Genome-Based Analysis of Genetic Diversity, Antimicrobial Susceptibility, and Virulence Gene Distribution in Salmonella Pullorum Isolates from Poultry in China.

Pullorum disease, caused by Salmonella enterica serovar Pullorum (S. Pullorum) infection, is a major pathogenic threat to the poultry industry. In this study, 40 S. Pullorum isolates from seven provinces of China were comprehensively analyzed in terms of antigenic type and antimicrobial susceptibility, and their drug-resistance genes and virulence genes were identified with whole-genome sequencing (WGS). We show that all these isolates were standard antigenic types, with ST92 the predominant genotype (92.5%). Disk diffusion assays revealed high resistance rates to streptomycin (92.5%), ciprofloxacin (82.5%), and ampicillin (80%), and the resistance rates to streptomycin, gentamicin, ampicillin, and cefotaxime were higher in isolates from sick chickens than in those from healthy chickens. In addition, gyrA mutations and eight acquired resistance genes were identified, with aac(6')-Iaa the most prevalent, followed by blaTEM1ß, sul2, and the GyrA S83F mutation. The resistance phenotypes to streptomycin, ampicillin, and ciprofloxacin correlated strongly with the presence of the aac(6')-Iaa resistance gene, blaTEM1ß resistance gene, and gyrA mutations, respectively. Analysis of the virulence genes showed that the isolates expressed numerous factors associated with secretion systems, including SPI-1 and SPI-2. Overall, this study extends our understanding of the epidemiology and antibiotic resistance of S. Pullorum in China.

Cellulase exhibited a therapeutic potential to inhibit Salmonella enterica serovar Typhi biofilm by targeting multiple regulatory proteins of biofilm.

Biofilm-mediated Salmonella enterica serovar Typhi (Salmonella Ser. Typhi) infections are a growing global health issue due to the formation of antibiotic resistance. The study aimed to discover some of the druggable target proteins of Salmonella Ser. Typhi biofilm and antibiofilm enzyme to prevent Salmonella Ser. Typhi biofilm-mediated infection. Enzymatic therapy has demonstrated effective therapeutic results against bacterial infections due to its specificity and high binding capacity to the target. Therefore, this study focused on the computational interaction between the cellulase enzyme and Salmonella Ser. Typhi biofilm targets proteins with help of the various computational experiments such as ADMET (absorption, distribution, metabolism, excretion, and toxicity), protein-protein interactions, MMGBSA, etc. Further, in vitro validations of the typhoidal biofilm and cellulose presence in Salmonella Ser. Typhi biofilm was conducted using Scanning Electron Microscopy (SEM), Fourier transform infrared spectroscopy, and Raman analysis. Additionally, a minimum biofilm inhibitory concentration assay for cellulase was conducted and find out the optimized cellulase concentration which showed its inhibitory effect on the Salmonella Ser. Typhi. The cellulase antibiofilm effect was analyzed with the help of SEM analysis. Further, the cellulose content in Salmonella Ser. Typhi was quantified before and after treatment of cellulase enzyme. As a result, 58.82 % cellulose content was decreased due to cellulase treatment in Salmonella Ser. Typhi. From the seven selected typhoidal biofilm regulatory proteins of Salmonella Ser. Typhi, we identified only five potential druggable targets: BcsA, CsgE, OmpR, CsgF, and CsgD. The BcsA protein is responsible for cellulose production in Salmonella Ser. Typhi biofilm. Consequently, cellulose worked as a fascinating drug target in Salmonella Ser. Typhi biofilm. Therefore, we used cellulase as a potential antibiofilm enzyme for target-based disruption of biofilm. The cellulase showed a high binding affinity with all five identified target proteins [BcsA(-205.62 kcal/mol) > CsgE(-108.20 kcal/mol) > OmpR(-107.58 kcal/mol) > CsgF(-73.74 kcal/mol) > CsgD(-66.61 kcal/mol)] in the protein-protein interaction analysis. Our computational analysis suggests that the cellulase enzyme may be used as a potential antibiofilm enzyme against Salmonella Ser. Typhi biofilm.

