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. 2009 May;6(4):407–415. doi: 10.1089/fpd.2008.0213

Emergence, Distribution, and Molecular and Phenotypic Characteristics of Salmonella enterica Serotype 4,5,12:i:–

Andrea I Moreno Switt 1, Yesim Soyer 1, Lorin D Warnick 2, Martin Wiedmann 1,✉
PMCID: PMC3186709  PMID: 19292687

Abstract

Salmonella spp. represent one of the most common causes of bacterial foodborne illnesses around the world. The species Salmonella enterica contains more than 2500 serotypes, and emergence of new human pathogenic Salmonella strains and serotypes represents a major public health issue. Salmonella enterica subsp. enterica serotype 4,5,12:i:– represents a monophasic variant of Salmonella Typhimurium, which has rarely been identified before the mid-1990s. The prevalence of this serotype among human salmonellosis cases has increased considerably since the mid-1990s and Salmonella 4,5,12:i:– currently (i.e., the first decade of the 2000s) represents one of the most common serotypes among human cases in many countries around the world. This paper discusses our current knowledge of the global ecology, epidemiology, transmission, and evolution of this emerging Salmonella serotype.

Introduction

Salmonella spp. represent a well-recognized foodborne bacterial pathogen, which causes a considerable number of illnesses and deaths worldwide. For example, in the United States Salmonella was estimated to represent the leading cause of foodborne illnesses due to bacterial pathogens in 2006 (CDC, 2007a). In addition, Salmonella has been estimated to cause more deaths due to foodborne illnesses than any other known pathogen in the United States (Mead et al., 1999). Symptoms of human nontyphoidal salmonellosis include enteritis as well as, less commonly, systemic manifestations, including septicemia (Mead et al., 1999). While the majority of human Salmonella infections appear to be foodborne, salmonellosis can also be acquired through contaminated drinking water, contact with infected animals, and direct human-to-human transmission. In addition to humans, Salmonella can also infect a variety of animals species, including mammals, birds, and reptiles.

The genus Salmonella currently includes two species, S. enterica and S. bongori. S. enterica is divided into subspecies I (enterica), II (salamae), IIIa (arizonae), IIIb (diarizonae), IV (houtenae), and VI (indica). Traditionally, characterization of Salmonella isolates uses serotyping, based on the Kauffmann–White scheme, for subtyping and strain differentiation (Kauffmann, 1965b; CDC, 2003; Foley et al., 2007); over 2500 Salmonella serotypes are currently known. Serotyping is based on antigenic variability of lipopolysaccharides (O antigen), flagellar proteins (H antigen), and capsular polysaccharides (Vi antigen). Most Salmonella strains are motile by means of peritrichous flagella, which can be encoded by two different flagellin genes on the bacterial chromosome (fliC and fljB); fliC and fljB expression is regulated through a mechanism called “phase variation.” The majority of the Salmonella serotypes are biphasic, meaning that they can express both genes (phase 1 and phase 2). Some Salmonella isolates and strains are monophasic though and may lack either phase 1 or phase 2 expression. For example, Salmonella serotype 4,5,12,i:– lacks expression of phase 2 flagella.

While the overall incidence of human salmonellosis appears to be fairly stable, e.g., in the United States (CDC, 2003), the incidence of infections caused by different serotypes and subtypes appears to change considerably over time (CDC, 2005). For example, the proportion of human Salmonella Typhimurium isolates in the United States that show the drug resistance phenotype ACSSuT (i.e., resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline), which is typical for Salmonella Typhimurium phage type DT104, increased from 9% in 1990 to 33% in 1996 (Hogue et al., 1997). Similarly, the frequency of multidrug-resistant (MDR) Newport among human Salmonella isolates in the United States appears to have increased from 1998 to 2001; in 1998 only 1% of Newport isolates characterized by the National Antimicrobial Resistance Monitoring System showed the MDR-AmpC phenotype, while 26% of Newport isolates from 2001 showed this phenotype (CDC, 2002). In addition to changes in the frequency of drug-resistant Salmonella subtypes, the frequency of different serotypes among human isolates also seems to change. For example, the frequency of Salmonella Newport among human isolates in the United States increased from 5% in 1997 to 10% in 2001 (CDC, 2002). On the other hand, the frequency of serotype Enteritidis among human clinical isolates in the United States decreased considerably from 21.9% in 1993 to 14.5% in 2003 (CDC, 2005). As detailed in this review, the monophasic serotype S. enterica subsp. enterica serotype 4,5,12:i:– appears to represent an emerging serotype with apparent worldwide distribution. Serotype 4,5,12:i:– currently is among the 10 most common serotypes associated with human infections in a number of countries, including the United States (based on data from 2005) (CDC, 2005) and Spain (based on data from 1998 to 2000) (Echeita et al., 2001). In addition, this serotype has been responsible for human outbreaks in California in 2004 (Norton et al., 2004), Luxemburg in 2006 (Mossong et al., 2007), and most recently, a 2007 multistate outbreak with more than 272 cases in the United States (CDC, 2007c).

