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Salmonella Typhimurium and Enteritidis are major causes of bloodstream infection in children in sub-Saharan Africa. This study assessed evidence for their zoonotic versus human reservoir. Index patients were children with blood culture confirmed Salmonella infection recruited during a microbiological surveillance study in Nanoro, rural Burkina between May 2013 and August 2014. After consent, their households were visited. Stool from household members and livestock (pooled samples per species) as well as drinking water were cultured for Salmonella. Isolates with identical serotype obtained from index patient and any household sample were defined as \"paired isolates\" and assessed for genetic relatedness by multilocus variable number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS). Twenty-nine households were visited for 32/42 (76.2%) eligible index patients: two households comprised two index patients each, and in a third household the index patient had a recurrent infection. Among the 32 index patients, serotypes were Salmonella Typhimurium (n = 26), Salmonella Enteritidis (n = 5) and Salmonella Freetown (n = 1). All Typhimurium isolates were sequence type (ST)313. Median delay between blood culture sampling and household visits was 13 days (range 6-26). Salmonella was obtained from 16/186 (8.6%) livestock samples (13 serotypes) and 18/290 (6.2%) household members (9 serotypes). None of the water samples yielded Salmonella. Paired Salmonella Typhimurium isolates were obtained from three households representing four index patients. MLVA types were identical in two pairs and similar in the third (consisting of two index patients and one household member). WGS showed a strong genetic relatedness with 0 to 2 core genome SNPs difference between pairs on a household level. Livestock samples did not yield any Salmonella Typhimurium or Salmonella Enteritidis, and the latter was exclusively obtained from blood culture. Other serotypes shared by human and/or livestock carriers in the same household were Salmonella Derby, Drac, Tennessee and Muenster. The current study provides further evidence of a human reservoir for invasive non-Typhoidal Salmonella (iNTS) in sub-Saharan Africa.

Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin–antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S . Enteritidis and S . Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect. The recalcitrance of many infections to antibiotic treatment may be due to the presence of ‘persisters’, or non-growing, antibiotic-tolerant bacteria. Here, the authors study the structures and functions of aminoacyl-tRNA acetyltransferase toxins, and their roles in persister formation in Salmonella .

This study was undertaken to survey the presence of Salmonella in 200 chicken samples collected from Mansoura, Egypt. Salmonella was detected in 16% (8/50), 28% (14/50), 32% (16/50) and 60% (30/50) of whole chicken carcasses, drumsticks, livers and gizzards, respectively, with an overall prevalence of 34% (68/200) among all samples. One hundred and sixty-six isolates were identified biochemically as Salmonella, and confirmed genetically by PCR, based on the presence of invA and stn genes. The spvC gene, however, was detected in only 25·3% (42/166) of the isolates. Isolates were serotyped as Salmonella Enteritidis (37·3%), S. Typhimurium (30·1%), S. Kentucky (10·8%), S. Muenster (8·4%), S. Virchow (4·8%), S. Anatum (4·8%), S. Haifa (1·2%), and four were non-typable. Antimicrobial susceptibility tests of the Salmonella isolates revealed that 100% were resistant to each of erythromycin, penicillin, and amoxicillin, while 98·8%, 96·4%, 95·2%, and 91·6% were resistant to nalidixic acid, sulphamethoxazole, oxytetracycline, and ampicillin, respectively. Multidrug resistance was evident for 92·8% of the isolates. The high contamination level of chicken meat with multidrug-resistant Salmonella can constitute a problem for public health.

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.

Salmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis. We compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enteritidis using contemporary proteomics. For each serovar, five clinical isolates (covering different geographical origins) and one reference strain were grown in vitro to the exponential phase. Levels of orthologous proteins quantified in all four serovars and within the typhoidal and non-typhoidal groups were compared and subjected to gene ontology term enrichment and inferred regulatory interactions. Differential expression of the core proteomes of the typhoidal serovars appears mainly related to cell surface components and, for the non-typhoidal serovars, to pathogenicity. Our comparative proteome analysis indicated differences in the expression of surface proteins between Salmonella Typhi and Paratyphi A, and in pathogenesis-related proteins between Salmonella Typhimurium and Enteritidis. Our findings may guide future development of novel diagnostics and vaccines, as well as understanding of disease progression.

Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S. Typhimurium and S. Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium ΔtolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications. In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20–70 °C) and pH levels (2–11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly ( p  < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

Salmonella is one of the most common causes of food-borne outbreaks and infection worldwide. Non-typhoidal Salmonella (NTS) infections are common and remain a significant public health problem among important bacterial foodborne diseases. The current study aimed to establish the Non typhoidal Salmonella infection and antimicrobial resistance status among selected patients at Morogoro Regional Referral Hospital (MRRH), Morogoro Region, Tanzania, to inform clinical care management and public health interventions. A cross-sectional study was conducted using medical records and samples were collected from hospitalised and outpatients between October and December 2021. A total of 153 participants were enrolled in the study and 132 consented to being sampled. The collected samples were analysed using standard microbiological techniques. The isolates were subjected to molecular genotyping, where Polymerase Chain Reaction (PCR) was performed targeting the 16S rDNA gene. PCR products were then submitted for sequencing to establish phylogenetic relatedness. Antimicrobial susceptibility testing and resistance genes screening were also conducted. The phylogenetic analysis identified two Salmonella serovars; Salmonella Enteritidis and Salmonella Typhimurium. The isolates were from four adults and seven children patients. The isolates were tested against six antimicrobial agents: tetracycline, trimethoprim, gentamycin, ciprofloxacin, ampicillin and cefotaxime. Further antimicrobial assays were performed by screening 10 antimicrobial resistance genes using PCR. Overall, the highest resistance was observed in ampicillin (100%), whereas the lowest resistance was recorded for ciprofloxacin and gentamicin (9.1%). In addition, four (36.4%) of the isolates were resistant to cefotaxime and three (27.3%) to tetracycline and trimethoprim. The isolates also exhibit the presence of resistance genes for sulfamethoxazole 1&2, tetracycline (tet) A&B, Beta-lactamase CTXM, Beta-lactamase TEM, Beta-lactamase SHV, Gentamycine, Acra and acc3-1 in different occurrences. The overall prevalence of Salmonella species in Morogoro region was 8.3% (11/132) with Salmonella Enteritidis and Salmonella Typhimurium being the only serovars detected from adults and children stool samples. Our investigation showed that both children and adults had been exposed to Salmonella spp. However, the occurrence of NTS was higher in children (5.3% (7/132) compared to adults (3.0% (4/132). To stop zoonotic infections and the development of antimicrobial resistance in the community, this calls for Infection Prevention and Control (IPC) and stewardship programmes on rational use of antimicrobials in both health facilities and at the community level.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)—the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot–apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from −20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.

Salmonella serovars are differentially able to infect chickens. The underlying causes are not yet fully understood. Aim of the present study was to elucidate the importance of Salmonella Pathogenicity Island 1 and 2 (SPI-1 and -2) for the virulence of two non-host-specific, but in-vivo differently invasive, Salmonella serovars in conjunction with the immune reaction of the host. Primary avian splenic macrophages were inoculated with Salmonella enterica sub-species enterica serovar (S.) Typhimurium and S. Infantis. The number and viability of intracellular bacteria and transcription of SPI-1 and -2 genes by the pathogens, as well as transcription of immune-related proteins, surface antigen expression and nitric oxide production by the macrophages, were compared at different times post inoculation. After infection, both of the Salmonella serovars were found inside the primary macrophages. Invasion-associated SPI-1 genes were significantly higher transcribed in S. Infantis- than S. Typhimurium-infected macrophages. The macrophages counteracted the S. Infantis and S. Typhimurium infection with elevated mRNA expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-12, IL-18 and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) as well as with an increased synthesis of nitric oxide. Despite these host cell attacks, S. Typhimurium was better able than S. Infantis to survive within the macrophages and transcribed higher rates of the SPI-2 genes spiC, ssaV, sifA, and sseA. The results showed similar immune reactions of primary macrophages after infection with both of the Salmonella strains. The more rapid and stronger transcription of SPI-2-related genes by intracellular S. Typhimurium compared to S. Infantis might be responsible for its better survival in avian primary macrophages.