Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica.

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing, and agriculture. In this study, we isolated 12 bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica was used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide (LPS), cellulose, and BtuB. Lipopolysaccharide was identified as a susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b which suggested that emergence of LPS mutations in culture with SPLA1a represented collateral sensitivity to SPLA5b. We show that bacteria-phage co-culture with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to single phage co-culture. Identification of host susceptibility and resistance genes and understanding infection dynamics are critical steps in the rationale design of phage cocktails against specific bacterial pathogens.IMPORTANCEAs antibiotic resistance continues to emerge in bacterial pathogens, bacterial viruses (phage) represent a potential alternative or adjunct to antibiotics. One challenge for their implementation is the predisposition of bacteria to rapidly acquire resistance to phages. We describe a functional genomics approach to identify mechanisms of susceptibility and resistance for newly isolated phages that infect and lyse Salmonella enterica and use this information to identify phage combinations that exploit collateral sensitivity, thus increasing efficacy. Collateral sensitivity is a phenomenon where resistance to one class of antibiotics increases sensitivity to a second class of antibiotics. We report a functional genomics approach to rationally design a phage combination with a collateral sensitivity dynamic which resulted in increased efficacy. Considering such evolutionary trade-offs has the potential to manipulate the outcome of phage therapy in favor of resolving infection without selecting for escape mutants and is applicable to other virus-host interactions.

Controlling the bacterial load of Salmonella Typhi in an experimental mouse model by a lytic Salmonella phage STWB21: a phage therapy approach.

BACKGROUND: Salmonella enterica serotype Typhi is one of the major pathogens causing typhoid fever and a public health burden worldwide. Recently, the increasing number of multidrug-resistant strains of Salmonella spp. has made this utmost necessary to consider bacteriophages as a potential alternative to antibiotics for S. Typhi infection treatment. Salmonella phage STWB21, isolated from environmental water, has earlier been reported to be effective as a safe biocontrol agent by our group. In this study, we evaluated the efficacy of phage STWB21 in reducing the burden of salmonellosis in a mammalian host by inhibiting Salmonella Typhi invasion into the liver and spleen tissue. RESULTS: Phage treatment significantly improved the survival percentage of infected mice. This study also demonstrated that oral administration of phage treatment could be beneficial in both preventive and therapeutic treatment of salmonellosis caused by S. Typhi. Altogether the result showed that the phage treatment could control tissue inflammation in mice before and after Salmonella infection. CONCLUSIONS: To the best of our knowledge, this is the first report of phage therapy in a mouse model against a clinically isolated Salmonella Typhi strain that includes direct visualization of histopathology and ultrathin section microscopy images from the liver and spleen sections.

Phages for treatment of Salmonella spp infection.

Salmonella, is one of the bacterial genera having more than 2500 serogroups is one of the most prominent food borne pathogen that is capable of causing disease out breaks among humans and animals. Recent reports clearly shows that this pathogen is evolved and it developed drug resistant towards most of the commercially available antibiotics. In order to overcome this emerging resistance, Bacteriophage therapy is one of the alternative solutions. It is more pathogen specific, high potency, and thereby highly safe for consumption. This chapter discuss about Rapid screening and Detection Methods Associated with Bacteriophage for Salmonella, commercially available phage products and regulatory status, Salmonella endolysins and future prospects of phage therapy.

Rhabdomyolysis and Acute Kidney Injury Associated with Salmonella Infection: A Report of 2 Cases.

BACKGROUND Rhabdomyolysis is a clinical syndrome characterized by elevated serum creatine kinase (CK) and myoglobin levels due to the breakdown of muscle fibers and is associated with symptoms such as myalgia, muscle swelling, and erythruria. Rhabdomyolysis has an array of potential causes, including Salmonella infection, although rare. We report 2 cases in which nontyphoidal salmonellae caused acute gastroenteritis complicated by rhabdomyolysis and myoglobinuric acute kidney injury (AKI). CASE REPORT Two male patients, aged 69 years and 62 years, presented to our hospital with sudden-onset fever, abdominal pain, and watery diarrhea. At the time of admission, the patients had elevated serum CK levels (32 225 U/L and 10 590 U/L, respectively) and serum creatinine levels (4.8 mg/dL and 8.8 mg/dL, respectively). Both patients also had elevated serum myoglobin concentrations with significant myoglobinuria. They were administered fluid therapy and intravenous empirical antibiotics (cefotaxime and metronidazole for Case 1, ciprofloxacin for Case 2). The patient in Case 2 underwent 3 sessions of hemodialysis due to persistent oliguria and exacerbation of metabolic acidosis. Salmonella B (Case 1) and Salmonella C (Case 2) were isolated from blood cultures. After about 2 weeks of inpatient care, both patients showed improvement of clinical symptoms and were discharged. CONCLUSIONS Patients with acute gastroenteritis induced by Salmonella infection can develop rhabdomyolysis and myoglobinuric AKI in rare cases. Timely administration of appropriate antibiotics and fluids is expected to produce a favorable prognosis. Furthermore, early initiation of hemodialysis after onset of oliguric AKI can improve clinical outcomes.