Serological Characterization of Salmonella 4,5,12:i:–

While a number of Salmonella serotypes are named (e.g., serotype Typhimurium, Newport), newly isolated serotypes are now typically designated by antigenic formulae with four components, including (i) subspecies designation (e.g., I), (ii) O antigen, (iii) phase 1H antigen, and (iv) phase 2 antigen; the three antigen designations are separated by colons (Brenner et al., 2000). According to this scheme, Salmonella Typhimurium would be described as I 4,5,12:i:1,2, indicating that this serotype belongs to subspecies I and carries the “4,5,12” O antigens, the “i” phase 1H antigen, and the “1,2” phase 2H antigens. Salmonella 4,5,12:i:– thus shares all O antigens and phase 1H antigens with Salmonella Typhimurium. Salmonella Lagos (4,5,12:i:1,5) also has the same O antigens and phase 1H antigens as Salmonella 4,5,12:i:–. Molecular subtype data showed considerable similarities between Salmonella 4,5,12:i:– and Salmonella Typhimurium, but not between Salmonella 4,5,12:i:– and Salmonella Lagos (Guerra et al., 2000; Echeita et al., 2001). The observations that Salmonella 4,5,12:i:– and Salmonella Typhimurium are genetically highly similar and have identical serotypes, except for the lack of phase 2 flagella in 4,5,12:i:–, lead to the hypothesis that Salmonella 4,5,12:i:– is a monophasic variant of Salmonella Typhimurium (Echeita et al., 2001). Importantly though, O factors 1, 5, and 27 may sometimes not be expressed in a given strain, suggesting that other serotypes that share O antigens 4 and 12 and phase 1 antigen i with Salmonella 4,5,12:i:– may be alternative ancestors of this serotype. Serotypes with related antigen profiles include Agama (4,12:i:1,6), Farsta (4,12:i:e,n,x), Tsevie (1,4,12:i:e,n,z15), Gloucester (1,5,12,27:i:l,w), Tumodi (1,4,12:i:z6), and an unnamed subspecies II serotype (4,12,27:i:z35) (Holt, 1984; Grimont and Weill, 2007). Unfortunately, isolates with these serotypes have not been included in comparative subtype and evolutionary studies on serotypes Typhimurium and 4,5,12:i:– (e.g., Echeita et al., 2001; Garaizar et al., 2002; Zamperini et al., 2007); and it is thus not currently possible to exclude these other rare serotypes as ancestors of Salmonella 4,5,12:i:–.

Worldwide Distribution of Salmonella 4,5,12:i:–

While there have been few reports of serotype 4,5,12:i:– in the peer-reviewed literature before the 1990s, isolates that appear to represent this serotype have occasionally been reported since at least the middle of the 20th century. For example, Edwards and Brunner (1946), both located at the Kentucky Agricultural Station in the United States, reported three Salmonella Typhimurium isolates that contained only phase 1 antigens; these isolates would now be designated as serotype 4,5,12:i:–. Unfortunately the country of isolation was not specifically detailed for these three isolates. In 1965, Kauffmann also reported a monophasic Salmonella Typhimurium isolate in a paper entitled “Monophasic Salmonella cultures for the preparation of H-serum” (Kauffmann, 1965a). While the rare isolation of this serotype before the 1990s may reflect a recent expansion and/or emergence of this serotype, it is important to acknowledge that 4,5,12:i:– isolates have been and still may be misclassified as Salmonella Typhimurium, leading to underreporting of this serotype. Serotype 4,5,12:i:– isolates also appear to often have been reported as “group B” or “subspecies I” (CDC, 2003).

Since the 1990s, isolation of Salmonella serotype 4,5,12:i:– has been reported in a variety of countries (Table 1).In Asia, serotype 4,5,12:i:– isolates obtained in 1993 in Thailand (Boonmar et al., 1998) included 52 isolates from humans patients with clinical illness and eight isolates from frozen chicken meat. Another study of Salmonella, isolated between 1991 and 1995 from patients with septicemia in Thailand, also found that Salmonella 4,5,12:i:– represented 8.2% of the 741 isolates from human blood samples that were characterized (Komolpis et al., 1999). In Asia, serotype 4,5,12:i:– has also been reported among human isolates in Taiwan (Chiu et al., 2006). Asian countries that are listed in the World Health Organization (WHO) Global Salmonella Surveillance system as having reported Salmonella 4,5,12:i:– include Thailand (2004 data) and Japan (2007 data) (WHO, 2008).

Table 1.

