Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil

Detecção de genes associados à virulência em cepas de Salmonella Enteritidis isoladas de frangos na região sul do Brasil

LIVESTOCK DISEASES

Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil

Detecção de genes associados à virulência em cepas de Salmonella Enteritidis isoladas de frangos na região sul do Brasil

Karen A. Borges^I,*; Thales Q. Furian^I; Anderlise Borsoi^II; Hamilton L.S. Moraes^I; Carlos T.P. Salle^I; Vladimir P. Nascimento^I

^ICentro de Diagnóstico e Pesquisa em Patologia Aviária (CDPA), Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 8824, Porto Alegre, RS 91540-000, Brazil

^IITuiuti Universidade do Paraná, Rua Sydnei Antônio Rangel 238, Curitiba, PR 82010-330, Brazil

ABSTRACT

Salmonella spp. are considered the main agents of foodborne disease and Salmonella Enteritidis is one of the most frequently isolated serovars worldwide. The virulence of Salmonella spp. and their interaction with the host are complex processes involving virulence factors to overcome host defenses. The purpose of this study was to detect virulence genes in S. Enteritidis isolates from poultry in the South of Brazil. PCR-based assays were developed in order to detect nine genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH and spvC) associated with the virulence in eighty-four isolates of S. Enteritidis isolated from poultry. The invA, hilA, sivH, sefA and avrA genes were present in 100% of the isolates; lpfA and sopE were present in 99%; agfA was present in 96%; and the spvC gene was present in 92%. It was possible to characterize the isolates with four different genetic profiles (P1, P2, P3 and P4), as it follows: P1, positive for all genes; P2, negative only for spvC; P3, negative for agfA; and P4, negative for lpfA, spvC and sopE. The most prevalent profile was P1, which was present in 88% of the isolates. Although all isolates belong to the same serovar, it was possible to observe variations in the presence of these virulence-associated genes between different isolates. The characterization of the mechanisms of virulence circulating in the population of Salmonella Enteritidis is important for a better understanding of its biology and pathogenicity. The frequency of these genes and the establishment of genetic profiles can be used to determine patterns of virulence. These patterns, associated with in vivo studies, may help develop tools to predict the ability of virulence of different strains.

Index terms:Salmonella Enteritidis, PCR, virulence profile, poultry.

RESUMO

Salmonella spp. estão entre os principais agentes causadores de doenças transmitidas por alimentos, e o sorovar Salmonella Enteritidis é o mais frequentemente isolado no mundo. A virulência de Salmonella spp. e a sua interação com o hospedeiro são processos complexos que envolvem fatores de virulência para sobreviver às defesas do hospedeiro. O objetivo deste estudo foi detectar genes de virulência em cepas de S. Enteritidis isoladas a partir de fontes avícolas no sul do Brasil. Ensaios de PCR foram desenvolvidos para a detecção de nove genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH e spvC) associados à virulência em oitenta e quatro amostras de S. Enteritidis. Os genes invA, hilA, sivH, sefA e avrA estavam presentes em 100% dos isolados; lpfA e sopE estavam presentes em 99%; agfA em 96%; e o gene spvC estava presente em 92%. Foi possível caracterizar os isolados em quatro perfis genéticos distintos (P1, P2, P3 e P4), sendo P1 positivo para todos os genes; P2 negativo apenas para spvC; P3 negativo para agfA e P4 negativo para lpfA, spvC e sopE. O perfil de maior frequência foi P1, presente em 88% dos isolados. Apesar de todos os isolados pertencerem ao mesmo sorovar, foi possível observar variações na presença de genes associados à virulência entre os mesmos. A caracterização dos mecanismos de virulência circulantes na população de Salmonella Enteritidis é importante para um maior entendimento da sua biologia e patogenicidade. A frequência destes genes e o estabelecimento de perfis genéticos podem ser utilizados para determinar os padrões de virulência dos isolados. Estes padrões, associados a estudos in vivo, podem auxiliar na elaboração de ferramentas que permitam predizer a capacidade de virulência das diferentes cepas.

