique in having qualities of plasmids and facilitate the transfer of endogenous plasmids from one organism to another (Alekshun and Levy, 2007) Integrons DNA sequencing of several unrelated antibiotic resistant genes revealed common upstream and downstream regions in the mid of 1980s (Cameron et al., 1986; Hall and Vockler, 1987; Ouellette et al., 1987; Sundstrom et al., 1988). These genetic determinants responsible for capturing and expression of resistance genes have been described as integrons. Since late 1980s integrons have been recognized as naturally occurring gene expression elements responsible for the recruitment and assembly of antibiotic resistance genes in clusters. Several studies have documented the prevalence of integrons in multidrug resistant serotypes of Salmonella and have found them to be widespread (Tosini et al., 1998; Brown et al., 2000; Daly and Fanning, 2000; Guerra et al., 2000; Havlickova et al., 2009). The presence of class 1 integron in S. Bareilly and S. Oslo was first reported by Khan et al. (2006). These integrons on transferable plasmids are considered to be the main mechanism for rapid spread of multidrug resistant phenotypes among Gram negative bacteria (Levings et al., 2005). These integrons are associated with multidrug resistance and such multidrug resistant integrons in Salmonella are a threat to empirical treatment. Integrons encode a site specific recombinase, the integrase, which efficiently promote the acquisition of exogenous genes (Hall and Collis, 1995). There are two groups of integrons, resistance integrons and super integrons. Genes known to be present in resistance integrons encode resistance to antibiotics while gene cassettes within the super integrons encode a variety of functions. Four classes of integrons are defined based on the homology of the integrase proteins (40-60 % amino acid identity) of Page 29

25 which only two are present in Salmonella (Fluit and Schmitz, 2004), with most clinical isolates belonging to class 1 type of integron (Martínez-Freijo et al., 1999). The upstream region of class 1 integrons consists of a 5 conserved segment containing an atti site, a common promoter sequence and the opposite strand contains an integrase gene (int) (Collis and Hall, 1995; Recchia and Hall, 1995), whereas, the 3 conserved segment contains a quaternary ammonium compound (antiseptic) resistance gene (qaceδ1), a sulphonamide resistance gene (suli), and an open reading frame (orf5) of unknown function (Hall and Stokes, 1993). About different gene cassettes located on integrons and associated with resistance genes have been characterized, and these are responsible for bacterial resistance to a broad spectrum of antimicrobial agents (Recchia and Hall, 1995). The class 1 integrase catalyses the site-specific recombination between atti of integron and a 59 base element of a mobile gene cassette, containing various antibiotic resistant genes and unknown open reading frames (Recchia and Hall, 1995) (Fig.8). With this structure, the class 1 integron is able of expressing gene cassettes that generally code for antibiotic resistance (Sabate and Prats, 2002). In particular, the Tn21 transposon, which is a member of the Tn3 family of transposable elements, has been associated with a class 1 integron, named In2. Tn21 and In2 have been found widely distributed in Gram negative bacteria (Liebert et al., 2000). Several studies have documented the prevalence of integrons in multidrug resistant serotypes of Salmonella and have found them to be widespread (Daly and Fanning, 2000; Guerra et al., 2000; Khan et al., 2006; Rodriguez et al., 2006; Havlickova et al., 2009; Deekshit et al., 2012). Class 2 integrons have been described within the Tn7 family of transposons (Radstrom et al., 1994). These integrons have a deletion rendering the integrase ineffective. Only a few class 2 integrons have been described in Salmonella (Fluit and Schmitz, 2004) while one class 3 Page 30

26 integron has been reported which is not associated with transposon but contains the bla IMP gene cassette conferring resistance to carbapenems (Arakawa et al., 1995). Fluit, 2005 Fig.8. Schematic representation of class 1 integron like structures consists of an integron and additional genes followed by qaceδ1 and suli genes present in the 3' of integrons Resistance of Salmonella to various groups of antibiotics Resistance to aminoglycosides There are three mechanisms of aminoglycoside resistance in bacteria; reduced uptake or decreased cell permeability, alteration at the ribosomal binding sites and production of aminoglycoside modifying enzymes leading to the enzymatic detoxification of drugs. In Salmonella, aminoglycoside resistance is mediated by modifying enzymes which attach certain groups to the aminoglycoside and destroy its antibacterial activity. Over 50 different modifying enzymes have been identified (Davies and Wright, 1997) and the genes encoding them are usually found on plasmids and transposons. There are three classes of aminoglycoside modifying enzymes: O-adenyltransferases (catalyses ATP-dependent adenylation of hydroxyl group), N-acetyltransferases (catalyses acetyl CoAdependent acetylation of an amino group) and O-phosphotransferases (catalyses ATP-dependent phosphorylation of a hydroxyl group). There are two alternatively used nomenclatures for the Page 31

