Battling Salmonella enteritidis infections: integrating proteomics and in vivo assessment of Galla Chinensis tannic acid.

Salmonella infections pose a significant threat to animal and human health. Phytochemicals present a potential alternative treatment. Galla chinensis tannic acid (GCTA), a hydrolyzable polyphenolic compound, inhibits bacterial growth and demonstrates potential as an alternative or supplement to antibiotics to prevent Salmonella infections. However, little is known about the antimicrobial mechanism of GCTA against Salmonella. Here, we revealed 456 differentially expressed proteins upon GCTA treatment, impacting pathways related to DNA replication, repair, genomic stability, cell wall biogenesis, and lipid metabolism using TMT-labeled proteomic analysis. TEM analysis suggested altered bacterial morphology and structure post-treatment. A Salmonella-infected-mouse model indicated that GCTA administration improved inflammatory markers, alleviated intestinal histopathological alterations, and reduced Salmonella enterica serovar Enteritidis (S. Enteritidis) colonization in the liver and spleen of Salmonella-infected mice. The LD50 of GCTA was 4100 mg/kg with an oral single dose, vastly exceeding the therapeutic dose. Thus, GCTA exhibited antibacterial and anti-infective activity against S. Enteritidis. Our results provided insight into the molecular mechanisms of these antibacterial effects, and highlights the potential of GCTA as an alternative to antibiotics.

Prevalence and antimicrobial susceptibility of Salmonella enteritidis and Salmonella typhimurium isolated from hen eggs and quail eggs in Karaj, Iran.

BACKGROUND AND AIM: Different Salmonella serotypes are considered one of the most important food pathogens in the world. Poultry meat and eggs are the primary carriers of Salmonella in human populations. This study aimed to estimate the Salmonella enteritidis and Salmonella typhimurium contamination rates of retail hen and quail eggs in Karaj, Iran. Moreover, the antimicrobial resistance patterns of the strains were evaluated, and the efficiency of the standard culture method and multiplex polymerase chain reaction (m-PCR) were compared. MATERIALS AND METHODS: In this descriptive cross-sectional study over 1 year (Jan-Dec 2022), 150 commercial and 150 backyard hen eggs and 300 commercial quail eggs, without cracks and fractures, were collected randomly from best selling groceries in Karaj city. All samples were examined for Salmonella contamination independently by standard culture and m-PCR approaches. A standard disc diffusion method was employed to assess the antimicrobial susceptibility of the strains against 18 antimicrobial agents. RESULTS: Out of 300 examined eggs, 2 S. enteritidis strains were isolated from the shell of backyard hen eggs. The same serotype was also detected in the contents of one of these two eggs. One S. typhimurium was isolated from the shell of a commercial hen egg. Overall, the Salmonella contamination of the shell and contents was 1% and 0.3%, respectively. Salmonella was not isolated from the eggshells or the contents of the quail eggs. There was complete agreement between the results of m-PCR and the standard culture methods. Among the 18 tested antibiotics, the highest resistance was recorded for colistin (100%), followed by nalidixic acid (75%). CONCLUSION: As most Salmonella spp. are associated with human food poisoning, continuous surveillance is required to effectively reduce the risk posed by contaminated poultry eggs. Furthermore, mandatory monitoring of antimicrobial use on Iranian poultry farms is recommended.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

The optimisation of Salmonella surveillance programmes for pullet and layer farms using local farm density as a risk factor.