Expression and biological activity of lytic proteins HolST-3 and LysST-3 of Salmonella phage ST-3.

Salmonella infection is a major public health concern. Several strategies for Salmonella infection prevention and control are currently available including vaccines and antibiotics. However, vaccines are expensive and inefficient, and the use of antibiotics can lead to antibiotic resistance. Thus, alternative strategies for the treatment of Salmonella remain warrant. In this study, recombinant holin HolST-3 and lysin LysST-3 from Salmonella phage ST-3 were expressed and purified, and their bactericidal properties were analyzed. HolST-3 and LysST-3 possessed a wider lysis spectrum and more efficient bactericidal effect than phage ST-3, and a synergistic bactericidal effect was observed when combined in vitro. In addition, we explored the bactericidal properties of HolST-3 and LysST-3 in vivo using zebrafish as a model organism, and found that the bactericidal effects of both HolST-3 and LysST-3 in vivo were comparable to those of cefotaxime, an antibiotic. This study provides a basis for the development of HolST-3 and LysST-3 as novel bactericidal agents for the prevention and treatment of infectious diseases caused by Salmonella spp.

Genome Analysis and Therapeutic Evaluation of a Novel Lytic Bacteriophage of Salmonella Typhimurium: Suggestive of a New Genus in the Subfamily Vequintavirinae.

Salmonella Typhimurium, a foodborne pathogen, is a major concern for food safety. Its MDR serovars of animal origin pose a serious threat to the human population. Phage therapy can be an alternative for the treatment of such MDR Salmonella serovars. In this study, we report on detailed genome analyses of a novel Salmonella phage (Salmonella-Phage-SSBI34) and evaluate its therapeutic potential. The phage was evaluated for latent time, burst size, host range, and bacterial growth reduction in liquid cultures. The phage stability was examined at various pH levels and temperatures. The genome analysis (141.095 Kb) indicated that its nucleotide sequence is novel, as it exhibited only 1-7% DNA coverage. The phage genome features 44% GC content, and 234 putative open reading frames were predicted. The genome was predicted to encode for 28 structural proteins and 40 enzymes related to nucleotide metabolism, DNA modification, and protein synthesis. Further, the genome features 11 tRNA genes for 10 different amino acids, indicating alternate codon usage, and hosts a unique hydrolase for bacterial lysis. This study provides new insights into the subfamily Vequintavirinae, of which SSBI34 may represent a new genus.

Salmonellosis Including Enteric Fever.

Salmonella is a gram-negative, motile, nonsporulating, facultative anaerobic bacillus, belongs to the family Enterobacteriaceae. The bacteria were first identified in 1884. It is transmitted through direct contact with an infected person or indirect contact by the consumption of contaminated food and water. More than 2500 serotypes of Salmonella enterica have been identified but less than 100 serotypes are known to cause infections in humans. S. enterica serovar typhi (S. typhi) and S. enterica serovar paratyphi (S. paratyphi A B C) cause enteric fever, whereas nontyphoidal Salmonella serotypes (NTS) cause diarrhea. NTS commonly presents with gastroenteritis and is a self-limiting disease. Enteric fever is a potentially life-threatening acute febrile systemic infection and is diagnosed by isolating a pathogen on culture. With the emergence of the extensive drug-resistant (XDR) S. typhi clone, limited treatment options are available. Vaccination of persons at risk, improvement of sanitation, promotion of food hygiene, and detection and control of chronic carriers are essential preventive control measures of enteric fever.

Microbiota transplantation from younger to older mice could restore lost immunity to effectively clear salmonella infection in Th2-biased BALB/c mice.

AIMS: The composition, overtly abundance, and diversity of gut microbiota, play a significant role in maintaining physiological homeostasis with age. Reports revealed that the gut microbial profile might be correlated with immunity and metabolism. It is, therefore, tantamount to know if an older individual can achieve the immunity and metabolic profile of a younger individual by receiving the gut microbiome of a younger individual. In the current report, we have studied the effects of cecal microbiota transplantation (CMT) from younger to older mice. MATERIALS AND METHODS: In this study, older BALB/c mice (23 weeks) received CMT from younger BALB/c mice (3 weeks). KEY FINDINGS: CMT recipient mice showed altered expressions of immune and tight junction protein genes in the colon of mice, while the non-CMT recipient mice did not. Older mice were treated with AVNM to make them compatible with CMT. Further data from metabolite studies revealed that AVNM treatment mainly affected the aromatic amino acid biosynthesis pathway while CMT mostly affected the metabolism of different carbohydrates. We repeated the analysis in C57BL/6 mice without any significant effects of CMT. SIGNIFICANCE: Results revealed that mice who received CMT showed more efficient restoration of gut microbiota than non-CMT recipient mice. CMT caused the alleviation of Salmonella infection and efficient recovery of the cecal index in the mice following antibiotics treatment.

