Enterica Serovar Typhimurium Research Articles

Murine Models of Salmonella Infection.

The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium-infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella-infected intestinal tissues.

Enterocin LD3 from Enterococcus hirae LD3 Inhibits the Growth of Salmonella entericasubsp. enterica serovar Typhimurium ATCC 13311 in Fruit Juice.

In order to prevent the growth of pathogens in food, bacteriocins produced by various probiotic lactic acid bacteria have been recognized as potential substitutes of chemical preservatives. In this study, enterocin LD3 was purified from the cell-free supernatant of a food isolate, Enterococcus hirae LD3 using multistep chromatography. In the fruit juice, lethal concentration (LC50) of enterocin LD3 was found to be 260µg/mL against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The cells treated with enterocin LD3 were red colour indicating dead cells after propidium iodide staining, while untreated cells were found blue after staining with 4', 6-diamidino-2-phenylindole. The mechanism of cell killing was analyzed using infrared spectrum of cells treated with enterocin LD3 which was found altered in the range of 1,094.30 and 1,451.82cm-1 corresponding to nucleic acids and phospholipids, respectively. The morphology of target cells were severely ruptured and lysed as observed under electron microscopy. Thus, the present study suggested that enterocin LD3 showed bactericidal activity against Salm. enterica subsp. enterica serovar Typhimurium ATCC 13311 and may be applied as a bio-preservative for the safety of fruit juices.

Nontyphoidal Salmonella Invasive Disease: Challenges and Solutions.

Nontyphoidal Salmonella are a leading cause of community-onset bacteremia and other serious infections in sub-Saharan African countries where large studies indicate that they are an uncommon cause of moderate-to-severe diarrhea. Approximately 535 000 nontyphoidal Salmonella invasive disease illnesses and 77 500 deaths were estimated to occur in 2017; 422 000 (78.9%) illnesses and 66 500 (85.9%) deaths in countries in sub-Saharan Africa. Lineages of Salmonella enterica serovar Typhimurium sequence type (ST) 313 and lineages of Salmonella enterica serovar Enteritidis ST11 dominate as causes of invasive disease. A major reservoir for these specific strains outside of humans has not been identified to date. Human fecal shedding of such strains is common in areas where nontyphoidal Salmonella invasive disease incidence is high. The case-fatality ratio of nontyphoidal Salmonella invasive disease is approximately 15%. Early diagnosis and treatment are needed to avert fatal outcomes. Antimicrobial resistance, including multiple drug resistance, decreased fluoroquinolone susceptibility, and resistance to third-generation cephalosporins, is increasing in prevalence and is likely to further compromise patient outcomes. Naturally acquired immunity against invasive disease develops in children aged >3 years in endemic areas, likely mediated in part by the sequential acquisition of T-cell immunity, followed by antigen-specific immunoglobulin G antibodies. Vaccines in preclinical or clinical development include live-attenuated S. enterica serovar Typhimurium, nontyphoidal S. enterica core and O-polysaccharide glycoconjugates, multiple antigen-presenting system complexes, and generalized modules for membrane antigens vaccines. The latter are in phase I trials in Europe and Africa. Both vaccine use, and other effective, evidence-based nonvaccine interventions, are needed to prevent and control nontyphoidal Salmonella invasive disease.

Repurposing the tyrosine kinase inhibitor nilotinib for use against intracellular multidrug-resistant Salmonella Typhimurium.