Human salmonellosis cases are often caused by Salmonella serovars Enteritidis and Typhimurium and associated with the consumption of eggs and egg products. Many countries therefore implemented general surveillance programmes on pullet and layer farms. The identification of risk factors for Salmonella infection may be used to improve the performance of these surveillance programmes. The aims of this study were therefore to determine 1) whether local farm density is a risk factor for the infection of pullet and layer farms by Salmonella Enteritidis and Typhimurium and 2) whether the sampling effort of surveillance programmes can be reduced by accounting for this risk factor, while still providing sufficient control of these serovars. We assessed the importance of local farm density as a risk factor by fitting transmission kernels to Israeli surveillance data during the period from June 2017 to April 2019. The analysis shows that the risk of infection by serovars Enteritidis and Typhimurium significantly increased if infected farms were present within a radius of approximately 4 km and 0.3 km, respectively. We subsequently optimized a surveillance programme that subdivided layer farms into low and high risk groups based on the local farm density with and allowed the sampling frequency to vary between these groups. In this design, the pullet farms were always sampled one week prior to pullet distribution. Our analysis shows that the risk-based surveillance programme is able to keep the between-farm R0 of serovars Enteritidis and Typhimurium below 1 for all pullet and layer farms, while reducing the sampling effort by 32% compared to the currently implemented surveillance programme in Israel. The results of our study therefore indicate that local farm density is an important risk factor for infection of pullet and layer farms by Salmonella Enteritidis and Typhimurium and can be used to improve the performance of surveillance programmes.

Comparative efficacy of difloxacin and nano-emulsion difloxacin as antibacterial agents against Salmonella enterica Serovar enteritidis in chickenss.

Background: Avian salmonellosis is a group of diseases caused by bacteria from the genus Salmonella with a negative impact on poultry, particularly chickens. In addition, salmonellosis is a global food-borne infection. Aim: The aim of this study was to evaluate the effect of nano-emulsion difloxacin (NED) and commercial difloxacin (CD) water supplement on broiler's growth, feed intake, and body weight, weight gain, growth rate, feed conversion ratio (FCR), and mortality rate (MR). The antibiotic sensitivity was determined both in-vivo and in-vitro for NED against Salmonella enterica Serovar enteritidis in chickens. Methods: 1500 one-day of age chicks were grouped into five groups as follows: group 1 (G1) control negative group, G2 control positive group (infected and not treated), G3 (infected and treated with CD, and G4 and G5 (infected and treated with NED at different doses). Samples, including the intestine, liver, and spleen were collected. Agar well diffusion test and minimum inhibitory concentrations were adopted. Histopathological lesions on different tissues were studied. During 35 days of the experiment, the feed intake, growth rate, growth gain, FCR, and MR were recorded daily. In addition, a variety of analytical techniques including transmission electron microscopic analysis, dynamic light scattering, UV-visible spectroscopy, and zeta-potential analysis were applied to characterize NED. Results: The agar well diffusion test indicated that NED was in-vitro effective against S. enteritidis isolates than CD. The minimum inhibitory concentration was recorded as NED inhibited bacterial growth till well 8 at a concentration of 0.78 µg/ml; on the other hand, the CD inhibited bacterial growth till well 6 at a concentration of 0.62 µg/ml. Growth performance and MRs in the groups treated with NED are significantly reduced. Conclusion: Treatment of broiler's drinking water with NED at doses of 0.5 and 1 ml instead of pure CD was able to enforce a new perspective, antibacterial efficacy, enhancing the productive performance, and reducing the MRs of broilers.

[Etiological characterization of invasive non-typhoid Salmonella strains in Guangdong Province from 2018 to 2022].