Termos de indexação:Salmonella Enteritidis, PCR, perfil de virulência, frangos.

INTRODUCTION

Salmonella spp. are considered the major cause of foodborne disease in humans, and Salmonella Enteritidis is the most frequently isolated serovar in Europe, South America and Asia (Vieira et al. 2009). Phylogenetic analyses show the influence of different factors in the existence and persistence of Salmonella spp in animals, such as cross-contamination among animals, environment and feed (Mello et al. 2011). The virulence of Salmonella spp. is associated with a combination of chromosomal and plasmid factors (Oliveira et al. 2003), and many studies have identified genes that encode these factors. Some virulence factors are associated with the cellular structure of the bacteria, such as fimbriae (Edwards & Puente 1998). The long polar fimbria (lpf operon) contributes to the affinity of the bacteria for Peyer's patches and adhesion to intestinal M cells (Bäumler & Heffron 1995, Bäumler et al. 1996). One of the main functions of aggregative fimbria (agf operon) is to promote the initial interaction of the bacteria with the intestine of the host and stimulate bacterial self-aggregation, resulting in higher rates of survival (Collinson et al. 1992, 1993). The Salmonella-encoded fimbria (sef operon) promotes a better interaction between the bacteria and the macrophages (Collinson et al. 1996).

Salmonella spp. pathogenicity islands (SPI) are of critical importance for Salmonella spp. virulence, once they encode a molecular apparatus called the type III secretion system (TTSS), which is able to inject bacterial effector proteins through bacterial and host membranes to interact with host cells (Marcus et al. 2000). The hilA gene encodes the central regulator HilA, which is necessary for the expression of the TTSS components. HilA is also required to invade epithelial cells and induce apoptosis of macrophages (Bajaj et al. 1996). The protein InvA is essential for epithelial invasion (Galán & Curtis III 1989) and AvrA is an effector protein of the TTSS complex that contributes to the virulence of Salmonella spp. by limiting the host's inflammatory responses through the inducement of cell apoptosis, especially of macrophages, and by the innibition of IL-8 and TNF-α (Collier-Hyames et al. 2002, Ben-Barak et al. 2006). Salmonella spp.'s outer proteins (Sops) contribute to the invasion by these bacteria through the generation of membrane deformations (Hardt et al. 1998) and the rearrangement of the cytoskeleton of the host cells (Galán & Zhou 2000). The sivH gene encodes an outer membrane protein associated with intestinal colonization (Kingsley et al. 2003). Other important Salmonella spp. virulence factors are found on virulence plasmids. All of the virulence plasmids share a highly conserved region designated spvRABCD (Salmonella plasmid virulence). The spv region promotes rapid growth and survival of Salmonella spp. within the host cells and it is important for systemic infection (Libby et al. 1997). The purpose of this study was to evaluate the virulence potential of S. Enteritidis isolates from poultry by detecting the presence of nine virulence-associated genes by polymerase chain reactions (PCRs), as well as to determine the distribution patterns of these genes.

MATERIALS AND METHODS

Bacterial isolates. This stduy was developed at the Diagnostic Center and Research in Avian Pathology (CDPA) of Federal University of Rio Grande do Sul (UFRGS). Eighty-four isolates of Salmonella Enteritidis were selected from CDPA collection. These bacteria were isolated between 1996 and 2010 from different avian sources in Rio Grande do Sul state in the south of Brazil. The sources included broiler systems and slaughterhouses; in addition we also used one sample of hatchery. Additional data of isolates (year and source of isolation) are shown in Table 1. A complete antigenic characterization and serovar identification were performed by the Enteric Pathogens Laboratory in the Oswaldo Cruz Institute Foundation (Fiocruz, Rio de Janeiro, Brazil). The bacterial isolates were kept frozen at -70°C in brain heart infusion broth and glycerol.

DNA extraction. The bacteria were retrieved from frozen culture stocks and cultured overnight at 37°C in brain heart infusion broth (Oxoid; Cambridge, United Kingdom). An aliquot of 1 mL of each bacterial culture was separated for DNA extraction by heat treatment as described by Borsoi et al. (2009).

