Integrated OMICs approach reveals energy metabolism pathway is vital for Salmonella Pullorum survival within the egg white.

Eggs, an important part of a healthy daily diet, can protect chicken embryo development due to the shell barrier and various antibacterial components within the egg white. Our previous study demonstrated that Salmonella Pullorum, highly adapted to chickens, can survive in the egg white and, therefore, be passed to newly hatched chicks. However, the survival strategy of Salmonella Pullorum in antibacterial conditions remains unknown. The overall transcripts in the egg white showed a large-scale shift compared to LB broth. The expression of common response genes and pathways, such as those involved in iron uptake, biotin biosynthesis, and virulence, was significantly changed, consistent with the other transovarial transmission serovar Enteritidis. Notably, membrane stress response, amino acid metabolism, and carbohydrate metabolism were specifically affected. Additional upregulated functionally relevant genes (JI728_13095, JI728_13100, JI728_17960, JI728_10085, JI728_15605, and nhaA) as mutants confirmed the susceptible phenotype. Furthermore, fim deletion resulted in an increased survival capacity in the egg white, consistent with the downregulated expression. The second-round RNA-Seq analysis of the Δfim mutant in the egg white revealed significantly upregulated genes compared with the wild type in the egg white responsible for energy metabolism located on the hyc and hyp operons regulated by FhlA, indicating the Δfim mutant cannot receive enough oxygen and switched to fermentative growth due to its inability to attach to the albumen surface. Together, this study provides a first estimate of the global transcriptional response of Salmonella Pullorum under antibacterial egg white and highlights the new potential role of fim deletion in optimizing energy metabolism pathways that may assist vertical transmission. IMPORTANCE: Pullorum disease, causing serious embryo death and chick mortality, results in substantial economic losses worldwide due to transovarial transmission. Egg-borne outbreaks are frequently reported in many countries. The present study has filled the knowledge gap regarding how the specific chicken-adapted pathogen Salmonella Pullorum behaves within the challenging environment of egg white. The deletion of the fim fimbrial system can increase survival in the albumen, possibly by reprogramming metabolism-related gene products, which reveals a new adaptive strategy of pathogens. Moreover, the comparison, including previous research on Salmonella Enteritidis, capable of vertical transmission, aims to provide diversified data sets in the field and further help to implement reasonable and effective measures to improve both food safety and animal health.

Salmonellosis outbreak archive in China: data collection and assembly.

Infectious disease outbreaks transcend the medical and public health realms, triggering widespread panic and impeding socio-economic development. Considering that self-limiting diarrhoea of sporadic cases is usually underreported, the Salmonella outbreak (SO) study offers a unique opportunity for source tracing, spatiotemporal correlation, and outbreak prediction. To summarize the pattern of SO and estimate observational epidemiological indicators, 1,134 qualitative reports screened from 1949 to 2023 were included in the systematic review dataset, which contained a 506-study meta-analysis dataset. In addition to the dataset comprising over 50 columns with a total of 46,494 entries eligible for inclusion in systematic reviews or input into prediction models, we also provide initial literature collection datasets and datasets containing socio-economic and climate information for relevant regions. This study has a broad impact on advancing knowledge regarding epidemic trends and prevention priorities in diverse salmonellosis outbreaks and guiding rational policy-making or predictive modeling to mitigate the infringement upon the right to life imposed by significant epidemics.

Genome degradation promotes Salmonella pathoadaptation by remodeling fimbriae-mediated proinflammatory response.