Objective: To understand the serotype distribution, drug resistance and molecular characterization of invasive non-typhoid Salmonella (iNTS) in Guangdong Province from 2018 to 2022 and provide scientific evidence for the prevention and treatment of blood flow infection caused by Salmonella. Methods: Serological identification, antimicrobial susceptibility testing, multilocus sequence typing (MLST), and whole genome sequencing were performed on Salmonella isolated from blood and stool samples in Guangdong from 2018 to 2022. Simultaneously, annotated the sequencing results for drug resistance genes and virulence factors by a microbial gene annotation system. Results: The 136 iNTS strains were divided into 25 serotypes, and Salmonella enteritidis accounted for 38.24% (52/136). The OR of other iNTS serotypes were calculated with Salmonella typhimurium as the control. The OR values of Oreninburg, Rysson, and Pomona serotypes were the highest, which were 423.50, 352.92, and 211.75, respectively. The drug resistance rate of iNTS was 0.74%-66.91%, which was lower than that of non-iNTS (3.90%-77.21%). The main iNTS of drug resistance were ampicillin and tetracycline, with resistance rates of 66.91% (91/136) and 50.00% (68/136), respectively, while the resistance rates to ciprofloxacin (5.88%,8/136), ceftazidime (5.88%,8/136), gentamicin (5.13%,7/136) and cefoxitin (0.74%, 1/136) were relatively low. iNTS carried a variety of drug-resistance genes and virulence factors, but no standard virulence factor distribution has been found. MLST cluster analysis showed that iNTS was divided into 26 sequence types, and ST11 accounted for 38.24% (52/136). Conclusions: The iNTS strains in Guangdong were dominated by Salmonella enteritidis, of which three serotypes, Oreninburg, Rison, and Pomona, may be associated with a higher risk of invasive infection during 2018 to 2022. iNTS was sensitive to clinical first-line therapeutic drugs (cephalosporins and fluoroquinolones), with highly diverse sequences and clear phylogenetic branches. ST11 was the local dominant clone group.

Chitosan-nanoparticle-based oral Salmonella enteritidis subunit vaccine elicits cross-protection against Salmonella typhimurium in broilers.

Non-typhoidal Salmonella infection is a significant health and economic burden in poultry industry. Developing an oral vaccine to induce robust mucosal immunity in the intestines of birds, especially cross protection against different Salmonella serotypes is challenging. Therefore, a potent oral vaccine platform that can mitigate different serotypes of Salmonella is warranted for the poultry industry. We reported earlier that the Salmonella enteritidis (SE) immunogenic outer membrane proteins (OMPs) and flagellin (FLA) entrapped in mannose chitosan nanoparticles (OMPs-FLA-mCS NPs) administered prime-boost (d-3 and 3-wk later) by oral inoculation elicits mucosal immunity and reduces challenge SE colonization by over 1 log10 CFU in birds. In this study, we sought to evaluate whether the SE antigens containing OMPs-FLA-mCS NPs vaccine induces cross-protection against Salmonella typhimurium (ST) in broilers. Our data indicated that the OMPs-FLA-mCS NPs vaccine induced higher cross-protective antibody responses compared to commercial Poulvac ST vaccine (contains a modified-live ST bacterium). Particularly, OMPs-FLA-mCS-NP vaccine elicited OMPs and FLA antigens specific increased production of secretory IgA and IgY antibodies in samples collected at both post-vaccination and post-challenge timepoints compared to commercial vaccine group. Notably, the vaccine reduced the challenge ST bacterial load by 0.8 log10 CFU in the cecal content, which was comparable to the outcome of Poulvac ST vaccination. In conclusion, our data suggested that orally administered OMPs-FLA-mCS-NP SE vaccine elicited cross protective mucosal immune responses against ST colonization in broilers. Thus, this candidate vaccine could be a viable option replacing the existing both live and killed Salmonella vaccines for birds.

Prevalence and characterization of non-typhoidal Salmonella in egg from grading and packing plants in Korea.