The increasing incidence of infections caused by multidrug-resistant Salmonella enterica has become a serious threat to global public health. Here, we found that the tyrosine kinase inhibitor nilotinib exhibits antibacterial activity against intracellular S. enterica serovar Typhimurium in RAW264.7 macrophages. Thus, we aimed to pharmacologically exploit the anti-intracellular Salmonella activity of nilotinib and to elucidate its mechanism of action. The antibacterial activity of the compounds was assessed by high-content analysis (HCA) and intracellular CFU, minimum inhibitory concentration (MIC), and bacterial growth assays. The cytotoxicity of the compounds was evaluated by HCA and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assays. The levels of cellular AMPK, phospho-AMPK, Atg7 and β-actin were determined by immunoblotting. The screen identified two small molecule compounds (SCT1101 and SCT1104) with potent activity against intracellular S. Typhimurium. Moreover, SCT1101 and SCT1104 enhanced the efficacy of ciprofloxacin and cefixime against intracellular S. Typhimurium. However, only SCT1101 exhibited activity against intracellular MDR and fluoroquinolone-resistant S. Typhimurium isolates. Subsequent mechanistic studies showed that neither of these nilotinib derivatives increased the phospho-AMPK level in RAW264.7cells. Neither the AMPK inhibitor compound C nor SBI-0206965 reversed the inhibitory effects of SCT1101 and SCT1104 on intracellular Salmonella. Furthermore, neither blockade of autophagy by 3-MA nor shRNA-mediated knockdown of Atg7 protein expression in RAW264.7cells affected the antibacterial activity of SCT1101 and SCT1104. The structure of nilotinib could be used to develop novel therapeutics for controlling MDR S. Typhimurium infections.

Phytochemicals: potential alternative strategy to fight Salmonella enterica serovar Typhimurium.

The rise of multidrug resistant (MDR) microorganisms is a great hazard worldwide and has made it difficult to treat many infectious diseases adequately. One of the most prevalent causes of outbreaks of foodborne illness worldwide is Salmonella. The ability of this and other harmful bacteria to withstand antibiotics has recently proven crucial to their effective control. Since the beginning of time, herbal medicines and phytochemicals have been employed for their potent antibacterial action and there is a growing trend toward the production of plant based natural products for the prevention and treatment of pathogenic infections. Numerous phytochemicals have been proven effective against the molecular determinants responsible for attaining drug resistance in pathogens like efflux pumps, membrane proteins, bacterial cell communications and biofilms. The medicinal plants having antibacterial activity and antibiotics combination with phytochemicals have shown synergetic activity against Salmonella enterica serovar Typhimurium. The inhibitory effects of tannins on rumen proteolytic bacteria can be exploited in ruminant nutrition. Improved control of the rumen ecology and practical use of this feed additive technology in livestock production will be made possible by a better knowledge of the modulatory effects of phytochemicals on the rumen microbial populations in combination with fermentation. This review focuses on the development of antibacterial resistance in Salmonella, the mechanism of action of phytochemicals and the use of phytochemicals against S. enterica serovar Typhimurium. The advances and potential future applications of phytochemicals in the fight against resistant are also discussed.

Evidence of international transmission of mobile colistin resistant monophasic Salmonella Typhimurium ST34

S. 4,[5],12:i:-, a monophasic variant of S. enterica serovar Typhimurium, is an important multidrug resistant serovar. Strains of colistin-resistant S. 4,[5],12:i:- have been reported in several countries with patients occasionally had recent histories of travels to Southeast Asia. In the study herein, we investigated the genomes of S. 4,[5],12:i:- carrying mobile colistin resistance (mcr) gene in Thailand. Three isolates of mcr-3.1 carrying S. 4,[5],12:i:- in Thailand were sequenced by both Illumina and Oxford Nanopore platforms and we analyzed the sequences together with the whole genome sequences of other mcr-3 carrying S. 4,[5],12:i:- isolates available in the NCBI Pathogen Detection database. Three hundred sixty-nine core genome SNVs were identified from 27 isolates, compared to the S. Typhimurium LT2 reference genome. A maximum-likelihood phylogenetic tree was constructed and revealed that the samples could be divided into three clades, which correlated with the profiles of fljAB-hin deletions and plasmids. A couple of isolates from Denmark had the genetic profiles similar to Thai isolates, and were from the patients who had traveled to Thailand. Complete genome assembly of the three isolates revealed the insertion of a copy of IS26 at the same site near iroB, suggesting that the insertion was an initial step for the deletions of fljAB-hin regions, the hallmark of the 4,[5],12:i:- serovar. Six types of plasmid replicons were identified with the majority being IncA/C. The coexistence of mcr-3.1 and blaCTX-M-55 was found in both hybrid-assembled IncA/C plasmids but not in IncHI2 plasmid. This study revealed possible transmission links between colistin resistant S. 4,[5],12:i:- isolates found in Thailand and Denmark and confirmed the important role of plasmids in transferring multidrug resistance.