Understanding changes in pathogen behavior (e.g. increased virulence, a shift in transmission channel) is critical for the public health management of emerging infectious diseases. Genome degradation via gene depletion or inactivation is recognized as a pathoadaptive feature of the pathogen evolving with the host. However, little is known about the exact role of genome degradation in affecting pathogenic behavior, and the underlying molecular detail has yet to be examined. Using large-scale global avian-restricted Salmonella genomes spanning more than a century, we projected the genetic diversity of Salmonella Pullorum (bvSP) by showing increasingly antimicrobial-resistant ST92 prevalent in Chinese flocks. The phylogenomic analysis identified three lineages in bvSP, with an enhancement of virulence in the two recently emerged lineages (L2/L3), as evidenced in chicken and embryo infection assays. Notably, the ancestor L1 lineage resembles the Salmonella serovars with higher metabolic flexibilities and more robust environmental tolerance, indicating stepwise evolutionary trajectories towards avian-restricted lineages. Pan-genome analysis pinpointed fimbrial degradation from a virulent lineage. The later engineered fim-deletion mutant, and all other five fimbrial systems, revealed behavior switching that restricted horizontal fecal-oral transmission but boosted virulence in chicks. By depleting fimbrial appendages, bvSP established persistent replication with less proinflammation in chick macrophages and adopted vertical transovarial transmission, accompanied by ever-increasing intensification in the poultry industry. Together, we uncovered a previously unseen paradigm for remodeling bacterial surface appendages that supplements virulence-enhanced evolution with increased vertical transmission.

Identification and Evaluation of Novel Antigen Candidates against Salmonella Pullorum Infection Using Reverse Vaccinology.

Pullorum disease, caused by the Salmonella enterica serovar Gallinarum biovar Pullorum, is a highly contagious disease in the poultry industry, leading to significant economic losses in many developing countries. Due to the emergence of multidrug-resistant (MDR) strains, immediate attention is required to prevent their endemics and global spreading. To mitigate the prevalence of MDR Salmonella Pullorum infections in poultry farms, it is urgent to develop effective vaccines. Reverse vaccinology (RV) is a promising approach using expressed genomic sequences to find new vaccine targets. The present study used the RV approach to identify new antigen candidates against Pullorum disease. Initial epidemiological investigation and virulent assays were conducted to select strain R51 for presentative and general importance. An additional complete genome sequence (4.7 Mb) for R51 was resolved using the Pacbio RS II platform. The proteome of Salmonella Pullorum was analyzed to predict outer membrane and extracellular proteins, and was further selected for evaluating transmembrane domains, protein prevalence, antigenicity, and solubility. Twenty-two high-scored proteins were identified among 4713 proteins, with 18 recombinant proteins successfully expressed and purified. The chick embryo model was used to assess protection efficacy, in which vaccine candidates were injected into 18-day-old chick embryos for in vivo immunogenicity and protective effects. The results showed that the PstS, SinH, LpfB, and SthB vaccine candidates were able to elicit a significant immune response. Particularly, PstS confers a significant protective effect, with a 75% survival rate compared to 31.25% for the PBS control group, confirming that identified antigens can be promising targets against Salmonella Pullorum infection. Thus, we offer RV to discover novel effective antigens in an important veterinary infectious agent with high priority.

Characterization of two-component system CitB family in Salmonella enterica serovar Gallinarum biovar Gallinarum.

Salmonella enterica serovar Gallinarum biovar Gallinarum is an avian-adapted pathogen causing fowl typhoid and leading to enormous economic loss in the global poultry industry. Two-component systems (TCSs) are crucial for bacteria survival, virulence, sensing and responding to the environment. 23 pairs of TCSs classified into five families were found in S. Gallinarum strain 287/91, of which the CitB family contains three pairs of TCSs, namely CitA/CitB, DcuS/DcuR and DpiB/DpiA, whose functions remained unaddressed. Thus, four mutants of S. Gallinarum strain U20, ΔcitAB (Δcit), ΔdcuSR (Δdcu), ΔdpiBA (Δdpi) and ΔcitABΔdcuSRΔdpiBA (Δ3), were constructed. The results suggested that the CitB family did not affect the growth or the metabolic capacities tested, while different TCSs exerted various effects on biofilm formation and antimicrobial resistance against multiple drug classes. Furthermore, the CitB family negatively impacted the tolerance of environmental stress, contributing to compromised virulence in chicken embryos and in vivo survival of S. Gallinarum. Collectively, this research provided new knowledge of how the CitB family is involved in the pathogenicity of S. Gallinarum.