Selected Peer-Reviewed Reports on Worldwide Isolation of Salmonella enterica Serovar 4,5,12:i:–

Year(s) of isolation Country Source Reference
1986–1987 Portugal Chicken Machado and Bernardo, 1990
1993–1994 Thailand Human, chicken meat Boonmar et al., 1998
1997 Spain Human, food Echeita et al., 1999
1991–2000 Brazil Human, food, animals Tavechio et al., 2004
1998–2000 United States Human, raw chicken meat Agasan et al., 2002
1998–2000 Spain Swine de la Torre et al., 2003
2000–2001 Thailand Human, frozen meat, foods Amavisit et al., 2005
2000–2003 Taiwan Human Chiu et al., 2006
2003–2004 Portugal Pig carcasses Vieira-Pinto et al., 2005
2004 United States Human, bovine Alcaine et al., 2006
Not available United States Bovine, poultry, nondomestic birds Zamperini et al., 2007
2006 Luxembourg Human, food, porcine Mossong et al., 2007
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One of the first of S. enterica subsp. enterica serovar 4,5,12:i:– isolates from Europe reported in the peer-reviewed literature was obtained from a chicken carcass in Portugal in 1986/87 (Machado and Bernardo, 1990). Subsequently, a considerable number of 4,5,12:i:– isolates have been reported from Spain, with the first reported isolation in this country in 1997 (Echeita et al., 1999). Since then, serotype 4,5,12:i:– appears to have become the most frequently encountered serotype in swine and the second most frequently encountered serotype in pork products in Spain (based on data from 2000 as reported by de la Torre et al., 2003); this observation has lead to the hypothesis that pigs may be a reservoir of this serotype (de la Torre et al., 2003). Isolation of serotype 4,5,12:i:– has also been reported for Luxemburg (Mossong et al., 2007), Portugal (Vieira-Pinto et al., 2005), and Germany (Guerra et al., 2004a) as well as Denmark, Bulgaria, and Slovakia, which are all listed in the WHO Global Salmonella Surveillance system as having reported Salmonella 4,5,12:i:– (WHO, 2008). In Luxembourg, serotype 4,5,12:i:– caused at least two Salmonella outbreaks in 2006, which appear to have been linked to consumption of contaminated pork (Mossong et al., 2007).

In the United States, serotype 4,5,12:i:– represented 0.2% of human clinical isolates in 1995; in 2004, 2.1% of human clinical isolates were classified as serotype 4,5,12:i:– (Grenne et al., 2006). Based on U.S. Centers for Disease Control and Prevention (CDC) reports, serotype 4,5,12:i:– was the 18th and 6th most common serotype recovered from human illness cases in 2002 and 2005, respectively (CDC, 2005). In the United States, serotype 4,5,12:i:– has also been isolated from different foods (e.g., raw ground chicken [Zamperini et al., 2007], chicken enchiladas [Norton et al., 2004]) and a variety of animal species, including chickens (Zamperini et al., 2007), cattle (Alcaine et al., 2005, 2006), nondomestic birds (Zamperini et al., 2007), and turtles (CDC, 2007b). Salmonellosis outbreaks in the United States have also been caused by Salmonella serotype 4,5,12:i:–, including a multistate outbreak in 2007 (linked to consumption of frozen poultry pie [CDC, 2007c]). Some salmonellosis cases in the United States caused by Salmonella serotype 4,5,12:i:– in 2006 (including two cases in Ohio and one case in Tennessee [CDC, 2007b]) were also linked to exposure to turtles infected with this serotype. Based on the WHO Global Salmonella Surveillance system (WHO, 2008), Canada has also reported isolation of Salmonella 4,5,12:i:– from human cases (2004 data) and Barbados has reported isolation of this serotype from animals and humans (2006 data).

Serotype 4,5,12:i:– has also been reported in South and Latin America. Among Salmonella isolated in the Brazilian state São Paulo between 1991 and 2000, 8.8% of human clinical isolates and 1.6 % of nonhuman isolates (representing predominantly food and animal isolates) were classified as serotype 4,5,12:i:– (Tavechio et al., 2004). Based on these data, serotype 4,5,12:i:– appears to have been one of the five most common Salmonella serotypes associated with human infections in São Paulo between 1991 and 2000 (Tavechio et al., 2004). According to the WHO Global Salmonella Surveillance WWW database (WHO, 2008), Chile and Costa Rica have also reported isolation of Salmonella 4,5,12:i:– from human cases (2007 data).

In summary, Salmonella serotype 4,5,12:i:– has been identified in a number of countries around the world since the early and mid-1990s. This serotype appears to specifically be responsible for a considerable number of human salmonellosis cases in different countries and has also been responsible for salmonellosis outbreaks in different continents. This serotype has also been isolated from a number of animal species (e.g., chickens, cattle, swine, and turtles) and food items (raw poultry, pork, and pork sausages). While at least some European studies suggest a common link of human infections with this serotype to pork and pork products, worldwide, serotype 4,5,12:i:– appears to be widely distributed and not characterized by a single reservoir, as supported by outbreaks and cases linked to poultry products and direct contact with turtles.