Salmonella pathogenesis-based In-silico design and immunoinformatic analysis of multi-epitope vaccine constructs in broiler veterinary medicine.

Salmonellosis, a zoonotic gastrointestinal disease, presents a significant global health burden with a high incidence rate. Transmission primarily occurs through the consumption of contaminated poultry products, although water and contact with asymptomatic animals are also vectors. The disease's pervasiveness has prompted international health organizations to advocate for robust prevention and control strategies. This study focuses on the in-silico design of a multi-epitope vaccine targeting Salmonella enterica serovar Typhimurium's fimH protein, a fimbriae component crucial for bacterial adhesion and pathogenicity. The vaccine construct was developed by identifying and synthesizing non-allergenic, antigenic, and non-toxic epitopes for both Cytotoxic T Lymphocytes and Helper T Lymphocytes. Adjuvants were incorporated to enhance immunogenicity, and the vaccine's structure was modeled using advanced bioinformatics tools. The proposed vaccine demonstrated promising antigenicity and immunogenicity profiles, with a favorable physical-chemical property analysis. The vaccine's structures, designed by computational analysis, suggests high likelihood to native protein configurations. Antigenicity and allergenicity assessments validate the vaccine's immunogenic potential and hypoallergenic nature. Physicochemical evaluations indicate favorable stability and solubility profiles, essential for vaccine efficacy. This comprehensive approach to vaccine design expressed in Chlorella vulgaris holds promises for effective salmonellosis control. The multi-epitope vaccine, designed through meticulous in-silico methods, emerges as a promising candidate for controlling salmonellosis. Its strategic construction based on the fimH protein epitopes offers a targeted approach to elicit a robust immune response, potentially curbing the spread of this disease in poultry.

Salmonella associated with agricultural animals exhibit diverse evolutionary rates and show evidence of recent clonal expansion.

Most foodborne salmonellosis outbreaks are linked to agricultural animal products with a few serovars accounting for most Salmonella isolated from specific animal products, suggesting an adaptation to the corresponding animal hosts and their respective environments. Here, we utilized whole-genome sequence (WGS) data to analyze the evolution and population genetics of seven serovars frequently isolated from ground beef (Montevideo, Cerro, and Dublin), chicken (Kentucky, Infantis, and Enteritidis), and turkey (Reading) in the United States. In addition, publicly available metadata were used to characterize major clades within each serovar with regard to public health significance. Except for Dublin, all serovars were polyphyletic, comprising 2-6 phylogenetic groups. Further partitioning of the phylogenies identified 25 major clades, including 12 associated with animal or environmental niches. These 12 clades differed in evolutionary parameters (e.g., substitution rates) as well as public health relevant characteristics (e.g., association with human illness, antimicrobial resistance). Overall, our results highlight several critical trends: (i) the Salmonella generation time appears to be more dependent on source than serovar and (ii) all serovars contain clades and sub-clades that are estimated to have emerged after the year 1940 and that are enriched for isolates associated with humans, agricultural animals, antimicrobial resistance (AMR), and/or specific geographical regions. These findings suggest that serotyping alone does not provide enough resolution to differentiate isolates that may have evolved independently, present distinct geographic distribution and host association, and possibly have distinct public health significance. IMPORTANCE: Non-typhoidal Salmonella are major foodborne bacterial pathogens estimated to cause more than one million illnesses, thousands of hospitalizations, and hundreds of deaths annually in the United States. More than 70% of Salmonella outbreaks in the United States have been associated with agricultural animals. Certain serovars include persistent strains that have repeatedly contaminated beef, chicken, and turkey, causing outbreaks and sporadic cases over many years. These persistent strains represent a particular challenge to public health, as they are genetically clonal and widespread, making it difficult to differentiate distinct outbreak and contamination events using whole-genome sequence (WGS)-based subtyping methods (e.g., core genome allelic typing). Our results indicate that a phylogenetic approach is needed to investigate persistent strains and suggest that the association between a Salmonella serovar and an agricultural animal is driven by the expansion of clonal subtypes that likely became adapted to specific animals and associated environments.

Assessment and genomic analysis of Salmonella and Campylobacter from different stages of an integrated no-antibiotics-ever (NAE) broiler complex: a longitudinal study.