Investigating the lipid-immune signaling axis in modulating the inflammatory response to Salmonella entericaserovar Typhimurium infections

Abstract Salmonella entericaserovar Typhimurium (STM) is an intracellular pathogen that causes gastroenteritis and can disseminate systemically through survival in phagocytes. Eicosanoids-derived bioactive lipids play critical roles in both the induction and resolution of inflammation during bacterial infections or other insults. A subset of these lipids are endogenous ligands for nuclear receptors/transcription factors Peroxisome proliferator-activated receptors (PPAR), which play major roles in fatty acid metabolism and modulation of immune functions (including macrophage MΦ polarization). To elucidate the role of PPARα, we infected streptomycin-pretreated C57BL/6 (wildtype) and Ppara−/−mice with STM (colitis model). We determined that Ppara−/−mice have increased eicosanoid production, decreased inflammatory gene expression and histopathology scores in cecal tissue, and reduced systemic dissemination of bacteria, compared to C57BL/6 mice. These data suggest that the activation of PPARα results in a deleterious immune response favoring STM infection. When polarizing MΦ to various subsets, STM infection enhanced M2b and M2c polarizations (anti-inflammatory and immunoregulatory) in a PPARα-dependent manner. Using liquid chromatography-mass spectrometry (LC/MS), we also determined that STM infection alters the fatty acid profiles in polarized MΦ. Specifically, C57BL/6 M2b-polarized STM-infected MΦ have decreased ceramide levels compared to uninfected control, which would inhibit programmed cell death and enhance bacterial intracellular survival. These findings provide important insights into how the reprogramming of immune metabolism affects host-pathogen interactions during bacterial infections. This work was supported by National Institute of Allergy and Infectious Disease R01 AI168550 (VT)

Eugenol alleviates Salmonella Typhimurium-infected cecal injury by modulating cecal flora and tight junctions accompanied by suppressing inflammation

BackgroundSalmonella enterica serovar Typhimurium (ST) mainly exists in poultry and poultry related products, which are common sources of human salmonellosis. So, ST is an important zoonotic pathogen that threatens public health and safety. Eugenol has been noted for its antibacterial and anti-inflammatory properties, and it is expected to develop into an antibacterial therapy in vivo. MethodsHerein, the effects of eugenol against ST infection in terms of intestinal flora, cecal tight junction, and cecal inflammation in broilers was evaluated in this study. ResultsThe results showed that oral eugenol pretreatment obviously relieved the histopathological changes and ultrastructural injury of cecum caused by ST infection. Further analysis found that eugenol lessened the number of ST in the cecum, restrained Proteobacteria and Ruminococcus, and kept the ratio of Firmicutes to Bacteroidetes (F/B), improved the relative abundance of Alistipes compared to the infection control. Moreover, eugenol sustained the expression of ZO-1, claudin-1, and occluding tight junction proteins, reduced the mRNA levels of myeloid differentiation factor 88 (MyD88), toll-like receptor-4 (TLR4) and inducible nitric oxide synthesis (iNOS), suppressed the phosphorylation of p65 and IκBα in the NF-κB signaling pathway, as well as the mRNA levels of TNF-α, IL-1β, IL-2, and IL-18 in the cecum tissue. ConclusionTaken together, eugenol could protect broilers against ST infection via maintaining intestinal flora, tight junctions involved in mucosal barrier function, then accordingly limiting the excessive inflammation, finally contributed to improving relative weight gains and survival rates of broilers.

Promising Probiotic Properties of the Yeasts Isolated from Rabilé, a Traditionally Fermented Beer Produced in Burkina Faso.