Genetic Characterization of Salmonella 4,5,12:i:–

Molecular subtyping and phylogenetic characterization of serotype 4,5,12:i:–

A number of studies have used different molecular subtyping methods (including pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and phage typing) to characterize Salmonella 4,5,12:i:– isolates (e.g., Echeita et al., 2001; Agasan et al., 2002; de la Torre et al., 2003; Amavisit et al., 2005). Findings from most of these studies indicate that Salmonella 4,5,12:i:– isolates are closely related to Salmonella Typhimurium, suggesting that 4,5,12:i:– is a monophasic variant of serotype Typhimurium. Phage types found among 4,5,12,i:– isolates from Spain include U302, DT 208, and DT 193, all phage types typical for Salmonella Typhimurium (de la Torre et al., 2003). Characterization of thirteen 4,5,12:i:– isolates from Spain (Echeita et al., 2001) found that these isolates, along with two Salmonella Typhimurium phage type DT104 and two U302 isolates, allowed for amplification of a 1000-bp fliB-fliA polymerase chain reaction (PCR) product (indicating the presence of an IS200 element downstream of fliB), while the same primers amplified a 250-bp fragment in Salmonella Lagos and a selection of isolates representing other serotypes (indicating the absence of the IS200 element downstream of fliB). Only the 4,5,12:i:– and Typhimurium DT104 and DT302 isolates yielded PCR products with another set of primers targeting a DT104 and U302 specific region; these primers did not yield a product with Salmonella Typhimurium LT2 or other Salmonella serotypes (Echeita et al., 2001). In a subsequent study, 16 of 23 serotype 4,5,12:i:– isolates from Spain were classified as phage type U302 (de la Torre et al., 2003); in addition at least some XbaI and BlnI PFGE types were found to be shared between 4,5,12:i:– and Typhimurium isolates, even though these two serotypes never shared the same combined XbaI/BlnI PFGE type (de la Torre et al., 2003). These data suggested that serotype 4,5,12:i:– isolates from Spain represent a variant of Salmonella Typhimurium and indicate that it may have emerged from an ancestor representing Salmonella Typhimurium U302 or a close relative to this phage type (Echieta et al., 2001; de la Torre et al., 2003). This is consistent with the observation that most Spanish 4,5,12:i:– isolates are phage type U302 (Echeita et al., 2001).

MLST-based characterization of 335 Salmonella isolates collected in New York state (USA), including 15 Salmonella Typhimurium and 18 Salmonella 4,5,12:i:– isolates, showed that all but one serotype 4,5,12:i:– isolate had the same sequence type 6 (ST6) that also represented the predominant ST among the characterized Salmonella Typhimurium isolates (Alcaine et al., 2006). ST6 was unique to serotypes Typhimurium 4,12:i:– and 4,5,12:i:–, supporting the initial findings based on characterization of Spanish isolates (de la Torre et al., 2003), that 4,5,12:i:– appears to have emerged from a Salmonella Typhimurium ancestor. More recent characterization of 32 serotype 4,5,12:i:– isolates from poultry, bovine, and nondomestic birds in Georgia (USA), along with characterization of selected Typhimurium isolates, also revealed a close genetic relationship between 4,5,12:i:– and Typhimurium isolates. Specifically, a number of 4,5,12:i:– isolates had XbaI and BlnI PFGE patterns that were identical or closely related to PFGE patterns found among Salmonella Typhimurium isolates (Zamperini et al., 2007). Characterization of different Salmonella isolates from Thailand (Amavisit et al., 2005) showed that 30 serotype 4,5,12:i:– isolates were positive in a duplex PCR assay that included a set of serotype Typhimurium specific mdh primers as well as a set of primers that target a Typhimurium phage type DT104 and U302 specific region. These data further support emergence of serotype 4,5,12:i:– from an ancestor representing Salmonella Typhimurium U302 or a close relative to this phage type.

In conclusion, characterization of Salmonella 4,5,12:i:– isolates from various countries consistently supports the hypothesis that this serotype has recently emerged from a Salmonella Typhimurium ancestor. At least for the Salmonella 4,5,12:i:– isolates from Spain and Thailand, the Typhimurium ancestor for 4,5,12:i:– appears to most likely be a strain closely related to Typhimurium phage type U302. Interestingly, most studies using discriminatory subtyping methods, such as PFGE, found that Salmonella 4,5,12:i:– isolates represent a considerable diversity of subtypes, even among isolates from a single given country. For example, at least 13 different XbaI PFGE types were found among 32 serotype 4,5,12:i:– isolates from Georgia (as determined by visual analysis of the PFGE patterns shown in Fig. 2 in Zamperini et al., 2007) and at least 11 XbaI PFGE types were found among 23 Spanish serotype 4,5,12:i:– isolates (de la Torre et al., 2003). These findings suggest considerable diversification of serotype 4,5,12:i:– after emergence (e.g., from a Salmonella Typhimurium ancestor) or possibly multiple independent emergence events.