Characterization of Two-Component System CitB Family in Salmonella Pullorum.

Salmonella enterica, serovar Gallinarum, biovar Pullorum, is an avian-specific pathogen which has caused considerable economic losses to the poultry industry worldwide. Two-component systems (TCSs) play an essential role in obtaining nutrients, detecting the presence of neighboring bacteria and regulating the expression of virulence factors. The genome analysis of S. Pullorum strain S06004 suggesting the carriage of 22 pairs of TCSs, which belong to five families named CitB, OmpR, NarL, Chemotaxis and LuxR. In the CitB family, three pairs of TCSs, namely CitA-CitB, DcuS-DcuR and DpiB-DpiA, remain unaddressed in S. Pullorum. To systematically investigate the function of the CitB family in S. Pullorum, four mutants, ΔcitAB (abbreviated as Δcit), ΔdcuSR (Δdcu), ΔdpiBA (Δdpi) and ΔcitABΔdcuSRΔdpiBA (Δ3), were made using the CRISPR/Cas9 system. The results demonstrated that the CitB family did not affect the growth of bacteria, the results of biochemical tests, invasion and proliferation in chicken macrophage HD-11 cells and the expression of fimbrial protein. But the mutants showed thicker biofilm formation, higher resistance to antimicrobial agents, enhanced tolerance to inhibition by egg albumen and increased virulence in chicken embryos. Moreover, the deletion of Dpi TCS was detrimental to survival after exposure to hyperosmotic and oxidative environments, as well as the long-term colonization of the small intestine of chickens. Collectively, we provided new knowledge regarding the possible role of the CitB family involved in the pathogenic processes of S. Pullorum.

A global dataset for prevalence of Salmonella Gallinarum between 1945 and 2021.

Pullorum disease and fowl typhoid are among the most significant poultry diseases worldwide. However, the global burden of these diseases remains unknown. Most importantly, the parameters contributing to the prevalence of Salmonella Gallinarum variants are not well documented. Therefore, in this study, we present a systematic review and meta-analysis of the global prevalence of Salmonella Gallinarum during 1945-2021. In total, 201 studies were identified for qualitative analysis (>900 million samples). The meta-analysis was subjected to over 183 screened studies. The global prevalence of S. Gallinarum (percentage of positive samples in total samples) was 8.54% (95% CI: 8.43-8.65) and showed a V-shaped recovery over time. Pullorum disease is most common in Asia, particularly in eastern China. Further investigations on chicken origin samples revealed significant differences in S. Gallinarum prevalence by gender, breed, raising mode, economic use, and growth stage, indicating a critical role of vertical transmission. Together, this study offered an updated, evidence-based dataset and knowledge regarding S. Gallinarum epidemics, which might significantly impact decision-making policy with targeted interventions.

Higher tolerance of predominant Salmonella serovars circulating in the antibiotic-free feed farms to environmental stresses.

To counteract the dramatic increase in antibiotic-resistant bacterial pathogens, many countries, including China, have banned the use of antibiotic-supplemented feed for farming animals. However, the exact consequences of this policy have not been systematically evaluated. Therefore, Salmonella isolates from farms that ceased using antibiotics 1-5 years ago were compared with isolates from farms that continue to use antimicrobials as growth promotors. Here, we used whole-genome sequencing combined with in-depth phenotypic assays to investigate the ecology, epidemiology, and persistence of multi-drug resistant (MDR) Salmonella from animal farms during the withdrawal of antibiotic growth promotors. Our results showed that the prevalence of Salmonella was significantly lower in antibiotic-free feed (AFF) farms compared to conventional-feed (CF) farms, even though all isolates obtained from AFF farms were MDR (>5 classes) and belonged to well-recognized predominant serovars. The additional phylogenomic analysis combined with principal component analysis showed high similarity between the predominant serovars in AFF and CF farms. This result raised questions regarding the environmental persistence capabilities of MDR strain despite AFF policy. To address this question, a representative panel of 20 isolates was subjected to disadvantageous environmental stress assays. These results showed that the predominant serovars in AFF and CF farms were more tolerant to stress conditions than other serovars. Collectively, our findings suggest that AFF helps eliminate only specific MDR serovars, and future guiding policies would benefit by identifying predominant Salmonella clones in problematic farms to determine the use of AFF and additional targeted interventions.