In recent years, research on yeasts as probiotics has gained more and more interest, which will allow the development of "new" products in the probiotics market. In this context, seventeen yeast strains isolated from Rabilé, a traditional beer produced in Burkina Faso, were assessed for their probiotic attributes. The yeast identification was performed by molecular methods, including PCR-RFLP and 5.8S-ITS region sequencing. Saccharomyces cerevisiae (14 strains) was the predominantly identified species, followed by Pichia kudriavzevii (2 strains) and Rhodotorula mucilaginosa (1 strain). Except for R. mucilaginosa, all yeast strains grew well at human temperature. The yeast strains showed high resistance when they were exposed to simulated gastrointestinal conditions. Auto-aggregation ability was between 70.20 ± 10.53% and 91.82 ± 1.96%, while co-aggregation with E. coli ranged from 24.92 ± 3.96% to 80.68 ± 9.53% and with S. enterica serovar Typhimurium from 40.89 ± 8.18% to 74.06 ± 7.94%. Furthermore, the hydrophobicity of isolated strains toward n-hexane was in the range from 43.17 ± 5.07% to 70.73 ± 2.42%. All yeast strains displayed high antioxidant capabilities, and the strains did not show hemolysis halos, such that they can be considered safe. Additionally, S. cerevisiae strains strongly inhibited the growth of foodborne pathogens. This is the first preliminary study to identify and characterize the yeast strains isolated from Rabilé with interesting probiotic properties.

Isolation and Screening of Antibacterial Activity of Actinomycetota from the medicinal plant, Anthemis pseudocotula Boiss

Antibiotic resistance is rising dramatically worldwide, and thus the production of new antibiotics is indispensable. Recent scientific initiatives have focused on the bioprospecting of microorganisms' secondary metabolites, with a particular focus on the look for natural products with antimicrobial properties derived from endophytes. All plant species, regardless of their type, are thought to anchor endophytic bacteria (EB). There are many potential uses for the natural therapeutic compounds made by EB in medicine, agriculture, and the pharmaceutical industry. To investigate antibacterial properties in this study, Actinomycetota (formerly, Actinobacteria) were isolated from Anthemis pseudocotula Boiss., identified, and underwent bioprospecting by morphological and molecular methods. Samples were collected from Ilam, Iran, and then divided into roots, leaves, stems, and flowers. After disinfection, they were cut into 2 mm pieces, cultured on casein agar culture medium, and incubated at 28°C for up to four weeks. Actinomycetota was identified using the polymerase chain reaction method targeting the 16S rRNA gene. To evaluate the antibacterial properties of the isolated Actinomycetota, the agar diffusion method was used. In parallel, the frequencies of biosynthetic gene clusters, including polyketide synthase (PKS-I and PKS-II) and nonribosomal peptide synthetase (NRPS) genes, were determined in the isolated Actinomycetota. Ninety bacteria were isolated from different parts of Anthemis flowers. Thirty-eight (42.2%) of these bacteria belonged to the phylum Actinomycetota, and out of these 38, 15 isolates (39.5%) had antibacterial properties. Of these, 11 isolates (73.3%) exhibited antibacterial effects against Staphylococcus aureus, 2 (13.3%) against Pseudomonas aeruginosa, 3 (20%) against Escherichia coli, and two isolates (13.3%) against Salmonella enterica sub-species of enterica serovar Typhimurium. The results of the molecular analysis of PKS-I, PKS-II, and NRPS genes showed that out of 38 isolated Actinomycetota strains, 23 isolates (60.5%) carried PKS-I gene, 6 (15.8%) harbored PKS-II gene, and 20 isolates (52.6%) had NRPS gene. This study indicates that Anthemis pseudocotula Boiss. has a number of active Actinomycetota that produce secondary metabolites with antibacterial properties.

Different Roles of Wild Boars and Livestock in Salmonella Transmission to Humans in Italy.

Wild boar (Sus scrofa) is the most widely distributed large wildlife mammal worldwide. To investigate the transmission of Salmonella enterica amongst wild boars (Sus scrofa), humans, and livestock, we compared via pulsed-field gel electrophoresis and whole genome sequences the isolates of S. enterica serovar Typhimurium (biphasic and monophasic variants) and Enteritidis collected from wild boars, food-producing animals, and human patients in Emilia-Romagna region (Northern Italy) between 2017 and 2020. Specifically, we analysed 2175 isolates originated from human (1832), swine (117), bovine (128), poultry (76), and wild boar (22). The genomic analyses showed that wild boars shared most of their lineages of biphasic Typhimurium with bovines and most of Enteritidis with poultry, whilst we did not find any lineage shared with swine. Moreover, almost 17% of human biphasic Typhimurium and Enteritidis belonged to genomic clusters including wild boar isolates, but the inclusion of bovine and poultry isolates in the same clusters and the peculiar spatial distribution of the isolates suggested that human cases (and wild boar infections) likely originated from bovines and poultry. Consequently, wild boars appear not to play a significant role in infecting humans with these serovars, but seem to get infected themselves from livestock, probably through the environment.