Egg washing guidelines vary across countries; however, since 2020, Korea has required that all eggs produced from farms with more than 10,000 laying hens must be washed through egg grading and packing (GP) plant. This study investigated the prevalence and characterization of non-typhoidal Salmonella in eggs after washing at GP plants. In total, 16,800 eggs were collected from 60 egg GP plants located inside commercial layer farms, and 840 pooled eggshell and egg contents were tested for Salmonella, respectively. Of the 60 GP plants tested, 11 (18.3%) and 12 (20.0%) plants were positive for Salmonella spp. In the eggshells and egg contents, respectively. In particular, High Salmonella prevalence in the eggshells and egg contents occurred most often in farms with laying hens older than 80 weeks (33.3% and 40.0%, respectively). However, among 840 pooled eggshells and egg content samples, only 19 (2.3%) of each sample type were positive only for non-typhoidal Salmonella spp. The most common Salmonella serovar in both eggshells and egg contents was S. Infantis, which was found in five (8.3%) of 60 GP plants for both samples types. The other Salmonella serovars detected in eggshells were S. Bareilly (5.0%), S. Agona (3.3%), S. Enteritidis (1.7%), and S. Montevideo (1.7%), whereas those detected in egg contents were S. Enteritidis (5.0%), S. Agona (3.3%), S. Newport (3.3%), S. Senftenberg (3.3%), and S. Derby (1.7%). Of the 19 virulence genes tested, 14 genes were detected in all Salmonella. Interestingly, the spvB gene was detected only in S. Enteritidis, and the sefC gene was detected only in S. Enteritidis and S. Senftenberg. Moreover, all S. Infantis isolates showed multidrug resistance (MDR) against five or more classes, and the other serovars only showed MDR against three to four classes or no MDR. These results suggest that comprehensive surveillance and advanced management approaches for egg GP plants are required to minimize egg contamination with non-typhoidal Salmonella.

Isolation, Identification, Antimicrobial Resistance, Genotyping, and Whole-Genome Sequencing Analysis of Salmonella Enteritidis Isolated from a Food-Poisoning Incident.

Salmonella enterica is a common pathogen in humans and animals that causes food poisoning and infection, threatening public health safety. We aimed to investigate the genome structure, drug resistance, virulence characteristics, and genetic relationship of a Salmonella strain isolated from patients with food poisoning. The pathogen strain 21A was collected from the feces of patients with food poisoning, and its minimum inhibitory concentration against commonly used antibiotics was determined using the strip test and Kirby-Bauer disk methods. Subsequently, WGS analysis was used to reveal the genome structural characteristics and the carrying status of resistance genes and virulence genes of strain 21A. In addition, an MLST-based minimum spanning tree and an SNP-based systematic spanning tree were constructed to investigate its genetic evolutionary characteristics. The strain 21A was identified by mass spectrometry as S. enterica, which was found to show resistance to ampicillin, piperacillin, sulbactam, levofloxacin, and ciprofloxacin. The WGS and bioinformatics analyses revealed this strain as Salmonella Enteritidis belonging to ST11, which is common in China, containing various resistance genes and significant virulence characteristics. Strain 21A was closely related to the SJTUF strains, a series strains from animal, food and clinical sources, as well as from Shanghai, China, which were located in the same evolutionary clade. According to the genetic makeup of strain 21A, the change G > A was found to be the most common variation. We have comprehensively analyzed the genomic characteristics, drug resistance phenotype, virulence phenotype, and genetic evolution relationship of S. Enteritidis strain 21A, which will contribute towards an in-depth understanding of the pathogenic mechanism of S. Enteritidis and the effective prevention and control of foodborne diseases.

Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages.

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336ΔfliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co-transcription. The deletion mutant ΔfliCΔdinJ increased cell death and IL-1ß secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1ß secretion in cells infected with ΔfliCΔdinJ compared to cells infected with ΔfliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3-/- and Nlrc4-/- mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1ß and IL-18 in vivo, contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.

CheV enhances the virulence of Salmonella Enteritidis, and the Chev-deleted Salmonella vaccine provides immunity in mice.

BACKGROUND: Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain's major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model. RESULTS: Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD50 values of C50336 and ΔcheV were 6.3 × 105 CFU and 1.25 × 107 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection. CONCLUSION: All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.

Antimicrobial Resistance and the Genomic Epidemiology of Multidrug-Resistant Salmonella enterica serovar Enteritidis ST11 in China.