27 genes encoding the same aminoglycoside modifying enzyme: one designation aph (3 )-1b refers to the type of modification (aminoglycoside phosphortransfer at the position 3 and the subtype of the gene 1b) and the other stra refers to the streptomycin resistance and the subtype of the gene A. Among the aad (aminogylcoside O-adenyltransferase) genes, only those which act at the position 3 (aada) and 2 (aadb) are known in Salmonella (Table 1). Streptomycin resistance is mediated by aada while aadb mediates gentamycin, kanamycin and tobramycin resistance. Various serotypes of Salmonella carrying aada1, aada2 (Chen et al., 2004; Randall et al., 2004), aada7 and aada21 (Doublet et al., 2004; Levings et al., 2005) genes have been identified. Among the genes conferring resistance to streptomycin, the aph (6)-Ia gene (stra) and the aph (6)-1d gene (strb) is reported to be widely distributed in Salmonella (Sundin and Bender, 1996) Resistance to β-lactam antibiotics Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics. Resistance to β-lactam antibiotics is mainly due to inactivation by β-lactamase enzymes and also due to decreased ability to bind penicillin binding protein (PBPs) and other mechanism such as permeability barrier in both Gram positive and Gram negative bacteria. About 400 different β-lactamases have been identified from both Gram positive and Gram negative bacteria. In Salmonella resistance to β- lactam antibiotic is mainly mediated by β-lactamase enzymes (Mitsuhashi et al., 1982) which inactivate the antibiotic. In Salmonella, there exists a wide variety of β-lactamases encoded by considerable number of genes (bla). At least 10 subgroups of β-lactamases have been identified encoding TEM-, SHV-, PSE-, OXA-, PER-, CTX-M-,CMY-, ACC-, DHA- or KPC Page 32

28 type β-lactamases (Table 1). Several studies have reported the presence of TEM- (Pasquali et al., 2005; Dierikx et al., 2010), SHV- type (Hasman et al., 2005), PSE-1 (Briggs and Fratamico, 1999; Boyd et al., 2002), OXA (Tosini et al., 1998) CTX-M (Di Conza et al., 2002; Hasman et al., 2005; Dierikx et al., 2010) and ACC- type β-lactamases (Dierikxa et al., 2010) in Salmonella Resistance to quinolones/fluoroquinolones High degree of fluoroquinolone resistance in Salmonella was first described in serovar Typhimurium DT204 during early 1990s in Germany (Heisig et al., 1993). Outbreaks of fluoroquinolone-resistant Salmonella infections have been reported in the United States, Taiwan, and Japan (Chiu et al., 2002). Bacterial resistance to fluoroquinolone is usually mediated by mutations in bacterial DNA gyrase (gyra and gyrb) and topoisomerase IV (parc and pare) genes, as well as by active efflux pumps (Hawkey, 2003). Quinolone resistance in Salmonella is one of the major problems around the world. In Salmonella quinolone resistance was attributed to the single point mutation in the gene coding for the A subunit of the gyrase gene and over expression of the quinolone efflux pumps. Plasmids that harbor quinolone resistant gene that encodes fluoroquinolone inactivating enzymes have also been discovered recently (Gay et al., 2006; Robicsek et al., 2006). Mutation in the quinolone resistance determining region (QRDR) of gyra at ser-83 resulting in alteration to Phe, Tyr, or Ala or at Asp-87 resulting in alteration to Gly, Asn, or Tyr are the most frequently observed mutations in nalidixic acid resistant strains of Salmonella (Griggs et al., 1996; Heurtin et al 1999; Liebana et al., 2002; Reche et al., 2002). Mutations at both amino acid residues Ser-83 and Asp-87 have been identified in clinical isolates of S. enterica serovar Typhimurium showing Page 33