Polymerase chain reaction (PCR). The PCRs were conducted in individual reactions using primers for the following genes: invA, hilA, avrA, agfA, lpfA, sefA, sopE, spvC and sivH. The sets of primer pairs, sizes of the PCR products and references used in the PCR assay are described in Table 2. All PCR mixtures (25μL) were performed with 2.5μL of 10X PCR buffer (Centro de Biotecnologia UFRGS; Porto Alegre, Rio Grande do Sul, Brazil), 1 U of Taq DNA polymerase (Centro de Biotecnologia UFRGS; Porto Alegre, Rio Grande do Sul, Brazil) and 2 μL of template DNA. The reagent concentrations, amplification conditions and number of cycles are described in Table 3. The cycling program was perfomed in the Esco Swift MaxPro thermal cycler (Esco, Singapore). The amplified products were separated by electrophoresis in a 1.2% agarose gel and stained with ethidium bromide. Fragments were transilluminated with UV light. Escherichia coli ATCC 25922 and Salmonella Enteritidis ATCC 13076 were used as negative and positive controls, respectively, for all PCR reactions, except for that of the agfA gene, for which Salmonella Typhimurium ATCC 14028 was used as a positive control. In all PCRs, a mixture of all constituents of the PCR mixed without the addition of extracted DNA was used as PCR control.

RESULTS

The Salmonella Enteritidis strains had different frequencies of the target genes, regardless the year and the source of isolation (Table 1). The invA, hilA, sivH, sefA and avrA genes were present in 100% (84/84) of the isolates. lpfA and sopE were present in 99% (83/84). Gene agfA was present in 96% (3/84) and the spvC gene was present in 92% (7/84). All isolates showed at least five virulence-associated genes. The results of the PCRs are summarized in Table 4. Based on the combination of present and absent genes, the S. Enteritidis were divided in four different gene profiles. In order to facilitate the analysis, these profiles were named P1, P2, P3 and P4. Regarding the profiles, among the 84 isolates analyzed, 88% (74/84) were categorized as P1 (positive for all genes), 7% (6/84) as P2 (spvC absent), 4% (3/84) as P3 (agfA absent) and 1% (1/84) as P4 (lpfA, sopE and spvC absent).

DISCUSSION

All South Brazilian Salmonella Enteritidis isolates were positive for invA and hilA; similar observations have been reported by other studies around the world (Amini et al. 2010, Campioni et al. 2012, Craciunas et al. 2012). It was expected that these genes would be detected in all of the isolates due to their importance for cell invasion. PCR is a useful tool for the rapid detection of Salmonella spp., and invA and hilA genes can be considered target genes for the detection of this genus.

Although results for the sopE and avrA genes were similar to the 100% frequency found in previous studies on Salmonella Enteritidis (Hopkins & Threlfall 2004), other works have found lower frequencies (Rahman et al. 2004, Streckel et al. 2004, Zou et al. 2011, Liu et al. 2012). This frequency variation could be caused by the recombinations that frequently occur in the location of these genes (Hopkins & Threlfall 2004). These findings are important, since changes in the repertoire of proteins, such as SopE and AvrA, can lead to changes in the ability of this serovar to adapt to new hosts and, consequently, the emergence of novel virulent strains (Prager et al. 2000). In our study, a high percentage (99%) of isolates had the avrA and sopE genes. However, only 17.1% and 9.7% of Salmonella Hadar isolates had the avrA and sopE genes, respectively (Cesco 2010). When comparing this data, it is clear that there is a difference in the pattern of proteins among different serovars. Some authors consider this high frequency of avrA gene is present only in serovars that are considered to be the most important etiological agents of salmonellosis (Ben-Barak et al. 2006). All of these isolates of S. Enteritidis were sivH gene positive. Although there are few studies on the frequency of this gene in populations of Salmonella spp., our results are similar to previous works (Kingsley et al. 2003). Many of these effectors proteins were shown to play an important role in Salmonella virulence. However, their absence in some isolates, such as sopE, suggests that they are not essential for invasive manifestation in the human host (Suez et al. 2013).