BACKGROUND: With the recent evolution of multidrug-resistant strains, the genetic characteristics of foodborne Salmonella enterica serovar Enteritidis and clinical isolates have changed. ST11 is now the most common genotype associated with S. Enteritidis isolates. METHODS: A total of 83 strains of S. Enteritidis were collected at the General Hospital of the People's Liberation Army. Of these, 37 were from aseptic sites in patients, 11 were from the feces of patients with diarrhea, and the remaining 35 were of chicken-origin. The minimum inhibitory concentration of S. Enteritidis was determined by the broth microdilution method. Genomic DNA was extracted using the QiAamp DNA Mini Kit, and whole-genome sequencing (WGS) was performed using an Illumina X-ten platform. Prokka was used for gene prediction and annotation, and bioinformatic analysis tools included Resfinder, ISFinder, Virulence Factor Database, and PlasmidFinder. IQ-TREE was used to build a maximum likelihood phylogenetic tree. The phylogenetic relationship and distribution of resistance genes was displayed using iTOL. Comparative population genomics was used to analyze the phenotypes and genetic characteristics of antibiotic resistance in clinical and chicken-origin isolates of S. Enteritidis. RESULTS: The chicken-origin S. Enteritidis isolates were more resistant to antibiotics than clinical isolates, and had a broader antibiotic resistance spectrum and higher antibiotic resistance rate. A higher prevalence of antibiotic-resistance genes was observed in chicken-origin S. Enteritidis compared to clinical isolates, along with distinct patterns in the contextual characteristics of these genes. Notably, genes such as blaCTX-M and dfrA17 were exclusive to plasmids in clinical S. Enteritidis, whereas in chicken-origin S. Enteritidis they were found in both plasmids and chromosomes. Additionally, floR was significantly more prevalent in chicken-origin isolates than in clinical isolates. Careful analysis revealed that the delayed isolation of chicken-origin S. Enteritidis contributes to accelerated gene evolution. Of note, certain resistance genes tend to integrate seamlessly and persist steadfastly within the chromosome, thereby expediting the evolution of resistance mechanisms against antibiotics. Our comparative analysis of virulence genes in S. Enteritidis strains from various sources found no substantial disparities in the distribution of other virulence factors. In summary, we propose that chicken-origin S. Enteritidis has the potential to cause clinical infections. Moreover, the ongoing evolution and dissemination of these drug-resistant genes poses a formidable challenge to clinical treatment. CONCLUSIONS: Constant vigilance is needed to monitor the dynamic patterns of drug resistance in S. Enteritidis strains sourced from diverse origins.

Epidemiological and Whole Genome Sequencing Analysis of Restaurant Salmonella Enteritidis Outbreak Associated with an Infected Food Handler in Jiangxi Province, China, 2023.

In China, Salmonella is one of the most frequent causes of bacterial gastroenteritis, and food handlers in restaurants as an important contaminated source were rarely reported. In May 2023, an outbreak of Salmonella enterica serovar Enteritidis infection in a restaurant in Jiangxi Province, China, was investigated. Cases were interviewed. Stool samples from cases, anal swabs from restaurant employees, suspicious raw food materials, and semifinished food were collected and examined. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed to determine the relatedness of the pathogen isolates. Antimicrobial resistance genes and virulence genes of isolates were analyzed by WGS. The antimicrobial profile of the isolates was detected by broth microdilution, which involved 20 different antibiotics. Among the 31 patrons, 26 showed gastrointestinal symptoms. Five Salmonella Enteritidis strains were isolated from patients (2), semifinished food (2), and food handler (1). The results of PFGE and single-nucleotide polymorphism showed that these five isolates were identical clones. These findings demonstrated that this outbreak was a restaurant Salmonella Enteritidis outbreak associated with an infected food handler. The rates of resistance to nalidixic acid and colistin and intermediate resistance to ciprofloxacin were 100%, 80%, and 100%, respectively. These outbreak isolates harbored point mutation gyrA p.D87G. The cause of inconsistency between the genotype and phenotype of resistance was deeply discussed. A total of 107 virulence genes were found in each isolate, with many being associated with Salmonella pathogenicity island (SPI)-1 and SPI-2. As an overlooked contamination source, infected food handlers can easily cause large-scale outbreaks. This outbreak highlighted that the government should enhance the training and supervision of food hygiene and safety for food handlers to prevent foodborne outbreaks.

