Biofilm formation, antimicrobial resistance genes, and genetic diversity of Salmonella enterica subspecies enterica serotype Enteritidis isolated from food and animal sources in Iran.

OBJECTIVES: Salmonella enterica serovar Entritidis is an important pathogen in foodborne diseases and causes gastroenteritis. Several studies have investigated the genetic diversity of the strains of this bacterium. However, our knowledge of the discriminatory power of the molecular methods is limited. METHODS: In total, 34 strains of S. enteritidis were isolated from food related to animals. Antibiotic resistance of the strains, antibiotic resistance genes, and biofilm formation capacity of the strains were evaluated. For the genetic analysis of the strains, PFGE was performed using AvrII restriction enzyme. RESULTS: Among the tested antibiotics, cefuroxime, nalidixic acid, and ciprofloxacin showed the highest resistance rates (79.4%, 47%, and 44.2%, respectively). Only three antibiotic-resistance genes were identified in these strains (blaTEM: 67.6%, tetA: 9%, and sul2: 3%). In total, 91% of the strains were biofilm producers. Clustering of strains using AvrII for 26 samples with the same XbaI PFGE profile showed that these strains were in one clone and had high homogeneity. CONCLUSIONS: In conclusion, it is better to use a combination of several typing methods for typing strains that are genetically very close so that the results are reliable.

Assessing potential determinants of the stagnating trend in Salmonella Enteritidis human infections in Europe and options for intervention: A multi-criteria decision analysis.

Background: After years of significant decline, the incidence of Salmonella enterica serotype Enteritidis (SE) human infections in Europe has started stagnating in recent years. The reasons for this stagnation remain largely unclear and are possibly multifactorial and interconnected in nature. We assessed and ranked several potential determinants of the stagnating SE trend in Europe, as well as different options for intervention at the level of poultry health and production, public health (infra)structure, and pathogen biology. Methods: A Multi-Criteria Decision-Analysis (MCDA) approach based on the Analytical Hierarchy Process was used. Through two separate surveys, a European panel of Salmonella experts first provided weights for several pre-defined criteria and subsequently scored different potential determinants and options for intervention (i.e. alternatives) against the criteria, during 2020-21. The weighting and scoring were based on Saaty's pairwise comparisons. The final ranking of the alternatives was derived from the summation of the products of each criterion weight with the score of the corresponding alternative. Sensitivity analyses were performed to assess the impact of different methodological choices, including European regions, and domains of expertise on the ranking of the determinants and options for intervention. Results: The first and second-ranked determinants of the stagnated trend in human SE infections were related to poultry health and production, namely "inadequacies of sampling programmes" and "premature relaxation of control measures". This ranking agreed with the ranking of the options for intervention, which were also those at the poultry health and production level, specifically "stricter biosecurity", "improving sampling", and "better/increased vaccination". Differences in rankings were observed among European regions and domains of expertise. Conclusions: The rankings of potential determinants and options for intervention for the stagnating SE trend in Europe pointed to the level of poultry health and production. Salmonella-control activities in poultry in Europe are harmonized across countries since many years, but the results of this study suggest that further improvements may be necessary for some countries. A multidisciplinary collaboration among veterinarians, public health professionals, and microbiologists is needed to further understand the origins of the stagnating SE trend and to identify effective interventions in order to reverse the trend, contextually in a given country, following a One Health approach.

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens.

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Integrated Whole-Genome Sequencing Infrastructure for Outbreak Detection and Source Tracing of Salmonella enterica Serotype Enteritidis.

As an important foodborne pathogen, Salmonella enterica serotype Enteritidis is recognized as one of the most common causes of human salmonellosis globally. Outbreak detection for this highly homogenous serotype, however, has remained challenging. Rapid advances in sequencing technologies have presented whole-genome sequencing (WGS) as a significant advancement for source tracing and molecular typing of foodborne pathogens. A retrospective analysis was conducted using Salmonella Enteritidis isolates (n = 65) from 11 epidemiologically confirmed outbreaks and a collection of contemporaneous sporadic isolates (n = 258) during 2007-2017 to evaluate the performance of WGS in delineating outbreak-associated isolates. Whole-genome single-nucleotide polymorphism (SNP)-based phylogenetic analysis revealed well-supported clades in concordance with epidemiological evidence and pairwise distances of ≤3 SNPs for all outbreaks. WGS-based framework of outbreak detection was thus proposed and applied prospectively to investigate isolates (n = 66) from nine outbreaks during 2018-2019. We further demonstrated the superior discriminatory power and accuracy of WGS to resolve and delineate outbreaks for pragmatic food source tracing. The proposed integrated WGS framework is the first in China for Salmonella Enteritidis and has the potential to serve as a paradigm for outbreak detection and source tracing of Salmonella throughout the stages of food production, as well as expanded to other foodborne pathogens.