Genetic basis of the monophasic phenotype in Salmonella 4,5,12:i:–

More than 50 genes are required for flagellar formation and function in Salmonella, these flagellar genes constitute at least 14 different operons. Most of operons are clustered in four regions on the chromosome. According to the relative position in the transcriptional hierarchy, the flagellar operons are grouped into three classes, including class 1 (represented by the flhD operon whose products are required for class 2 expression), class 2 (which contains the operons responsible for formation of basal structure and the hook-basal-body complex), and class 3 (which contains the operons responsible for filament formation, flagellar rotation, and chemotaxis) (Ikebe et al., 1999). Flagellar phase variation in Salmonella Typhimurium involves genes in the operon class 3 (Ikebe et al., 1999) and entails transcription of either fliC or fljB, which both encode flagellin proteins. Cellular expression of FliC is called phase 1 and cellular expression of FljB is called phase 2. Flagellar phase variation is caused by the reversible inversion of a DNA segment (i.e., the H segment), which contains the promoter for fljB (Yamamoto et al., 2006). The H segment is flanked by inverted repeat sequences, hixL and hixR (Fig. 1), between which site-specific recombination occurs, leading to H inversion. This recombination event is catalyzed by a DNA invertase encoded by hin, which is located within the H segment. The gene fljA, which encodes a negative regulator for fliC expression, is located downstream of fljB. When the H segment is in the “on” orientation, both fljB and fljA are transcribed, and fliC is consequently repressed by FljA (Fig. 1). When the H segment is in the off orientation, both fljB and fljA genes are not transcribed, the fliC gene is expressed (Aldridge et al., 2006; Yamamoto et al., 2006). This phase switching occurs at a rate of 10−3 to 10−5 per cell generation (Yamamoto et al., 2006).

FIG. 1.

FIG. 1.

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Flagellar phase variation in Salmonella (this figure is reproduced, with permission, from Aldridge et al. [2006]). The reversible, Hin-mediated inversion of the H segment (located between hixL and hixR) results in the inversion of a promoter driving the expression of fljB (which encodes the phase 2 flagellin) and fljA. fljA encodes an inhibitor of fliC, which in turn encodes phase 1 flagellin. The Hin recombinase in conjunction with the Fis protein catalyzes a site-specific recombination reaction between the hixL and hixR recombination sites. The upper part of this figure shows the promoter configuration during phase 2 flagella expression; fljBA is transcribed allowing for production of FljB and FljA, which inhibits fliC transcription. The bottom part of this figure shows the promoter configuration during phase 1 flagella expression; the promoter for fljBA is inverted and thus cannot facilitate fljAB transcription; fliC is transcribed because FljA is not produced and thus cannot inhibit fliC transcription.

Isolates of serotype 4,5,12:i:– are phenotypically characterized by a lack of phase 2 flagella expression. Genetic characterization, using microarrays, of four Spanish 4,5,12:i:– isolates revealed a large chromosomal deletion, which spanned 16 genes including hin, fljB, and fljA (Garaizar et al., 2002). These findings provided the initial identification of the genetic basis for the lack of phase 2 flagella in serotype 4,5,12:i:–. Characterization with PCR-based and colony blot approaches of 30 serotype 4,5,12:i:– isolates from poultry, bovine, and nondomestic birds in Georgia (USA) also found that a number of these isolates had partial or complete deletions of fljB, the structural gene for phase 2 flagella. Interestingly, the fljB deletions in these 4,5,12:i:– isolates from Georgia represented different deletion patterns, including apparent deletion of the entire fljB in some isolates as well as partial deletion of fljB in other isolates (Zamperini et al., 2007). All but one of these isolates were characterized by presence of hin though. Recently, a genome sequence of a 4,5,12:i:– animal isolate (CVM23701) has become available (Rosovitz et al., 2007); a preliminary analysis (Soyer et al., unpublished data) of this sequence (GenBank accession number ABAO010000014) suggests a deletion of a multigene fragment, including fljA and fljB with retention of an intact hin, which is 100% identical to the hin sequence in Salmonella Typhimurium.

Overall, the findings of studies summarized above suggest that different mutations and deletions can be responsible for the lack of phase 2 flagella expression in naturally occurring 4,5,12,i:– isolates. Specifically, at least some of the 4,5,12,i:– isolates from Spain appear to be characterized by deletion of a large fragment (between STM2757 and STM2774), including hin (Garaizar et al., 2002), while most of the isolates from the United States characterized thus far seem to be typified by deletions that eliminate fljB but maintain hin (Zamperini et al., 2007; Soyer et al., unpublished data). These findings may support a model that suggests multiple independent deletion events that led to emergence of 4,5,12,i:– from Salmonella Typhimurium ancestors in different locations. Alternatively, the evolution of 4,5,12,i:– may represent a single emergence event, followed by subsequent rearrangements and/or additional deletions in the region surrounding the fljAB operon, resulting in different 4,5,12,i:– lineages.