Utilizing fab fragment-conjugated surface plasmon resonance-based biosensor for detection of Salmonella Enteritidis.

Although antibodies, a key element of biorecognition, are frequently used as biosensor probes, the use of these large molecules can lead to adverse effects. Fab fragments can be reduced to allow proper antigen-binding orientation via thiol groups containing Fab sites that can directly penetrate Au sites chemically. In this study, the ability of the surface plasmon resonance (SPR) sensor to detect Salmonella was studied. Tris(2-carboxyethyl)phosphine was used as a reducing agent to obtain half antibody fragments. Sensor surface was immobilized with antibody, and bacteria suspensions were injected from low to high concentrations. Response units were changed by binding first reduced antibody fragments, then bacteria. The biosensor was able to determine the bacterial concentrations between 103 and 108 CFU/mL. Based on these results, the half antibody fragmentation method can be generalized for faster, label-free, sensitive, and selective detection of other bacteria species.

Lack of correlation between growth, stress, and virulence phenotypes in strains of Salmonella enterica serovar Enteritidis, S. Typhimurium DT104, S. 4,12, b:- and S. Liverpool.

Strains of Salmonella Enteritidis (SEnt, n = 10) and S. Typhimurium (STm, n = 11), representing clones with high impact on human health, and strains of S. 4,12: b:- (S412B n = 11) and S. Liverpool (SLiv, n = 4), representing clones with minor impact on human health were characterized for 16 growth, stress, and virulence phenotypes to investigate whether systematic differences exist in their performance in these phenotypes and whether there was correlation between performance in different phenotypes. The term serotype was not found to be predictive of a certain type of performance in any phenotype, and surprisingly, on average, strains of SEnt and STm were not significantly better in adhering to and invading cultured intestinal cells than the less pathogenic types. Forest analysis identified desiccation tolerance and the ability to grow at 42°C with high salt as the characters that separated serovars with low human health impact (S412B/SLiv) from serovars with high human health impact (SEnt/STm). The study showed that variation in phenotypes was high even within serovars and correlation between phenotypes was low, i.e. the way that a strain performed phenotypically in one of the tested conditions had a low predictive value for the performance of the strain in other conditions.

Modelling the low temperature growth boundaries of Salmonella Enteritidis in raw and pasteurized egg yolk, egg white and liquid whole egg: Influence of the initial concentration.

Salmonella is the most frequently reported cause of foodborne outbreaks with known origin in Europe, with eggs and egg products standing out as the most frequent food source (when it was known). The growth and survival of Salmonella in eggs and egg products have been extensively studied and, recently, it has been reported that factors such as the initial concentration and thermal history of the egg product can also influence its growth capability. Therefore, the objective of this study was to define the boundary zones of the growth/no growth domain of Salmonella Enteritidis (4 strains) as a function of temperature (low temperature boundary) and the initial concentration in different egg products. A series of polynomial logistic regression equations were successfully adjusted, allowing the study of these factors and their interaction on the probability of growth of S. Enteritidis in these products. Results obtained indicate that the minimum growth temperatures of Salmonella Enteritidis are higher in egg white (9.5-18.3 °C) than in egg yolk (7.1-7.8 °C) or liquid whole egg (7.2-7.9 °C). Results also demonstrate that in raw liquid whole egg and raw and pasteurized egg white, the minimum growth temperature of Salmonella Enteritidis does depend on the initial concentration. Similarly, the previous thermal history of the egg product only influenced the minimum growth temperature in some of them. On the other hand, large differences in the minimum growth temperatures among strains were observed in some products (up to approx. 6 °C in egg white). Finally, it should be noted that none of the strains grew at 5 °C under any of the conditions assayed. Therefore, storage of egg products (particularly whole liquid egg and egg yolk) below this temperature might be regarded/proposed as a good management approach. Our experimental approach has allowed us to provide a more accurate prediction of S. Enteritidis minimum growth temperatures in egg products by taking into account additional factors (initial concentration and thermal history) while also providing a quantification of the intra-specie variability. This would be of high relevance for improving the safety of egg products.