Transfer RNA halves are found as nicked tRNAs in cells: evidence that nicked tRNAs regulate expression of an RNA repair operon.

Transfer RNA fragments are proposed to regulate numerous processes in eukaryotes, including translation inhibition, epigenetic inheritance, and cancer. In the bacterium Salmonella enterica serovar Typhimurium, 5' tRNA halves ending in 2',3' cyclic phosphate are proposed to bind the RtcR transcriptional activator, resulting in transcription of an RNA repair operon. However, since 5' and 3' tRNA halves can remain base paired after cleavage, the 5' tRNA halves could potentially bind RtcR as nicked tRNAs. Here we report that nicked tRNAs are ligands for RtcR. By isolating RNA from bacteria under conditions that preserve base pairing, we show that many tRNA halves are in the form of nicked tRNAs. Using a circularly permuted tRNA that mimics a nicked tRNA, we show that nicked tRNA ending in 2',3' cyclic phosphate is a better ligand for RtcR than the corresponding 5' tRNA half. In human cells, we show that some tRNA halves similarly remain base paired as nicked tRNAs following cleavage by anticodon nucleases. Our work supports a role for the RNA repair operon in repairing nicked tRNAs and has implications for the functions proposed for tRNA fragments in eukaryotes.

Design and off-target prediction for antisense oligomers targeting bacterial mRNAs with the MASON web server.

Antisense oligomers (ASOs), such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (make antisense oligomers now), a web server for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON's off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10-mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq data set from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed that our findings are not unique to Salmonella We believe that MASON's off-target assessment will improve the design of specific PNAs and other ASOs.

Antimicrobial Resistance and Mechanisms of Azithromycin Resistance in Nontyphoidal Salmonella Isolates in Taiwan, 2017 to 2018.

Antimicrobial resistance was investigated in 2,341 nontyphoidal Salmonella (NTS) isolates recovered from humans in Taiwan from 2017 to 2018 using antimicrobial susceptibility testing. Azithromycin resistance determinants were detected in 175 selected isolates using PCR and confirmed in 81 selected isolates using whole-genome sequencing. Multidrug resistance was found in 47.3% of total isolates and 96.2% of Salmonella enterica serovar Anatum and 81.7% of S. enterica serovar Typhimurium isolates. Resistance to the conventional first-line drugs (ampicillin, chloramphenicol, and cotrimoxazole), cefotaxime and ceftazidime, and ciprofloxacin was found in 32.5 to 49.0%, 20.3 to 20.4%, and 3.2% of isolates, respectively. A total of 76 (3.1%) isolates were resistant to azithromycin, which was associated with mph(A), erm(42), erm(B), and possibly the enhanced expression of efflux pump(s) due to ramAp or defective ramR. mph(A) was found in 53% of the 76 azithromycin-resistant isolates from 11 serovars and located in an IS26-mph(A)-mrx(A)-mphR(A)-IS6100 unit in various incompatibility plasmids and the chromosomes. erm(42) in S. enterica serovar Albany was carried by an integrative and conjugative element, ICE_erm42, and in S. enterica serovar Enteritidis and S. Typhimurium was located in IS26 composite transposons in the chromosomes. erm(B) was carried by IncI1-I(α) plasmids in S. Enteritidis and S. Typhimurium. ramAp was a plasmid-borne ramA, a regulatory activator of efflux pump(s), found in only S. enterica serovar Goldcoast. Since the azithromycin resistance determinants are primarily carried on mobile genetic elements, they could easily be disseminated among human bacterial pathogens. The ramAp-carrying S. Goldcoast isolates displayed azithromycin MICs of 16 to 32 mg/L. Thus, the epidemiological cutoff value of ≤16 mg/L of azithromycin proposed for wild-type NTS should be reconsidered. IMPORTANCE Antimicrobial resistance in NTS isolates is a major public health concern in Taiwan, and the mechanisms of azithromycin resistance are rarely investigated. Azithromycin and carbapenems are the last resort for the treatment of invasive salmonellosis caused by multidrug-resistant (MDR) and extensively drug-resistant Salmonella strains. Our study reports the epidemiological trend of resistance in NTS in Taiwan and the genetic determinants involved in azithromycin resistance. We point out that nearly half of NTS isolates from 2017 to 2018 are MDR, and 20% are resistant to third-generation cephalosporins. The azithromycin resistance rate (3.1%) for the NTS isolates from Taiwan is much higher than those for the NTS isolates from the United States and Europe. Our study also indicates that azithromycin resistance is primarily mediated by mph(A), erm(42), erm(B), and ramAp, which are frequently carried on mobile genetic elements. Thus, the azithromycin resistance determinants could be expected to be disseminated among diverse bacterial pathogens.