The objective of this study was to determine prevalence and perform genomic analysis of Salmonella spp. and Campylobacter spp. isolated from different stages of an integrated NAE broiler complex. Environmental samples were screened with 3M-Molecular Detection System (MDS) and MDS positive samples were further processed for confirmation of results and identification. Core genome-based phylogenies were built for both bacteria isolated from this study along with selected NCBI genomes. The odds ratios and 95% confidence limits were compared among stages and sample types (α < 0.05) using multivariable model. Based on MDS results, 4% and 18% of total samples were positive for Salmonella spp. and Campylobacter spp. respectively. The odds of Salmonella detection in hatchery samples were 2.58 times as likely as compared to its detection in production farms' samples (P = 0.151) while the odds of Campylobacter detection in production farms' samples were 32.19 times as likely as its detection in hatchery (P = 0.0015). Similarly, the odds of Campylobacter detection in boot swabs, soil, water, and miscellaneous samples were statistically significant (P < 0.05) as compared with fly paper as reference group. The serovars identified for Salmonella were Typhimurium, Barranquilla, Liverpool, Kentucky, Enteritidis, Luciana, and Rough_O:r:1,5. For Campylobacter, the species identified were Campylobacter jejuni and Campylobacter coli. Phylogeny results show close genetic relatedness among bacterial strains isolated from different locations within the same stage and between different stages. The results show possibility of multiple entry points of such bacteria entering broiler complex and can potentially contaminate the final raw product in the processing plant. It suggests the need for a comprehensive control strategy with strict biosecurity measures and best management practices to minimize or eliminate such pathogens from the poultry food chain.

Genomic insight into the diversity of Glaesserella parasuis isolates from 19 countries.

Glaesserella parasuis is a commensal bacterial organism found in the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease, which causes severe economic losses in the swine industry. This study aimed to better understand the epidemiological characteristics of this opportunistic pathogen. We investigated the prevalence and distribution of sequence types (STs), serovars, antimicrobial resistance genes (ARGs), and potential virulence factors (VFs) in 764 G. parasuis isolates collected from diseased and healthy pigs from 19 countries, including China. Multilocus sequence typing showed a high degree of variation with 334 STs, of which 93 were not previously recognized. Phylogenetic analysis revealed two major clades distinguished by isolation year, source, country, and serovar. The dominant serovars of G. parasuis were serovars 4 (19.50%), 7 (15.97%), 5/12 (13.87%), and 13 (12.30%). Serovar 7 gradually became one of the dominant serovars in G. parasuis with more VFs and fewer ARGs. Serovars 4 and 5/12 were the most frequent serovars in diseased pigs, whereas serovars 2, 8, and 11 were predominant in healthy pigs. Serovars 7 and 13 possessed more VFs than the other serovars. This study provides novel insights into the global prevalence and epidemiology of G. parasuis and valuable clues for further investigation into the pathogenicity of G. parasuis, which will facilitate the development of effective vaccines.IMPORTANCEGlaesserella parasuis is a clinically important gram-negative opportunistic pathogen, which causes serious financial losses in swine industry on a global scale. No vaccine is known that provides cross-protection against all 15 serovars; furthermore, the correlation between serovar and virulence is largely unknown. This study provides a large number of sequenced strains in 19 countries and compares the genomic diversity of G. parasuis between diseased and healthy pigs. We found a slight change in the dominant serovar of G. parasuis in the world, with serovar 7 gradually emerging as one of the predominant serovars. The observed higher average number of VFs in this particular serovar strain challenges the previously held notion that serovar 7 is non-virulent, indicating a more complex virulence landscape than previously understood. Our analysis indicating that six ARGs [tet(B), sul2, aph(3')-Ia, aph (6)-Id, blaROB-1, and aph(3'')-Ib] are likely to be transmitted horizontally in their entirety. By analyzing VFs, we provided an improved understanding of the virulence of G. parasuis, and these key findings suggest that vaccine development will be challenging.

Complete genome of a rare Salmonella enterica subsp. enterica serovar Hessarek from human stool.