Ecological prevalence, genetic diversity, and multidrug resistance of Salmonella enteritidis recovered from broiler and layer chicken farms.

Salmonella is a significant foodborne pathogen that has a significant impact on public health, and different strains of multidrug resistance (MDR) have been identified in this genus. This study used a combination of phenotypic and genotypic approaches to identify distinct Salmonella species collected from poultry broiler and layer farms, and antibiotic sensitivity testing was performed on these species. A total of 56 Salmonella isolates were serotyped, and phenotypic antibiotic resistance was determined for each strain. The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) method was also used to provide a genotypic description, from which a dendrogram was constructed and the most likely phylogenetic relationships were applied. Salmonella isolates were detected in 20 (17%) out of 117 samples collected from small-scale broiler flocks. Salmonella isolates were classified as MDR strains after showing tolerance to 4 antibiotics, but no resistance to cloxacillin, streptomycin, vancomycin, or netilmicin was observed. From a genotypic perspective, these strains lack dfrD, parC, and blasfo-1 resistant genes, while harboring blactx-M, blaDHA-L, qnrA, qnrB, qnrS, gyrA, ermA, ermB, ermC, ermTR, mefA, msrA, tet A, tet B, tet L, tet M resistance genes. The genotyping results obtained with ERIC-PCR allowed isolates to be classified based on the source of recovery. It was determined that Salmonella strains displayed MDR, and many genes associated with them. Additionally, the ERIC-PCR procedure aided in the generation of clusters with biological significance. Extensive research on Salmonella serotypes is warranted, along with the implementation of long-term surveillance programs to monitor MDR Salmonella serotypes in avian-derived foods.

Layer-by-layer assembly of chitosan/alginate thin films containing Salmonella enterica bacteriophages for antibacterial applications.

The emergence of antibiotic resistant bacteria and the ineffectiveness of routine treatments inspired development of alternatives to biocides for antibacterial applications. Bacteriophages are natural predators of bacteria and are promising alternatives to antibiotics. This study presents fabrication of a Salmonella enterica bacteriophage containing ultra-thin multilayer film composed of chitosan and alginate and demonstrates its potential as an antibacterial coating for food packaging applications. Chitosan/alginate film was prepared through layer-by-layer (LbL) self-assembly technique. A bacteriophage, which belongs to Siphoviridae morphotype (MET P1-001_43) and infects Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), was post-loaded into chitosan/alginate film. The LbL growth, stability, and surface morphology of chitosan/alginate film as well as phage deposition into multilayers were analysed through ellipsometry, QCM-D and AFM techniques. The bacteriophage containing multilayers showed antibacterial activity at pH 7.0. In contrast, anti-bacterial activity was not observed at acidic conditions. We showed that wrapping a Salmonella Enteritidis contaminated chicken piece with aluminium foil whose surface was modified with phage loaded chitosan/alginate multilayers decreased the number of colonies on the chicken meat, and it was as effective as treating the meat directly with phage solution.

The quorum sensing molecule C12-HSL promotes biofilm formation and increases adrA expression in Salmonella Enteritidis under anaerobic conditions.

Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bacteria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 °C, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 °C. Biofilm formation was found not to be influenced by the atmospheric conditions at 28 °C. However, it was reduced at 37 °C under anaerobiosis. C12-HSL enhanced biofilm formation at 37 °C under anaerobiosis and increased the expression of the adrA and luxS genes, suggesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions.

Evaluation of the in vitro and in vivo efficiency of in-feed bacteriophage cocktail application to control Salmonella Typhimurium and Salmonella Enteritidis infection in broiler chicks.

RESEARCH HIGHLIGHTS: Bacteriophage (BP) cocktail was partially resistant to different temperatures and pH values.The BP cocktail showed lytic effects on different Salmonella isolates.The BP cocktail reduced Salmonella colonization in the internal organs of broilers.