Differential Phenotypic Diversity among Epidemic-Spanning Salmonella enterica Serovar Enteritidis Isolates from Humans or Animals

Abstract

Nontyphoidal salmonellae are major causes of food-borne disease worldwide. In Uruguay, Salmonella enterica serovar Enteritidis was the most commonly isolated serovar throughout the last decade, with a marked epidemic period between 1995 and 2004. In a previous study, we conducted comparative genomics of 29 epidemic-spanning S. Enteritidis field isolates, and here we evaluated the pathogenic potential of the same set of isolates using several phenotypic assays. The sample included 15 isolates from human gastroenteritis, 5 from invasive disease, and 9 from nonhuman sources. Contrary to the genetic homogeneity previously observed, we found great phenotypic variability among these isolates. One-third of them were defective in at least one assay, namely, 10 isolates were defective in motility, 8 in invasion of Caco-2 cells, and 10 in survival in egg albumen. Twelve isolates were tested for invasiveness in 3-day-old chickens, and five of these were significantly less invasive than the reference strain. The two oldest preepidemic isolates were reduced in fitness in all assays, providing a plausible explanation for the previous negligible incidence of S. Enteritidis in Uruguay and supporting the view that the introduction or emergence of a more virulent strain was responsible for the marked rise of this serovar. Further, we found differences in fitness among the isolates which depended on the source of isolation.

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

“…These data support our hypothesis that not all isolates of S. Enteritidis recovered from poultry are equally pathogenic. Although the molecular mechanisms underlying the differences in the virulence of S. Enteritidis remain poorly understood, both flagella and TTSS proteins encoded by virulence-associated genes located on SPI-1 and SPI-2 are known to play a major role in the pathogenesis of S. Enteritidis infection in cultured epithelial cells and in chicken and mouse challenge models (Allen-Vercoe & Cogan et al, 2004;Dibb-Fuller et al, 1999;Methner & Barrow, 1997;van Asten et al, 2000van Asten et al, , 2004Yim et al, 2010). We observed that isolates with low invasiveness also had impaired secretion of SPI-1-encoded TTSS proteins (SipA and SipD) or motility-associated proteins (FlgK, FljB and FlgL) encoded by flagellar TTSS (Fig.…”

“…Most other studies to characterize the pathogenic potential of S. Enteritidis strains have primarily focused on isolates recovered from either human clinical cases or nonpoultry-associated environmental sources (Betancor et al, 2009;Lu et al, 1999;Pan et al, 2009;Solano et al, 1998Solano et al, , 2001; Yim et al, 2010). Humphrey et al (1996) showed that human isolates of S. Enteritidis PT4 with enhanced heat and acid tolerance were more virulent in mice and more invasive in chickens compared with poultryassociated strains of S. Enteritidis.…”

“…Enteritidis is the most genetically homogeneous serotype of Salmonella (Hudson et al, 2001;Liebana et al, 2001;Morales et al, 2005;Porwollik et al, 2005;Swaminathan et al, 2001). Despite limited genomic diversity, variation in phenotypic traits, including the ability to form biofilm, grow to high cell density, produce high-molecular-mass LPS and survive within egg albumin, has been commonly observed among field isolates of S. Enteritidis (Clavijo et al, 2006;Gast & Holt, 1995Guard-Petter, 1998;Guard-Petter et al, 1999;Jain & Chen, 2007;Lock & Board, 1992;Pan et al, 2009;Solano et al, 1998;Yim et al, 2010). S. Enteritidis strains also vary in their virulence potential in poultry, irrespective of phage types (Poppe et al, 1993) and even within clonal lineages (Olsen et al, 1999;Yim et al, 2010).…”

“…Despite limited genomic diversity, variation in phenotypic traits, including the ability to form biofilm, grow to high cell density, produce high-molecular-mass LPS and survive within egg albumin, has been commonly observed among field isolates of S. Enteritidis (Clavijo et al, 2006;Gast & Holt, 1995Guard-Petter, 1998;Guard-Petter et al, 1999;Jain & Chen, 2007;Lock & Board, 1992;Pan et al, 2009;Solano et al, 1998;Yim et al, 2010). S. Enteritidis strains also vary in their virulence potential in poultry, irrespective of phage types (Poppe et al, 1993) and even within clonal lineages (Olsen et al, 1999;Yim et al, 2010). Dhillon et al (1999) tested six strains of S. Enteritidis and showed that mortalities in experimentally challenged 1-day-old chickens ranged from 0 to 23 %, whereas Barrow (1991) reported that the mortalities caused by six strains of S. Enteritidis varied from 20 to 96 %.…”

Naturally Occurring Motility-Defective Mutants of Salmonella enterica Serovar Enteritidis Isolated Preferentially from Nonhuman Rather than Human Sources

“…Motility tests were performed as described by Yim et al (43). Briefly, 2 l of overnight cultures grown in LB broth were spotted onto the surface of an LB plate containing 0.3% agar (and arabinose when indicated) and incubated for 6 h at 37°C.…”