We present a complete genome of Salmonella enterica subsp. enterica serovar Hessarek isolated from a human stool from an outbreak linked to egg consumption in South Australia. Orientation of the rrn operon and characteristics of the Salmonella virulence plasmid indicates that this serovar is virulent toward humans and birds.

Prevalence and Genomic Characterization of Multidrug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 Circulating - Beijing Municipality, China, 2016-2023.

Introduction: Highly fluoroquinolone-resistant Salmonella enterica serovar Kentucky (S. Kentucky) of sequence type (ST) 198 has emerged as a global multidrug-resistant (MDR) clone, posing a threat to public health. Methods: Whole genome sequencing and antibiotic susceptibility testing was used to characterize the population structure and evolutionary history of 54 S. Kentucky isolates recovered from food and human clinical cases in Beijing from 2016 to 2023. Results: All 54 S. Kentucky ST198 isolates exhibited resistance to quinolones, carrying point mutations in the quinolone resistance-determining regions (gyrA_S83F and parC_S80I). Resistance to other antibiotics (folate pathway inhibitors, cephems, aminoglycosides, phenicols, rifamycin, fosfomycin, macrolides, and tetracyclines), mediated by the sul1, sul2, dfrA14, bla CTX-M, bla TEM-1B, aac(3)-Id, aadA2, aadA7, aph(3')-I, aph(3'')-Ib, rmtB, floR, arr-2, fosA, mph(A), and tet(A) genes, was also observed in different combinations. The Beijing S. Kentucky ST198 evolutionary tree was divided into clades 198.2-1 and 198.2-2, which were further differentiated into three subclades: 198.2-2A, 198.2-2B, and 198.2-2C. Compared with the extended-spectrum ß-lactamase-encoding gene bla CTX-M-14b in 198.2-1, the co-existence of bla CTX-M-55 and bla TEM-1B, as well as chromosomally located qnrS1, was detected in most 198.2-2 isolates, which showed more complex MDR phenotypes. S. Kentucky ST198 outbreak isolates derived from two predominant clonal sources: 198.2-1 with cgST236434 and 198.2-2A with cgST296405. Conclusions: The S. Kentucky population in Beijing is genetically diverse, consisting of multiple co-circulating lineages that have persisted since 2016. Strengthening surveillance of food and humans will aid in implementing measures to prevent and control the spread of AMR.

Salmonella Peritonitis Secondary to Gastroduodenal Perforation: An Unusual Presentation of Enteric Fever.

Salmonella-induced peritonitis, secondary to spontaneous gastrointestinal perforation, is a rare but potentially life-threatening condition. We present a case of a 62-year-old female with a history of systemic hypertension, who presented with diffuse abdominal pain and altered bowel habits. Initial evaluation suggested acute gastroenteritis, but worsening symptoms led to emergent exploratory laparotomy, revealing a gastric/duodenal perforation. Peritoneal fluid analysis and culture confirmed Salmonella Paratyphi A infection. The patient underwent an emergency laparotomy with omental patch repair and received intravenous ceftriaxone, leading to a full recovery. This case underscores the importance of considering Salmonella infection in the differential diagnosis of peritonitis, prompt surgical intervention, and appropriate antimicrobial therapy for optimal management and outcomes. Further research on epidemiological trends, host-pathogen interactions, and antibiotic resistance should be explored. Clinical studies should refine diagnostic criteria and treatment protocols, while animal models can aid in understanding pathophysiology and vaccine development for Salmonella peritonitis. Public health interventions and environmental studies will enhance prevention and control strategies.

[Detection of Salmonella and Enumeration of Hygienic Indicator Bacteria in Dried Wood Ear Mushrooms].