We are not aware of any comparative phenotypic experiments characterizing the virulence of wild-type Typhimurium and 4,5,12,i:– isolates. Genetically engineered Salmonella Typhimurium mutants missing fljB (having flagellar expression patterns similar to serotype 4,5,12,i:–) have shown a reduced ability to induce IL-8 secretion in tissue culture cells (Gewirtz et al., 2001). In another study, Salmonella mutants expressing FliC (but not FljB), i.e., constructs with flagellar expression patterns similar to serotype 4,5,12,i:–, were recovered in greater number from blood and spleen of infected mice and cause higher mortality, as compared to strains expressing FljB (but not FliC) (Ikeda et al., 2001). While there is thus some evidence that genetically engineered Salmonella Typhimurium strains lacking phase 2 flagella show virulence-associated phenotypes distinct from parent strains expressing phase 2 flagella, future experiments with wild-type Typhimurium and 4,5,12,i:– isolates are needed to further probe the virulence characteristics of serotype 4,5,12,i:– strains.

Drug Resistance of Salmonella 4,5,12:i:–

Drug resistance patterns

Interestingly, the drug resistance profiles of the Salmonella 4,5,12:i:– isolates recovered around the world range from pansusceptible to multidrug resistance. Resistance phenotypes in this section will be reported using abbreviations for the main antibiotics, including ampicillin (A), chloramphenicol (C), kanamycin (K), streptomycin (S), sulfamethoxazole (Su), tetracycline (T), trimethoprim (Tm), gentamicin (G), and nalidixic acid (NA).

Characterization of 122 serotype 4,5,12:i:– isolates from humans (114 isolates) and chicken meat (8 isolates) collected in 1993 and 1994 in Thailand found a number of MDR isolates, along with pansusceptible isolates (38% of human and 75% of food isolates were pansusceptible). The most common multidrug resistance patterns among these isolates from Thailand included ACKGSuTm (22 isolates) and ACKGSuTm with additional resistance to nalidixic acid (NA) (20 strains) (Boonmar et al., 1998); these two resistance types are very similar with resistance to nalidixic acid typically conferred by point mutations in gyrA and gyrB (Giraud et al., 2006). Some of the earliest reported isolates (i.e., those isolated in Thailand in 1993 and 1994) thus represented a mix of MDR and pansusceptible types. Characterization of 271 human and 17 food isolates with serotype 4,5,12:i:– that were collected in Spain in 1998 and 1999 revealed that all of these isolates showed a multidrug resistance phenotype (generally ACSuGSTTm, with a few isolates showing sensitivity to tetracycline [T]) (Echeita et al., 1999). Serotype 4,5,12:i:– isolates from two human outbreaks in Luxemburg in 2006 also showed a multidrug resistance phenotype (ASSuT) (Mossong et al., 2007).

Among 369 serotype 4,5,12:i:– isolates collected in Brazil between 1991 and 2000, 8% of human and 5% of nonhuman isolates (i.e., isolates from foods and animals) were susceptible to all the tested agents (Tavechio et al., 2004). A total 55% and 62.5% of human and nonhuman isolates, respectively, were reported as showing intermediate resistance to one or more antibiotics (typically tetracycline and streptomycin). A total of 37% and 31% of human and nonhuman isolates, respectively, showed resistance to between 1 and 13 of the antimicrobial agents tested; a considerable number of isolates (18.5% and 13.6% of human and nonhuman isolates, respectively, were resistant only to tetracycline). In total, 27 of the 4,5,12,i:– isolates from Brazil displayed multiresistance to three or more antimicrobials, two of these isolates were reported as resistant to 13 antimicrobial agents, including netilmicin, tetracycline, chloramphenicol, gentamicin, kanamycin, ampicillin, cephalothin, sulfonamides, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, streptomycin, amikacin, and nalidixic acid (Tavechio et al., 2004). Among 114 human serotype 4,5,12:i:– isolates obtained in the United States between 1996 and 2003, 82% were pansusceptible and 18% were resistant to at least one antimicrobial agent, three isolates showed an ACSSuT resistance type, and four isolates showed ceftiofur resistance (Grenne et al., 2006). Another study, which evaluated 68 human serotype 4,5,12:i:– isolates collected between 1998 and 2000 in New York city, found that 38% of these isolates were susceptible to all antimicrobial agents tested; 34% of isolates showed intermediate resistance to one or two antibiotics (streptomycin, sulfamethoxazole, tetracycline). A total of 28% of these isolates from New York city showed resistance to one or more antimicrobial agents, with only four isolates (5.9%) showing resistance to four or more antimicrobial agents (Agasan et al., 2002).

Overall, based on the data published to date, the majority of serotype 4,5,12,i:– isolates from Europe (i.e., Spain and Luxemburg) appear to show a multidrug resistance phenotype, while the majority of 4,5,12,i:– isolates from North and South America (i.e., United States, Brazil) appear to be pansusceptible or resistant to only a few antimicrobial drugs.

Mechanism of antimicrobial resistance among serotype 4,5,12,i:– isolates

Salmonella spp. isolates can carry a number of different antibiotic resistance genes, which may be located on either the chromosome or on plasmids (Michael et al., 2006; Miriagou et al., 2006; Alcaine et al., 2007). In addition, point mutations in chromosomal genes can confer resistance to selected antimicrobial agents (e.g., fluoroquinolones, nalidixic acid). A number of studies have identified the specific resistance genes and genetic mechanisms associated with antimicrobial drug resistance phenotypes in different Salmonella serotypes (Alcaine et al., 2007).