Autologous hematopoietic stem cell product contaminated with Salmonella due to occult salmonellosis in an asymptomatic donor.

Recent advancements in infectious disease testing methods and pathogen reduction technologies have greatly reduced the incidence of microbial contamination of allogeneic blood products. Despite this significant reduction, contamination of autologous cellular therapy products remains a challenging issue, as many of these mitigation strategies are not feasible for such products. Most microorganisms isolated from cellular therapy products are Gram-positive normal skin flora, and studies have demonstrated that adverse effects are infrequent when these contaminated products are infused. However, no prior report has documented an autologous hematopoietic stem cell (HSC) or other cellular therapy product contaminated with Salmonella bacteria-a pathogenic Gram-negative organism. We present the first known case of Salmonella contaminating an HSC product secondary to occult salmonellosis in the donor, and discuss the implications of this contaminating organism and the therapeutic dilemma posed by this scenario.

Genomic Epidemiology of Multidrug-Resistant Nontyphoidal Salmonella in Young Children Hospitalized for Gastroenteritis.

Nontyphoidal Salmonella (NTS) gastroenteritis in children remains a significant burden on health care and constitutes a majority of all admissions for Salmonella infections in public hospitals in Hong Kong. In this prospective study, 41% of 241 children hospitalized with gastroenteritis from three public hospitals during 2019 were culture confirmed to have NTS infection. These Salmonella isolates were whole-genome sequenced and in silico predicted for their serovars/serotypes using the Salmonella In Silico Typing Resource (SISTR) and SeqSero1, and the antimicrobial resistance (AMR) genes were determined. Phylogenetic analysis revealed three major clades belonging to Salmonella enterica serovar Enteritidis sequence type 11 (ST11) (43%), multidrug-resistant (MDR) S. Typhimurium ST19 (12%) and its monophasic variant ST34 (25%), and mostly singletons of 15 other serovars. MDR S. Typhimurium and its variant were more common in infants <24 months of age and possessed genotypic resistance to five antimicrobial agents, including ampicillin (A), chloramphenicol (C), aminoglycosides (Am), sulfonamides (Su), and tetracyclines (T). Older children were more often infected with S. Enteritidis, which possessed distinct genotypic resistance to AAmSu and fluoroquinolones. In addition, 3% of the isolates possessed extended-spectrum beta-lactamase (ESBL) CTX-M genes, while one isolate (1%) harboring the carbapenemase gene blaNDM-1 was identified. Our findings provide a more complete genomic epidemiological insight into NTS causing gastroenteritis and identify a wider spectrum of determinants of resistance to third-generation beta-lactams and carbapenems, which are often not readily recognized. With high rates of multidrug-resistant NTS from studies in the Asia-Pacific region, the rapid and reliable determination of serovars and resistance determinants using whole-genome sequencing (WGS) is invaluable for enhancing public health interventions for infection prevention and control. IMPORTANCE Nontyphoidal Salmonella (NTS) gastroenteritis is a foodborne disease with a large global burden. Antimicrobial resistance (AMR) among foodborne pathogens is an important public health concern, and multidrug-resistant (MDR) Salmonella is prevalent in Southeast Asia and China. Using whole-genome sequencing, this study highlights the relationship of the MDR Salmonella serotypes and the diverse range of Salmonella genotypes that contaminate our food sources and contribute to disease in this locality. The findings update our understanding of Salmonella epidemiology and associated MDR determinants to enhance the tracking of foodborne pathogens for public health and food safety.

Investigation of Salmonella Phage-Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets.

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus-host infection networks have been used to characterize the "predator-prey" interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage-bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

Salmonella Osteomyelitis in Adults: A Systematic Review.