An outbreak of Salmonella Stanley in the United States associated with dried wood ear mushrooms imported from China prompted us to conduct serotyping of Salmonella isolated from dried wood ear mushrooms in voluntary testing, and quantitative test for Salmonella along with enumeration of hygienic indicator bacteria in positive samples in order to evaluate the risk of Salmonella outbreak from dried wood ear mushrooms. The major serovars of Salmonella isolates obtained from 20 samples were as follows: O3,10 group-London (n=3) and Weltevreden (n=5) etc, totaling 9 strains; O4 serogroup-Saintpaul (n=2), Stanley (n=1), Typhimurium (including monophasic variant; n=3), totaling 6 strains. O7 serogroup (Potsdam) and O8 serogroup (Newport) were one strain each. Qualitative and quantitative tests for Salmonella were conducted on 10 samples with remaining amounts. As a result, one sample was 220 MPN/g, six samples were<0.6 MPN/g, and three samples were negative for Salmonella per 25 g. The mean aerobic bacterial counts and coliforms in these samples were 7.8 and 6.1 log10 CFU/g, respectively. Furthermore, qualitative test for Salmonella and enumeration of hygienic indicator bacteria were conducted on dried wood ear mushroom products (33 domestic and 30 imported products) retailed in Japan. No samples showed positive for Salmonella per 25 g, and the mean aerobic bacterial counts and coliforms were approximately 2 log10 CFU/g lower than those in the 10 samples where Salmonella was isolated during voluntary testing. While no Salmonella was detected in domestically retailed wood ear mushrooms products, the serovars associated with foodborne diseases were isolated from voluntary testing samples. It indicates that potential for consumption of Salmonella contaminated wood ear mushrooms, which is at risk of causing food poisoning.

Colonization, spread and persistence of Salmonella (Typhimurium, Infantis and Reading) in internal organs of broilers.

Transfer of Salmonella to internal organs of broilers over a 35 d grow-out period was evaluated. A total of 360 one-day old chicks were placed in 18 floor pens of 3 groups with 6 replicate pens each. On d 0, broilers were orally challenged with a cocktail of Salmonella (equal population of marked serovars; nalidixic acid-resistant S. Typhimurium, rifampicin-resistant S. Infantis, and kanamycin-resistant S. Reading) to have 3 groups: L (low; ∼2 log CFU/bird); M (medium; ∼5 log CFU/bird); and H (High; ∼8 log CFU/bird). On d 2, 7 and 35, 4 birds/pen were euthanized and ceca, liver, and spleen samples were collected aseptically. Gizzard samples (4/pen) were collected on d 35. The concentration of Salmonella in liver and spleen were transformed to binary outcomes (positive and negative) and fitted in glm function of R using cecal Salmonella concentrations (log CFU/g) and inoculation doses (L, M, and H) as inputs. On d 2, H group showed greater (P ≤ 0.05) cecal colonization of all 3 serovars compared to L and M groups. However, M group showed greater (P ≤ 0.05) colonization of all 3 serovars in the liver and spleen compared to L group. Salmonella colonization increased linearly in the ceca and quadratically in the liver and spleen with increasing challenge dose (P ≤ 0.05). On d 35, L group had greater (P ≤ 0.05) S. Infantis colonization in the ceca and liver compared to M and H groups (P ≤ 0.05). Moreover, within each group on d 35, the concentration of S. Reading was greater than those of S. Typhimurium and S. Infantis for all 3 doses in the ceca and high dose in the liver and gizzard (P ≤ 0.05). Salmonella colonization diminished in the ceca, liver, and spleen during grow-out from d 0 to d 35 (P ≤ 0.05). On d 35, birds challenged with different doses of Salmonella cocktail showed a similar total Salmonella spp. population in the ceca (ca. 3.14 log CFU/g), liver (ca. 0.54 log CFU/g), spleen (ca. 0.31 log CFU/g), and gizzard (ca. 0.42 log CFU/g). Estimates from the fitted logistic model showed that one log CFU/g increase in cecal Salmonella concentration will result in an increase in relative risk of liver and spleen being Salmonella-positive by 4.02 and 3.40 times (P ≤ 0.01), respectively. Broilers from H or M group had a lower risk (28 and 23%) of being Salmonella-positive in the liver compared to the L group when the cecal Salmonella concentration is the same (P ≤ 0.05). Oral challenge of broilers with Salmonella spp. with various doses resulted in linear or quadratic increases in Salmonella colonization in the internal organs during early age and these populations decreased during grow-out (d 35). This research can provide guidance on practices to effectively mitigate the risk of Salmonella from chicken parts and enhance public health.