Genome-Based Assessment of Antimicrobial Resistance and Virulence Potential of Isolates of Non-Pullorum/Gallinarum Salmonella Serovars Recovered from Dead Poultry in China.

Paratyphoid avian salmonellosis is considered one of the leading causes of poultry death, resulting in significant economic losses to poultry industries worldwide. In China, especially in Shandong province, the leading producer of poultry products, several recurrent outbreaks of avian salmonellosis have been reported during the last decade where the precise causal agent remains unknown. Moreover, the establishment of earlier and more accurate recognition of pathogens is a key factor to prevent the further dissemination of resistant and/or hypervirulent clones. Here, we aim to use whole-genome sequencing combined with in silico toolkits to provide the genomic features of the antimicrobial resistance and virulence potential of 105 regionally representative non-Pullorum/Gallinarum Salmonella isolates recovered from dead poultry between 2008 and 2019 in Shandong, China. Additionally, phenotypic susceptibility to a panel of 15 antibiotics representing 11 classes was assessed by the broth microdilution method. In this study, we identified eight serovars and nine multilocus sequence typing (MLST) types, with Salmonella enterica serovar Enteritidis sequence type 11 (ST11) being the most prevalent (84/105; 80%). Based on their phenotypic antimicrobial resistance, 77.14% of the isolates were defined as multidrug resistant (≥3 antimicrobial classes), with the detection of one S. Enteritidis isolate that was resistant to the 11 classes. The highest rates of resistance were observed against nalidixic acid (97.14%) and ciprofloxacin (91.43%), followed by ampicillin (71.43%), streptomycin (64.77%), and tetracycline (60%). Genomic characterization revealed the presence of 41 resistance genes, with an alarmingly high prevalence of blaTEM-1B (60%), in addition to genomic mutations affecting the DNA gyrase (gyrA) and DNA topoisomerase IV (parC) genes, conferring resistance to quinolones. The prediction of plasmid replicons detected 14 types, with a dominance of IncFIB(S)_1 and IncFII(S)_1 (87.62% for both), while the IncX1 plasmid type was considered the key carrier of antimicrobial resistance determinants. Moreover, we report the detection of critical virulence genes, including cdtB, rck, sodCI, pef, and spv, in addition to the typical determinants for Salmonella pathogenicity island 1 (SPI-1) and SPI-2. Furthermore, phylogenomic analysis revealed the detection of three intra-farm and five inter-farm transmission events. Overall, the detection of Salmonella isolates presenting high antimicrobial resistance and harboring different critical virulence genes is of major concern, which requires the urgent implementation of effective strategies to mitigate non-Pullorum/Gallinarum avian salmonellosis. IMPORTANCE Avian salmonellosis is one of the leading global causes of poultry death, resulting in substantial economic losses in China (constituting 9% of overall financial losses). In Shandong province, a top poultry producer (30% of the overall production in China, with 15% being exported to the world), extensive outbreaks of avian salmonellosis have been reported in the past decade where the causal agents or exact types remain rarely addressed. From approximately 2008 to 2019, over 2,000 Salmonella strains were isolated and identified from dead poultry during routine surveillance of 95 poultry farms covering all 17 cities in Shandong. Approximately 1,500 isolates were confirmed to be of non-Pullorum/Gallinarum Salmonella serovars. There is an urgent need to understand the mechanisms behind the implication of zoonotic Salmonella serovars in systemic infections of poultry. Here, we analyzed populations of clinically relevant isolates of non-Pullorum/Gallinarum Salmonella causing chicken death in China by a whole-genome sequencing approach and determined that antimicrobial-resistant Salmonella Enteritidis remained the major cause in the past decades.