It was verified that all isolates presented had at least two of the fimbrial genes analyzed in this study, highlighting the importance of fimbriae in the infection process. It is possible that there are additive effects of the adhesins Lpf and Agf in the colonization of the intestine and systemic virulence (Wagner & Hensel 2011). The high frequency of lpfA and agfA were similar to other data obtained by previous works that studied different serovars (Doran et al. 1993, Borsoi et al. 2009, Cesco 2010). Besides being important in the adhesion during the infection process, the agfA gene is also associated with biofilm formation (Barnhart & Chapman 2006, Yoo et al. 2013). Our results show this gene is present in isolates from carcasses, which can pose greater risk of contamination on the slaughterhouses. The negative isolates may have lost the gene during their evolution. Studies concerning the frequency of these genes are important in tracking the adaptation of different serovars of Salmonella spp. to an increasing number of hosts because the acquisition and loss of fimbrial genes are involved in this process (Bäumler et al. 1997). The high frequency of sefA is consistent with previous findings (Amini et al. 2010, Craciunas et al. 2012), and it can be considered a target gene to identify the serovar S. Enteritidis by PCR (Amini et al. 2010).

Our results for the virulence plasmid gene spvC were similar to those found by other authors (Oliveira et al. 2003, Castilla et al. 2006, Amini et al. 2010). There are also other studies that have found lower frequencies for this gene in strains of avian origin (Okamoto et al. 2009, Derakhshandeh et al. 2013, Moussa et al. 2013). It is possible that the presence of this gene is related to the host from which the sample was isolated (Amini et al. 2010). Amini et al. (2010) compared the frequencies of spvC in strains isolated from humans (100%) and cattle (90%), which are not similar to those found in S. Enteritidis strains isolated from poultry. Comparing different serovars, it was observed that 92% of S. Enteritidis strains had the spvC gene, whereas only 28% of S. Typhimurium strains (Moussa et al. 2013) and 0% of S. Hadar strains (Cesco 2010) were positive for the gene. The different frequencies found for this gene showed that the virulence of Salmonella Enteritids alternates among the plasmid and chromosomal factors, according to the genetic profile of each isolate.

Despite the antigenic similarities among the S. Enteritidis isolates used in this study, the gene pattern was not the same for all bacteria. Although all the serovars of Salmonella spp. can be considered as potentially pathogenic, there are some differences in their virulence (Karasova et al. 2009). The highest frequency of P1 profile demonstrate that these genes are widely distributed in the population of Salmonella spp. The presence of more than one genetic profile may suggest acquisitions or deletions of genes in different clones, which could promote different levels of strain adaptation to the host (Bäumler et al. 1997, Prager et al. 2000, Moussa et al. 2013).

It is known that Salmonella spp. isolates lost and acquire new virulence factors over time in order to adapt to new hosts or to the environment (Bäumler et al. 1997, Suez et al. 2013). Currently, the foremost challenges are determining how Salmonella acquires virulence factors and what the most important genetic traits conferring virulence to Salmonella spp. The knowledge of these characteristics allows a better approach in the study of Salmonella virulence and hence the development of strategies to reduce this virulence. Studies involving the exact involvement of each gene in the pathogenesis of this bacterium would be possible to establish criteria for predicting the virulence of this microorganism.

CONCLUSION

The understanding of the virulence of Salmonella spp. requires several steps. Nevertheless, the results of this study support, through the provision of protocols and gene profiles, the premise that there is a genetic differentiation in isolates from the same serovar, which provides a basis for criteria to determine possible variations for in vivo virulence of different strains, as well as for further studies in phylogenetic analysis.

Received on June 27, 2013.

Accepted for publication on October 26, 2013.

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Publication Dates

• Publication in this collection
  18 Feb 2014
• Date of issue
  Dec 2013

History

• Received
  27 June 2013
• Accepted
  26 Oct 2013