Curing of a field strain of Salmonella enterica serovar Infantis isolated from poultry from its highly stable pESI like plasmid.

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944. This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.

Serovar and sequence type distribution and phenotypic and genotypic antimicrobial resistance of Salmonella originating from pet animals in Chongqing, China.

A total of 334 Salmonella isolates were recovered from 6,223 pet rectal samples collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas. Forty serovars were identified that included all strains except for one isolate that did not cluster via self-agglutination, with Salmonella Typhimurium monophasic variant, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Pomona, and Salmonella Give being the predominant serovars. Fifty-one sequence types were identified among the isolates, and ST198, ST11, ST19, ST451, ST34, and ST155 were the most common. The top four dominant antimicrobials to which isolates were resistant were sulfisoxazole, ampicillin, doxycycline, and tetracycline, and 217 isolates exhibited multidrug resistance. The prevalence of ß-lactamase genes in Salmonella isolates was 59.6%, and among these isolates, 185 harbored blaTEM, followed by blaCTX-M (66) and blaOXA (10). Moreover, six PMQR genes, namely, including qnrA (4.8%), qnrB (4.2%), qnrD (0.9%), qnrS (18.9%), aac(6')-Ib-cr (16.5%), and oqxB (1.5%), were detected. QRDR mutations (76.6%) were very common in Salmonella isolates, with the most frequent mutation in parC (T57S) (47.3%). Furthermore, we detected six tetracycline resistance genes in 176 isolates, namely, tet(A) (39.5%), tet(B) (8.1%), tet(M) (7.7%), tet(D) (5.4%), tet(J) (3.3%), and tet(C) (1.8%), and three sulfonamide resistance genes in 303 isolates, namely, sul1 (84.4%), sul2 (31.1%), and sul3 (4.2%). Finally, we found 86 isolates simultaneously harboring four types of resistance genes that cotransferred 2-7 resistance genes to recipient bacteria. The frequent occurrence of antimicrobial resistance, particularly in dogs and cats, suggests that antibiotic misuse may be driving multidrug-resistant Salmonella among pets.IMPORTANCEPet-associated human salmonellosis has been reported for many years, and antimicrobial resistance in pet-associated Salmonella has become a serious public health problem and has attracted increasing attention. There are no reports of Salmonella from pets and their antimicrobial resistance in Chongqing, China. In this study, we investigated the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella strains isolated from pet fecal samples in Chongqing. In addition, ß-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs in Salmonella isolates were examined. Our findings demonstrated the diversity of serovars and sequence types of Salmonella isolates. The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrug-resistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans. Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizontally between strains.

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.

The usefulness of nanopore sequencing in whole-genome sequencing-based genotyping of Listeria monocytogenes and Salmonella enterica serovar Enteritidis.

Bacterial genotyping through whole-genome sequencing plays a crucial role in disease surveillance and outbreak investigations in public health laboratories. This study assessed the effectiveness of Oxford Nanopore Technologies (ONT) sequencing in the genotyping of Listeria monocytogenes and Salmonella enterica serovar Enteritidis. Our results indicated that ONT sequences, generated with the R10.4.1 flow cell and basecalled using the Dorado 0.5.0 Super Accurate 4.3 model, exhibited comparable accuracy to Illumina sequences, effectively discriminating among bacterial strains from outbreaks. These findings suggest that ONT sequencing has the potential to be a promising tool for rapid whole-genome sequencing of bacterial pathogens in public health laboratories for epidemiological investigations. IMPORTANCE: This study unveils that Oxford Nanopore Technologies sequencing, by itself, holds the potential to serve as a whole-genome sequencing-based genotyping tool in public health laboratories, enabling routine subtyping of bacterial isolates for disease surveillance and outbreak investigations.

Metabolite profiling and bioactivity guided fractionation of Lactobacillaceae and rice bran postbiotics for antimicrobial-resistant Salmonella Typhimurium growth suppression.