Moisture Content of Bacterial Cells Determines Thermal Resistance of Salmonella enterica Serotype Enteritidis PT 30.

Salmonella spp. are resilient bacterial pathogens in low-moisture foods. There has been a general lack of understanding of critical factors contributing to the enhanced thermal tolerance of Salmonella spp. in dry environments. In this study, we hypothesized that the moisture content (XW ) of bacterial cells is a critical intrinsic factor influencing the resistance of Salmonella spp. to thermal inactivation. We selected Salmonella enterica serotype Enteritidis PT 30 to test this hypothesis. We first produced viable freeze-dried S. Enteritidis PT 30, conditioned the bacterial cells to different XW s (7.7, 9.2, 12.4, and 15.7 g water/100 g dry solids), and determined the thermal inactivation kinetics of those cells at 80°C. The results show that the D-value (the time required to achieve a 1-log reduction) decreased exponentially with increasing XW We further measured the water activities (aw) of the freeze-dried S. Enteritidis PT 30 as influenced by temperatures between 20 and 80°C. By using those data, we estimated the XW of S. Enteritidis PT 30 from the published papers that related the D-values of the same bacterial strain at 80°C with the aw of five different food and silicon dioxide matrices. We discovered that the logarithmic D-values of S. Enteritidis PT 30 in all those matrices also decreased linearly with increasing XW of the bacterial cells. The findings suggest that the amount of moisture in S. Enteritidis PT 30 is a determining factor of its ability to resist thermal inactivation. Our results may help future research into fundamental mechanisms for thermal inactivation of bacterial pathogens in dry environments.IMPORTANCE This study established a logarithmic relationship between the thermal death time (D-value) of S. Enteritidis PT 30 and the moisture content (XW ) of the bacterial cells by conducting thermal inactivation tests on freeze-dried S Enteritidis PT 30. We further verified this relationship using literature data for S. Enteritidis PT 30 in five low-moisture matrices. The findings suggest that the XW of S. Enteritidis PT 30, which is rapidly adjusted by microenvironmental aw, or relative humidity, during heat treatments, is the key intrinsic factor determining the thermal resistance of the bacterium. The quantitative relationships reported in this study may help guide future designs of industrial thermal processes for the control of S. Enteritidis PT 30 or other Salmonella strains in low-moisture foods. Our findings highlight a need for further fundamental investigation into the role of water in protein denaturation and the accumulation of compatible solutes during thermal inactivation of bacterial pathogens in dry environments.

Phage therapy as an approach to control salmonella enterica serotype enteritidis infection in mice

Abstract INTRODUCTION: Salmonella enterica serotype Enteritidis (S. Enteritidis) is a cause of food-borne human illness. Given the prevalence of antibiotic resistance of Salmonella Enteritidis and the lack of antibiotic efficacy in future years, its replacement with other agents is necessary. One of the most useful agents is bacteriophages. METHODS S. Enteritidis was identified using a multiplex polymerase chain reaction assay. The effective bacteriophages were isolated from hospital wastewater samples. The effects of the bacteriophages were evaluated both in vitro and in vivo. RESULTS The phage SE20 belonged to the Podoviridae family, and the genome size was 40 kb. The evaluation of phage SE20 at variable pH ranges showed its susceptibility to pH < 3 and pH > 12. The animal model showed that mice infected with S. Enteritidis developed hepatomegaly and splenomegaly, but did not experience gastrointestinal complications after receiving the bacteriophages. CONCLUSIONS The results of this study suggest that phage SE20 is a promising candidate for controlling salmonellosis caused by Salmonella Enteritidis.

Immune Response Against Salmonella Enteritidis Is Unsettled by HIV Infection.

The human immunodeficiency virus (HIV) is responsible for more than 2 million new infections per year and opportunistic infections such as Salmonella spp. Gastroenteritis is an important cause of mortality and morbidity in developing countries. Monocytes and macrophages play a critical role in the innate immune response against bacterial infections. However during HIV infection the virus can infect these cells and although they are more resistant to the cytopathic effects, they represent an important viral reservoir in these patients. Our aim was to evaluate the monocyte functions from HIV-1 infected patients after in vitro exposition to Salmonella Enteritidis. Our results suggest impairment of monocytes phagocytic and microbicidal activity in HIV-1 non-treated patients, which was more evident in women, if compared with men. Moreover, monocytes from HIV-1 infected and non-treated patients after stimulation with the bacteria, produced more pro-inflammatory cytokines than monocytes from HIV-treated patients, suggesting that HIV-1 infected patients have their functions unbalanced, once in the presence of an opportunistic infection in vitro.

Antimicrobial resistance, virulence genes and genetic relatedness of Salmonella enterica serotype Enteritidis isolates recovered from human gastroenteritis in Tehran, Iran.