While only a few studies exist so far on antimicrobial resistance genes found among antimicrobial drug–resistant Salmonella serotype 4,5,12:i:– isolates, the available data may help to develop an initial understanding of the evolution and emergence of serotype 4,5,12:i:–, including 4,5,12:i:– MDR strains. While initial characterization of serotype 4,5,12:i:– isolates from Spain reported the presence of multiple (two to four) small cryptic plasmids as well as either a 140-kb spvC (Salmonella plasmid virulence gene)-positive or 120-kb spvC-negative plasmid (Guerra et al., 2000; Echeita et al., 2001), these studies did not test either these plasmids or the chromosome for the presence of antimicrobial resistance genes. Further characterization of MDR 4,5,12:i:– isolates from Spain with an ACGSSuTSTm resistance phenotype revealed the presence of multiple previously described resistance genes, including blaTEM-1 (encoding a broad spectrum β-lactamase that provides resistance to penicillin and amino-penicillin such us ampicillin), aac(3)-IV and aadA2 (encoding modified aminoglycoside enzymes that can inactivate gentamicin and streptomycin by modifying different residues in the active sites of these drugs), cmlA (encoding an efflux pump that mediates resistance to chloramphenicol), sul1 and sul2 (both encoding an dihydropteroate synthase that is resistant to sulfonamides), dfrA12 (encoding an dihydrofolate reductase that is resistant to trimethoprim), and tetA (encoding an efflux pump that mediates resistance to tetracycline) (Guerra et al., 2001, 2004b). These studies (Guerra et al., 2001, 2004b) further found that dfrA12 and aadA2 appear to be located on a class 1 integron in the 4,5,12:i:– isolates characterized, while blaTEM-1, cmlA, aac(3)-IV, and tetA were mapped to the large 120- or 140-kb plasmid previously described in Spanish 4,5,12:i:– isolates (Guerra et al., 2000). Guerra et al. (2004b) found evidence that the class 1 integron carrying dfrA12 and aadA2 is also located on a plasmid in the Spanish 4,5,12,i:– isolates characterized. Interestingly, Guerra et al. (2004b) did not find the same combination of resistance genes found in the two 4,5,12,i:– isolates they characterized among any of the eight MDR Salmonella Typhimurium isolates (including four DT104 isolates) they characterized.

While blaTEM-1, cmlA, aadA2, dfrA12, sul1, and sul2 have all previously been found in Salmonella Typhimurium isolates (Michael et al., 2006), blaTEM-1, cmlA, and dfrA12 have not been typically found in the MDR Salmonella DT104 (which typically carries blaPSE-1, floR, tetG, aadA2, and sul1) suggesting that the antibiotic resistance gene clusters found in the Spanish serotype 4,5,12,i:– isolates are unlikely to be related to the resistance genes in the pandemic DT104 strain (Boyd and Hartl, 1998; Guerra et al., 2004a, 2004b).

While MDR Salmonella DT193 typically carry blaTEM-1 along with sul1 and sul2, they carry dfrA1 and aadA1 and tetB (Gebreyes and Altier, 2002; Miriagou et al., 2006) and thus are also unlikely to represent the source of the antibiotic resistance gene clusters found in the Spanish serotype 4,5,12,i:– isolates. MDR Salmonella Newport, another MDR subtype that appears to have emerged in the North America in the late 1990s (Bird et al., 2002; Poppe et al., 2005), has been reported to carry the resistance genes blaCMY, flost, strA, strB, sul2, and tetA on a plasmid (Poppe et al., 2005). Except for sul2 and tetA, these genes are different from those identified in the Spanish 4,5,12,i:– isolates, and MDR Salmonella Newport thus also appears to be unlikely to be the source of the resistance gene cluster reported in the Spanish 4,5,12,i:– isolates. Interestingly, the resistance gene profile most closely related to the resistance gene found in the Spanish 4,5,12,i:– isolates was found in a human Salmonella Cholerasuis isolate for which a complete genome sequence was determined (Chiu et al., 2005). This isolate carried a number of resistance genes on a plasmid, including blaTEM-1, aadA2, cmlA, and sul1, which have been found in 4,5,12,i:– isolates, as well as other resistance genes that have not yet been reported in 4,5,12,i:– isolates. It is thus unlikely that the specific plasmid in Salmonella Cholerasuis isolate was transferred to the ancestor of the Spanish MDR 4,5,12,i:– isolates. The resistance clusters in this Cholerasuis isolate and in the Spanish MDR 4,5,12,i:– isolates may be related though and share a common ancestor.