Probiotic-fermented supplements (postbiotics) are becoming increasingly explored for their activity against antibiotic-resistant enteropathogens. Prebiotics are often incorporated into postbiotics to enhance their efficacy, but due to strain differences in probiotic activity, postbiotic antimicrobial effects are poorly understood. To improve postbiotic antimicrobial efficacy, we investigated and compared metabolite profiles of postbiotics prepared with three lactic acid bacteria strains (L. fermentum, L. paracasei, and L. rhamnosus) cultured with and without rice bran, a globally abundant, rich source of prebiotics. At their minimum inhibitory dose, L. fermentum and L. paracasei postbiotics + rice bran suppressed S. Typhimurium growth 42-55% more versus their respective probiotic-alone postbiotics. The global, non-targeted metabolome of these postbiotics identified 109 metabolites increased in L. fermentum and L. paracasei rice bran postbiotics, including 49 amino acids, 20 lipids, and 12 phytochemicals metabolites. To identify key metabolite contributors to postbiotic antimicrobial activity, bioactivity-guided fractionation was applied to L. fermentum and L. paracasei rice bran-fermented postbiotics. Fractionation resulted in four L. fermentum and seven L. paracasei fractions capable of suppressing S. Typhimurium growth more effectively versus the negative control. These fractions were enriched in 15 metabolites that were significantly increased in the global metabolome of postbiotics prepared with rice bran versus postbiotic alone. These metabolites included imidazole propionate (enriched in L. fermentum + rice bran, 1.61-fold increase; L. paracasei + rice bran 1.28-fold increase), dihydroferulate (L. fermentum + rice bran, 5.18-fold increase), and linoleate (L. fermentum + rice bran, 1.82-fold increase; L. paracasei + rice bran, 3.19-fold increase), suggesting that they may be key metabolite drivers of S. Typhimurium growth suppression. Here, we show distinct mechanisms by which postbiotics prepared with lactic acid bacteria and rice bran produce metabolites with antimicrobial activity capable of suppressing S. Typhimurium growth. Probiotic strain differences contributing to postbiotic antimicrobial activity attract attention as adjunctive treatments against pathogens.

Antimicrobial resistance profile in Salmonella enterica serovar Choleraesuis isolates from diseased pigs in Taiwan.

This study investigated antimicrobial resistance in Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates from diseased pigs in Taiwan (2015-2020). Among 272 isolates, florfenicol (96.7%), enrofloxacin (96.3%), doxycycline (91.2%), gentamicin (84.6%), and tiamulin (80.5%) exhibited high resistance. 99.3% of the isolates were resistant to at least one antibiotic, and 97.8% of the isolates were multidrug resistant. This study illustrated that S. Choleraesuis isolates exhibited high resistance to antimicrobials currently used in the Taiwanese swine industry.

In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain.

The application of live attenuated Salmonella Typhimurium vaccines has significantly helped control Salmonella in poultry products. Because the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) scores all Salmonella as positive, regardless of serovar, attenuated vaccine strains that are identified at processing contribute negatively toward Salmonella performance standards. This study was designed to determine the incidence of a live attenuated Salmonella serovar Typhimurium vaccine identified in broiler products by FSIS and to develop a PCR assay for screening of isolates. Salmonella Typhimurium short-read sequences from broiler samples uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection database by the USDA-FSIS from 2016 to 2022 were downloaded and assembled. These were analyzed using the Basic Local Alignment Search Tool (BLAST) with a sequence unique to field strains, followed by a sequence unique to the vaccine strain. The PCR assays were developed against field and vaccine strains by targeting transposition events in the crp and cya genes and validated by screening Salmonella serovar Typhimurium isolates. Between 2016 and 2022, 1708 Salmonella Typhimurium isolates of chicken origin were found in the NCBI Pathogen Detection database, corresponding to 7.99% of all Salmonella identified. Of these, 104 (5.97%) were identified as the vaccine strain. The PCR assay differentiated field strains from the vaccine strain when applied to isolates and was also able to detect the vaccine strain from DNA isolated from mixed serovar overnight Salmonella enrichment cultures. Live attenuated Salmonella vaccines are a critical preharvest tool for Salmonella control and are widely used in industry. With forthcoming regulations that will likely focus on Salmonella Typhimurium, along with other serovars, there is a need to distinguish between isolates belonging to the vaccine strain and those that are responsible for causing human illness.

