Elsevier

LWT

Volume 73, November 2016, Pages 375-382
LWT

Behavior variability of Salmonella enterica isolates from meat-related sources

https://doi.org/10.1016/j.lwt.2016.06.036Get rights and content

Highlights

  • Great behavior variability of Salmonella isolates was observed.

  • Growth kinetic parameters of isolates showed strain-dependent.

  • The fold-change of nine virulence genes showed isolate-to-isolate variation.

  • Low expression of virulence genes and high growth rate was found in Sinstorf.

  • A high diversity in repeat length and space numbers of CRISPR was observed.

Abstract

Salmonella enterica has been concerned globally due to its serious contamination in the food industry and serious risks to human health. In this study, the diversity of growth, genotypic virulence and clustered regularly interspaced short palindromic repeats (CRISPR) locus of 17 Salmonella enterica isolates from meat-related sources was evaluated. The results showed that the tested isolates exhibited significant differences (p < 0.05) in the growth kinetic behaviors, the growth rate was independent on serotype, and the fast-growing pattern was observed in Sinstorf serotype. The fold-change of nine genes involved in six virulence units showed isolate-to-isolate variation (p < 0.05), virulence genes associated with SPI-2 and SPI-4 displayed high expression. Relative lower expression levels of tested genes were obviously found in serotypes of Sinstorf, Stanley and Aberdeen. A considerable high diversity in repeats length and space numbers of both CRISPR-1 and CRISPR-2 among tested isolates was also observed, even within serotypes, which was also confirmed by the phylogenetic relationships. No significant relationship between the expression levels of each tested gene and the numbers of CRISPR spaces was observed. The information indicated that the variability among isolates of Salmonella with respect to their behavioral aspects is extensive, and assessment of strain variability is quite indispensable.

Introduction

Salmonella is recognized as one of the globally widespread food-borne pathogens. Although the total number of Salmonella outbreaks within the EU decreased markedly by 38.1% from 2008 to 2013, Salmonella was still the most frequently detected causative agent in the food-borne outbreaks reported in the EU (22.5%, EFSA-ECDC, 2015) and in the US (15.29%, CDC, 2016). Human acute gastroenteritis characterized by emesis and diarrhea was commonly linked to Salmonella infection associated with over 2500 serotypes. Most serovars preferred variety of habitats including humans and animals, disease cases associated with serovar/host combination (e.g. Typhimurium and child) have been confirmed in hospital (Krumkamp et al., 2016). Typhimurium and Enteritidis were the global predominant serotypes of Salmonella confirmed in outbreaks and food recalls. Most serotypes (except Typhimurium and Enteritidis) responsible for major outbreaks from Europe belonged to Hadar and Infantis, whereas most serotypes from North America belonged to Newport and Heidelberg (Vieira et al., 2009).

Salmonella infection is usually caused by cells invasion, which is closely related to virulence units including pathogencity island, plasmids, lipopolysaccharides and fimbriae. Each virulence unit includes many virulence genes, such as invA and hilA in type III secretion system of pathogencity island, spvR in plasmid and fimA in fimbriae. Given that genes encoding virulence ability evolved and varied at a high rate due to environment pressure of habitats even for the same strain, studies have demonstrated that the diversity of Salmonella strains or serotypes played a critical role in the virulence of cells (Shah et al., 2011, Yim et al., 2010). McWhorter, Davos, and Chousalkar (2015) recently found that the in vitro invasive capacity of 17 Salmonella strains, determined using the gentamicin protection assay with the human intestinal epithelial cell line Caco-2, varied greatly with serotypes, and the survival percent of mice infected with Typhimurium was significant less than that of all other mice infected with Infantis, Lille, Montevideo and Oranienburg. The global transcriptome differences of six S. Enteritidis strains, previously characterized as high-pathogenicity and low-pathogenicity strains confirmed both in vitro and in vivo virulence assays, have also been revealed by Shah (2014) with RNA Sequencing. These studies suggested that it is essential to expand the knowledge of strain-to-strain or serotype-to-serotype variation in the virulence of food-associated Salmonella isolates.

Clustered regularly interspaced short palindromic repeats (CRISPR), which was first discovered in Escherichia coli in 1987, are present in about 50% of the sequenced bacteria including Salmonella. CRISPR region generally contains a newly discovered family of repeated DNA sequences characterized by direct repeats separated by variable sequences known as “spacers”. Two CRISPR locus, which can provide insights into pathogen evolutionary, genetic diversity and transmission (e.g., vertical or horizontal) rates of a system, have been found in Salmonella. (Pettengill et al., 2014). Diversity of CRISPR locus have been explored as a subtyping tool within Salmonella, identifying strain differentiation at the serotype or sub-serotype level (Liu et al., 2011, Shariat et al., 2013b, Shariat et al., 2013a), and also could be used for tracking the farm-to-fork spread of the most prevalent serotypes of Salmonella during outbreak investigations. The direct or indirect relationships between CRISPRs and physiological characteristics of pathogens, such as virulence, attachment, commensalism, stress response and antibiotic resistance, have been recently revealed (Jiang et al., 2015, Belkum et al., 2015, Louwen et al., 2014, Zegans et al., 2009). It is quite essential to obtain more details insight into the diversity of CRISPR locus of Salmonella. The present study was therefore carried out to gain insights into the growth, virulence and CRISPR characteristics of meat-related Salmonella isolates, which would be of benefit for epidemiological investigations and microbial risk assessment in food.

Section snippets

Strains and incubation medium

A standard Salmonella strain (ATCC 14028) and 16 isolates of Salmonella enterica collected from meat-related sources in 2014, were tested in this study. The details of isolates information is shown in Table 1. Stock cultures were maintained frozen at −70 °C in tryptone soy broth (TSB, Luqiao Technology Co. Ltd., Beijing, China) containing 40% glycerol. Prior to experiments, each isolate was activated twice on tryptone soy agar (TSA, Luqiao Technology Co. Ltd., Beijing, China) incubated at 37 °C

Growth kinetic parameters

Due to the good agreement fitted between experimental and predicted values, the modified Gompertz equation is commonly regarded as an appropriate tool for describing in vitro bacterial growth (Diez-Garcia et al., 2012). Therefore, growth data of the tested Salmonella isolates were each fitted to the modified Gompertz equation in this study. More than 0.99 of R2 values were observed for each tested strain (Table 1), indicating the goodness of fitting for each growth curve. The comparative growth

Acknowledgments

This study was supported by the project funded by the grants from Natural Science Foundation of Jiangsu Province in China (BK20150678) and National Natural Science Foundation of China (31501574).

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