OBJECTIVES: Salmonella enterica serotype Enteritidis is a major serotype associated with human salmonellosis. The main objective of this study was to determine the antibiotic susceptibility patterns and the presence of virulence-associated genes among S. Enteritidis strains isolated from patients with gastroenteritis in Tehran, Iran. METHODS: Over a period of 14 months (May 2015 to July 2016), 44 S. Enteritidis isolates recovered from clinical sources were characterised for antimicrobial susceptibility and virulence genes. Possible genetic relatedness among the strains was also assessed using pulsed-field gel electrophoresis (PFGE). RESULTS: Salmonella Enteritidis isolates showed high rates of resistance to ciprofloxacin (90.9%) and nalidixic acid (77.3%). Of the 44 S. Enteritidis isolates, 30 (68.2%) were resistant to three or more antibiotics. Twenty-two different antimicrobial resistance patterns were detected among the isolates. The most frequent resistance type was antibiotype 14 (resistance to ciprofloxacin, cefuroxime and nalidixic acid), occurring in 8 (18.2%) of the isolates. Notably, all of the isolates carried invA, sefA, sipA and sopE2 virulence genes. Furthermore, 17 virulence profiles were observed among the strains. The most common virulence profile was VP1 (n=17; 38.6%), harbouring all of the virulence genes. Two distinct PFGE patterns were observed among 44S. Enteritidis isolates. There was no association between virulence profiles or antibiotypes and PFGE clusters. CONCLUSIONS: Overall, this study provides valuable information on the virulence gene content, antibiotic resistance and genetic diversity of S. Enteritidis isolated from human sources in Iran.

Genetic diversity and virulence genes of Salmonella enterica subspecies enterica serotype Enteritidis isolated from meats and eggs.

Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) is one of the leading causes of food-borne gastroenteritis associated with the consumption of contaminated food products of animal origin. Little is known about the genetic diversity and virulence content of S. Enteritidis isolated from poultry meats and eggs in Iran. A total of 34 S. Enteritidis strains were collected from different food sources of animal origin in Tehran from May 2015 to July 2016. All of the S. Enteritidis strains were serotyped, antimicrobial susceptibility tested, and characterized for virulence genes. Pulsed-field gel electrophoresis (PFGE) was also applied for comparison of genetic relatedness. All of the strains harbored invA, hilA, ssrA, sefA, spvC, and sipA genes. A high prevalence of resistance against certain antibiotics such as cefuroxime (79.4%), nalidixic acid (47%), and ciprofloxacin (44.2%) was also observed. Regarding PFGE, S. Enteritidis strains from different sources showed considerable overlap, suggesting the lack of diversity among these isolates. Moreover, no correlation between virulence profiles or antibiotypes and PFGE clusters was observed. In conclusion, our study provided valuable information on virulence gene content, antibiotic resistance, and genetic diversity of S. Enteritidis isolated from food sources.

Genetically Similar Isolates of Salmonella enterica Serotype Enteritidis Persistent in China for a Long-Term Period.

Salmonella enterica serotype Enteritidis (S. Enteritidis) is an important causative agent of nontyphoidal salmonellosis in human populations. In this study, we collected 72 S. Enteritidis strains from 2004 to 2014 in Ningbo, mid-east China. Of the 72 strains, we identified a dominant clone of 58 strains recovered from patient's feces (n = 48), blood (n = 1), pleural effusion (n = 1), chickens (n = 3), and dessert cakes (n = 5) by pulsed-field gel electrophoresis (PFGE) and variable-number of tandem repeat analysis (MLVA). The profile arrangements of MLVA were SE1-SE2-SE3-SE5-SE6-SE8-SE9: 4-4-3-11-10-1-3. These dominant strains were susceptible to ampicillin, chloramphenicol, tetracycline, ciprofloxacin, gentamicin, cefotaxime and trimethoprim-sulfamethoxazole, and resistant to nalidixic acid. Additionally, all isolates harboured virulence genes invA, sipA, sopE, and spvB when tested by PCR. Our results reveal that genetically similar S. Enteritidis strains which accounted for several outbreaks as well as blood infection and pleural cavity infection are prevalent in China for a long-term period. This situation calls for further attention in the prevention and control of foodborne disease caused by Salmonella species.

Salmonella enterica serotype enteritidis in French Polynesia, South Pacific, 2008-2013.

Outbreaks of Salmonella enterica serotype Enteritidis infections associated with eggs occurred in French Polynesia during 2008-2013. Molecular analysis of isolates by using clustered regularly interspaced short palindromic repeat polymorphisms and multilocus variable-number tandem-repeat analysis was performed. This subtyping made defining the epidemic strain, finding the source, and decontaminating affected poultry flocks possible.

Analysis of Salmonella enterica serotype Enteritidis isolated from human and chickens by repetitive sequence-PCR fingerprinting, antibiotic resistance and plasmid profiles

A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.