Overall, the limited data available on the antimicrobial resistance genes found in serotype 4,5,12,i:– isolates constrain our ability to understand the evolution of MDR 4,5,12,i:– strains and the natural history of the antimicrobial resistance gene clusters found in these strains. While characterization of the antimicrobial resistance genes in additional 4,5,12,i:– isolates, including from countries other than Spain, is needed, the data available to date suggest that the resistance gene clusters found in the Spanish 4,5,12,i:– isolates originated from an ancestor other than an MDR Salmonella Typhimurium. Whether acquisition of the resistance genes in the Spanish 4,5,12,i:– isolates occurred before or after the loss of the phase 2 flagella expression also remains to be determined.

Conclusions

Salmonella 4,5,12:i:–, which has only rarely been reported among Salmonella isolated before 1993, has been found in human clinical cases, different animal species, and foods in countries located on different continents, including Europe, Asia, and South and North America. Molecular subtyping data and phylogenetic analyses consistently support Salmonella 4,5,12:i:– being closely related to Salmonella Typhimurium and most likely representing a monophasic variant that emerged from a Salmonella Typhimurium ancestor through deletions and/or mutations of the genes responsible for phase 2 flagella expression. Other rare Salmonella serotypes (e.g., Agama, Farsta, Tsevie, Tumodi, Gloucester, and unnamed subspecies II) (Grimont and Weill, 2007) do share many O antigens and the phase 1H antigens with both Salmonella 4,5,12:i:– and Typhimurium though and may thus have been ancestors for some emergence events. Comparative molecular subtype characterization of isolates representing all these serotypes will be necessary to further clarify the emergence and evolution of Salmonella 4,5,12:i:–.

While the deletions and mutations linked to the loss of phase 2 flagella appear to differ among most Spanish and North American 4,5,12:i:– isolates (most North American isolates apparently retained a copy of hin, while this gene apparently is absent from most Spanish isolates), it is not yet clear whether the natural history of 4,5,12:i:– represents a single emergence event followed by subsequent diversification and pandemic spread or whether it represents multiple independent emergence events. The observation that European 4,5,12:i:– isolates appear to predominately represent an MDR phenotype, while Asian and North and South American isolates appear only rarely to be MDR may support the hypothesis of two independent emergence events leading to an MDR “European” lineage and a non-MDR lineage that may be more commonly found in Asia and the Americas. Further studies are thus clearly needed to better understand the evolution and emergence of Salmonella 4,5,12:i:–.

Regardless of the specific events leading to the evolution and emergence of Salmonella 4,5,12:i:–, it is clear that this serotype is evolutionary successful as supported by its worldwide distribution and the fact that it has become one of the 5–10 most common Salmonella serotypes responsible for human infections in different countries. Thus, a critical question is whether this emerging serotype has specific characteristics that facilitate its rapid spread and ecological success. While the MDR phenotype found among some strains (e.g., those apparently predominant in Europe) may provide one selective advantage for this emergent subtype, serotype 4,5,12:i:– isolates also are commonly reported among human cases in the United States and Brazil, despite the observation that most 4,5,12:i:– isolates in these countries are not MDR. It is thus tempting to speculate that characteristics other than an antimicrobial drug resistance phenotype at least partially account for the ecological success of serotype 4,5,12,i:–. For example, while phase variation between two flagellin types generally is believed to be an important virulence mechanism allowing the pathogen to evade the immunity of the host, by producing a subpopulation with different flagellin antigens (Ikeda et al., 2001), monophasic variants lacking the second flagellar phase may have a selective advantage; for example, by completely silencing expression of flagellar antigens that may be recognized by the immune system. Further studies are also needed to test this hypothesis and to identify other genetic and phenotypic characteristics that may provide a selective advantage for serotype 4,5,12,i:– isolates.

Importantly, initial genomic microarray studies (Garaizar et al., 2002) have already identified gene deletions other than those in the region responsible for phase 2 flagella expression in selected 4,5,12,i:– isolates, which may contribute to phenotypic characteristics impacting transmission and virulence. Further analyses of the recently completed genome sequence for a 4,5,12,i:– isolate from the United States (Rosovitz et al., 2007) will provide additional opportunities to probe the evolution of Salmonella 4,5,12:i:–.

Finally, it will be critical for public health systems worldwide to continue and expand systems that monitor emergence and frequency of different Salmonella serotypes, including 4,5,12,i:–. These continued efforts will be critical not only to identify emerging new Salmonella strains, but also to allow for an improved understanding of various factors that may be responsible for or contribute to emergence of new strains of Salmonella, which continues to be responsible for most deaths due to known foodborne pathogens in many countries around the world.

Acknowledgments

Andrea Moreno was supported by National Research Initiative Competitive Grant 2006-55212-17250 from the U.S. Department of Agriculture (USDA) Cooperative State Research, Education, and Extension Service Epidemiological Approaches to Food Safety program awarded to Dr. Randall Singer. Research on Salmonella in the authors' laboratories is supported by a number of sources including two USDA Special Research Grants (34459-15625-05 and 34459-16952-06 to Martin Wiedmann) and federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. N01-AI-30054 (to Lorin Warnick). The authors thank Kevin Cummings and Laura Spoor for help with collecting information used for this review.

Disclosure Statement

No competing financial interests exist.

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