The aim of this systematic review was to characterize the clinical features of adults with Salmonella osteomyelitis and summarize diagnosis and treatment methods to provide guidance for clinicians. This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We conducted a literature search in the PubMed, EMBASE, and Cochrane Library databases. Article screening and data extraction were performed by two reviewers individually. All the included studies were independently evaluated by two reviewers using the Methodological Index for Non-Randomized Studies (MINORS) criteria. A total of 67 articles published between 1970 and 2019 were selected, which include 69 patients with an average age of 47.5 years (range, 18-79).The majority of cases (47.76%) occurred in immunocompetent adults without common risk factors. Aspiration and biopsy cultures were all positive in Salmonella osteomyelitis patients who underwent aspiration or biopsy. All infections were monomicrobial, and a total of 12 different serotypes were identified. The three most commonly reported Salmonella serotypes were Salmonella typhi (19 cases), Salmonella typhimurium (12 cases), and Salmonella enteritidis (11 cases). Only 12 of the 67 cases in our data (17.91%) had diarrhea symptoms, and 44 of the 67 cases (65.67%) had fever symptoms. Fifty-nine of the 67 cases (88.06%) had local inflammatory manifestations, such as erythema, swelling, and tenderness in the affected area. The commonly reported involved sites were the vertebrae, femur, and tibia. Antibiotic therapy alone was utilized in 30 cases, and 24 patients (80.00%) were eventually cured. In total, 75.68% of patients achieved satisfactory results after treatment with surgery and antibiotics. Third-generation cephalosporins were most commonly utilized, and antibiotic treatment was administered for an average of 11.3 weeks (95% CI, 8.31-14.37 weeks). Salmonella osteomyelitis should be considered in patients without any common risk factors. Aspiration or biopsy can facilitate the identification of pathogens to guide antibiotic choice. Empirical therapy with a third-generation cephalosporin is recommended until the susceptibility of the strain is determined.

Isolation and Characterisation of Bacteriophages with Activity against Invasive Non-Typhoidal Salmonella Causing Bloodstream Infection in Malawi.

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in Sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa.

Generation of TCRα-transduced T cells for adoptive transfer therapy of salmonellosis in mice.

Adoptive transfer therapy has great potential to treat diseases such as cancer as well as autoimmune and infectious diseases. Identification of chain-centric T cell receptors (TCRs) with the dominant-active antigen-specific α-chains (TCRα) can significantly improve the efficacy of adoptive cell therapy while reducing time, labor, and costs of generation of TCR-modified antigen-specific T cells. This protocol describes how to generate salmonella-specific TCRα-modified mouse T cells by retroviral transduction and evaluate their functional activity in vivo in the mouse model of salmonellosis. For complete details on the use and execution of this protocol, please refer to Kalinina et al. (2020).

The complete genome of lytic Salmonella phage vB_SenM-PA13076 and therapeutic potency in the treatment of lethal Salmonella Enteritidis infections in mice.

S. Enteritidis continues to be the most common pathogen of farm animals and a major public health burden worldwide. Using bacteriophages is a potential alternative to antibiotics against S. Enteritidis infection. In this study, the genome analysis of the lytic phage vB_SenM-PA13076 (PA13076) infecting S. Enteritidis revealed a linear, double-stranded DNA genome, which comprised of 52,474 bp and contained 69 ORFs. It belongs to the order Caudovirales; family Myoviridae, genus unclassified. The genes coded for DNA packaging, phage structural proteins, lysis components, DNA recombination, regulation, modification, and replication. No bacterial virulence or drug-resistance genes were detected. The phage PA13076 protected mice from a lethal dose of S. Enteritidis 13076Amp (5 × 108 CFU) by reducing the concentration of bacterial cells in blood, intestine, liver, spleen, and kidney. The phage PA13076 achieved at least 2.5 log reductions of S. Enteritidis cells in infected mice within 24 h (P < 0.05) when compared to the organs of control mice. The data also indicated that phage PA13076 could rapidly enter the blood and four organs of infected mice, remaining therein at concentrations of>104 PFU/g for at least 72 h. These results show that phage PA13076 has definite potential as an antibacterial therapeutic agent for attenuating S. Enteritidis infections.

Bacteriophage ZCSE2 is a Potent Antimicrobial Against Salmonella enterica Serovars: Ultrastructure, Genomics and Efficacy.

Developing novel antimicrobials capable of controlling multidrug-resistant bacterial pathogens is essential to restrict the use of antibiotics. Bacteriophages (phages) constitute a major resource that can be harnessed as an alternative to traditional antimicrobial therapies. Phage ZCSE2 was isolated among several others from raw sewage but was distinguished by broad-spectrum activity against Salmonella serovars considered pathogenic to humans and animals. Lytic profiles of ZCSE2 against a panel of Salmonella were determined together with low temperature activity and pH stability. The morphological features of the phage and host infection processes were characterized using a combination of transmission electron and atomic force microscopies. Whole genome sequencing of ZCSE2 produced a complete DNA sequence of 53,965 bp. No known virulence genes were identified in the sequence data, making ZCSE2 a good candidate for phage-mediated biological control purposes. ZCSE2 was further tested against S. Enteritidis in liquid culture and was observed to reduce the target bacterium to below the limits of detection from initial concentrations of 107-108 Colony Forming Units (CFU)/mL. With a broad host-range against pathogenic Salmonella serovars, phage ZCSE2 constitutes a potential tool against a major cause of human and animal disease.

Phage Therapy as an Approach to Control Salmonella enterica serotype Enteritidis Infection in Mice.

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