29 high level resistance to fluoroquinolones (e.g., MIC of ciprofloxacin: 32 μg/ml) (Heisig 1993). Some Salmonella isolates have an additional mutation at codon 464 of gyrb (Ser to Phe) and a fourth mutation in the QRDR of parc, which led to the amino acid change of Ser80 to Ile (Guerra et al., 2003; Baucheron et al., 2004). Over expression of the efflux pump is also one of the major problem of quinolone resistance in Salmonella. At least six major groups of active drug efflux pump transporters have been identified in prokaryotes to date viz. ATPbinding cassette (ABC), major facilitator superfamily (MFS), small multidrug resistance (SMR), multi antimicrobial resistance (MAR), resistance nodulation division (RND) and multidrug and toxic compound extrusion (MATE) (van Bambeke et al., 2000). Efflux pumps that contribute to antibiotic resistance have been described in a number of clinically important bacteria like Salmonella (AcrAB-TolC) (Buckley et al., 2006), Campylobacter jejuni (CmeABC) and Escherichia coli (AcrAB-TolC, AcrEF-TolC, EmrB, and EmrD). Although target gene mutations and efflux pumps are two known mechanisms associated with fluoroquinolone resistance in bacteria, the additive or synergistic contribution of the two mechanisms in emerging fluoroquinolone resistance is not clear in Salmonella Resistance to phenicols In Salmonella, the predominant resistance mechanism to chloramphenicol is by the enzymatic inactivation of the type A or type B chloramphenicol acetyltransferases (CAT) as well as export of chloramphenicol by the specific efflux proteins. Consequently there are two types of cat genes, cata and catb that encode two types of CatA protein (Table 1). The gene cata1 was detected on Transposon 9 in different serotypes like S. Typhi, S. Typhimurium DT104, S. Agona and S. Derby. The cata2 has been detected on a multiresistant plasmid from Page 34

30 S. Choleraesuis (Chiu et al., 2005) and S. Enteritidis (Chen et al., 2004; Randall et al., 2004). Three different types of catb (catb2, catb3 and catb8) have been found in Salmonella. All catb resistance genes are located upon gene cassettes identified as class I multiresistance integrons (Ahmed et al., 2005). The gene flor encoding proteins that export florphenicol, also designated as flo, flost or pp-flo, plays an important role as a part of SGI-1 associated multiresistant gene cluster (Briggs and Fratamico, 1999). The chloramphenicol exporter gene cmla is a cassette borne gene found in plasmid located class I integrons in S. Typhimurium. Recently, a new cmla4 variant has been identified in a plasmid borne class I integron of S. Agona (Michael et al., 2006a) Resistance to sulphonamides Sulphonamides are the first antibiotics developed for large scale clinical use which target dihydropteroate synthase (DHPS). Sulphonamide resistance in Gram negative bacilli generally arises from the acquisition of either of the two genes sul1 and sul2, encoding forms of dihydropteroate synthase that are not inhibited by the drug (Enne et al., 2001). The sul1 gene is normally found linked to other resistance genes in class 1 integrons, while sul2 is usually located on small non conjugative plasmids or large transmissible multiresistance plasmids (Enne et al., 2001). The recently described sul3 has been detected together with sul1 on a large multiresistance plasmid from S. Choleraesuis (Chiu et al., 2005). In Salmonella, all three sulphonamide resistance genes (sul1, sul2 and sul3) are reported (Table 1). PCR analysis has revealed the presence of sul1 in many Salmonella serovars (Chen et al., 2004; Randall et al., 2004; Michael et al., 2006b; Deekshit et al., 2012). sul2 has been physically linked to streptomycin resistant genes stra-strb found on plasmids and has been isolated from R- Page 35

31 plasmids of Salmonella (Chu et al., 2001). In more than 70 typical class 1 integrons isolated so far, the sequences of the sul1 gene are identical. These integrons have been isolated from various bacteria of diverse ecological niches such as human E. coli (Enne et al., 2001), S. Choleraesuis (Chu et al., 2001) and serovar Typhimurium DT104 (Ng et al., 1999) Resistance to tetracyclines The tetracyclines, first discovered in the 1940s, are the family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl trna to the ribosomal acceptor (A) site. Mechanisms encoded by tetracycline resistant genes include energy dependent efflux, ribosomal protection and enzymatic inactivation. Currently, at least 38 classes of tetracycline resistant genes have been described from a variety of bacteria (Roberts, 2005) (Table 1). Of these, 23 genes code for efflux proteins, 11 for ribosomal protection proteins, three for inactivating enzyme and one for unknown function (Roberts, 2005; Zhang et al., 2009). Among these genes classes, A to D and G are most frequently detected in the Enterobacteriaceae (Roberts, 1996; Chopra and Roberts, 2001). However, the detection rates of teta and tetb was higher than tetc and tetd from the bacteria isolated from aquatic environments, which included Salmonella and other Enterobacteriaceae (Tao et al., 2010). Of the 35 tet genes, only five that are reported in Salmonella are, teta, tetb (Frech and Schwarz, 2000), tetc (Bernardi and Bernardi, 1984), tetd (Allard et al., 1993) and tetg (Briggs and Fratamico, 1999). All these genes code for a membrane associated efflux protein consisting of 12 transmembrane segments which can export tetracycline, oxytetracycline, chlortetracycline and doxycycline (Michael et al., 2006a). The teta gene has been found on the plasmid and the chromosome, while tetb, tetc and tetd have been detected on the chromosome of different Page 36