Detección in silico y por PCR de una cepa vacunal viva atenuada de Salmonella Typhimurium. La aplicación de vacunas vivas atenuadas contra Salmonella Typhimurium ha ayudado significativamente a controlar Salmonella en productos avícolas. Debido a que el Servicio de Inspección de Seguridad Alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA-FSIS) califica todas las Salmonella como positivas, independientemente del serovar. Las cepas atenuadas de la vacuna que se identifican en el procesamiento contribuyen negativamente a los estándares de desempeño de Salmonella. Este estudio fue diseñado para determinar la incidencia de una vacuna viva atenuada de Salmonella serovar Typhimurium identificada en productos de pollo de engorde por el FSIS y para desarrollar un ensayo de PCR para la detección de aislados. Se recolectaron y ensamblaron secuencias de lectura corta de Salmonella Typhimurium de muestras de pollos de engorde introducidas en la plataforma de detección de patógenos del Centro Nacional de Información Biotecnológica (NCBI) por el USDA-FSIS entre los años 2016 al 2022. Estos se analizaron utilizando la herramienta de búsqueda de alineación local básica con una secuencia exclusiva para las cepas de campo, seguida de una secuencia exclusiva para la cepa vacunal. Los ensayos de PCR se desarrollaron contra cepas de campo y vacunales centrándose en eventos de transposición en los genes crp y cya y se validaron mediante la detección de aislados de Salmonella serovar Typhimurium. Entre 2016 y 2022, se encontraron 1708 aislados de Salmonella Typhimurium de origen avícola en el sistema de detección de patógenos del NCBI, lo que corresponde al 7.99 % de todas las Salmonellas identificadas. De ellas, 104 (5.97%) fueron identificadas como cepa vacunal. El ensayo de PCR diferenció las cepas de campo de la cepa de la vacuna cuando se aplicó a los aislados y también fue capaz de detectar la cepa de la vacuna a partir del ADN aislado de cultivos de enriquecimiento por toda la noche de Salmonella con serovares mixtos. Las vacunas vivas atenuadas contra Salmonella son una herramienta fundamental para el control de Salmonella y se utilizan ampliamente en la industria. Con las próximas regulaciones que probablemente se centrarán en Salmonella Typhimurium, junto con otros serovares, es necesario distinguir entre los aislados que pertenecen a la cepa vacunal y los que son responsables de causar enfermedades humanas.

Genetic diversity and antimicrobial susceptibility of Streptococcus suis from diseased Swiss pigs collected between 2019 - 2022.

Streptococcus suis is an important pathogen causing severe disease in pigs and humans, giving rise to economic losses in the pig production industry. Out of 65 S. suis isolates collected from diseased pigs in Switzerland between 2019 and 2022, 57 isolates were thoroughly examined by phenotypic and whole genome sequence (WGS) based characterization. The isolates' genomes were sequenced allowing for a comprehensive analysis of their distribution in terms of serovar, sequence type (ST), clonal complex (CC), and classical virulence markers. Antimicrobial resistance (AMR) genes were screened, and phenotypic susceptibility to eight classes of antimicrobial agents was examined. Serovar 6, devoid of any resistance genes, was found to be most prevalent, followed by serovars 1, 3, 1/2, and 9. Thirty STs were identified, with ST1104 being the most prevalent. Serovar 2 and serovar 1/2 were associated with CC1, potentially containing the most virulent variants. Based on single nucleotide polymorphism (SNP) analyses, fifteen isolates belonged to one of seven putative transmission clusters each consisting of two or three isolates. High phenotypic AMR rates were detected for tetracyclines (80%) and macrolides (35%) and associated with the resistance genes tet(O) and erm(B), respectively. In contrast, susceptibility to ß-lactam antibiotics and phenicols was high. Determination of phenotypic AMR profiling, including the minimum inhibitory concentrations (MICs) of the tested antimicrobial agents, sets a baseline for future studies. The study provides valuable insights into the genetic diversity and antimicrobial susceptibility of Swiss S. suis isolates, facilitating the identification of emerging clones relevant to public health concerns.

ß-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi.

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a ß-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of ß-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of ß-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from ß-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that ß-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of ß-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.