Antibacterial activity of cinnamon oil against multidrug-resistant Salmonellaserotypes and anti-quorum sensing potential

The antimicrobial resistance due to Salmonella and other foodborne pathogenic microorganisms, witnessed in recent years, has become a significant health concern. Bacteria use chemical signals to communicate each other and regulate their behavior including virulence. Depending increased antibiotic resistance, new drug development strategies being investigating and the usability of active ingredients of various medicinal and aromatic plants is especially tested as an alternative. The aim of this study was to determine the anti-quorum sensing (QS) activity of cinnamon oil (CO) on pigment production of Chromobacterium violaceum and to evaluate antibacterial activity against multidrug resistant (MDR) S. enterica serotypes. Anti QS activity was tested using biosensor strain and antibacterial activity was determined by microdilution method according to EUCAST standards. CO was found effective on QS mechanisms of Chromobacterium violaceum with pigment inhibition. Nineteen foodborne pathogens isolated from different poultry/cow sourced foods and serotyped as S. enterica serovar Infantis (15), Kentucky (1), Newport (1), Telaviv (1), and Typhimurium (1). S. enterica Infantis strains were found resistant to three or more antibiotics and categorized as MDR. The results concluded that CO has strong antibacterial activity against all S. enterica serotypes with MIC between 0.125 µg ml-1 and 1.0 µg ml-1. This research demonstrates CO is a potential candidate for developing new antimicrobial agents, antiseptic solutions or natural food preservatives against MDR Salmonella isolates also a potential as an anti-QS agent.

Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

Factors Required for Adhesion of Salmonella enterica Serovar Typhimurium to Lactuca sativa (Lettuce).

Salmonella enterica serovar Typhimurium is a major cause of foodborne gastroenteritis. Recent outbreaks of infections by S. enterica serovar Typhimurium are often associated with non-animal-related food, i.e., vegetables, fruits, herbs, sprouts, and nuts. One main problem related to the consumption of fresh produce is the minimal processing, especially for leafy green salads. In this study, we focused on butterhead lettuce (Lactuca sativa) to which S. enterica serovar Typhimurium adheres at higher rates compared to Valerianella locusta, resulting in prolonged persistence. Here, we systematically analyzed factors contributing to adhesion of S. enterica serovar Typhimurium to L. sativa leaves. Application of a reductionist, synthetic approach, including the controlled surface expression of specific adhesive structures of S. enterica serovar Typhimurium, one at a time, enabled the identification of relevant fimbrial and nonfimbrial adhesins, the O-antigen of lipopolysaccharide, the flagella, and chemotaxis being involved in binding to L. sativa leaves. The analyses revealed contributions of Lpf fimbriae, Sti fimbriae, autotransported adhesin MisL, T1SS-secreted BapA, intact lipopolysaccharide (LPS), and flagella-mediated motility to adhesion of S. enterica serovar Typhimurium to L. sativa leaves. In addition, we identified BapA as a potential adhesin involved in binding to V. locusta and L. sativa leaf surfaces. IMPORTANCE The number of produce-associated outbreaks by gastrointestinal pathogens is increasing and underlines the relevance to human health. The mechanisms involved in the colonization of, persistence on, and transmission by, fresh produce are poorly understood. Here, we investigated the contribution of adhesive factors of S. enterica serovar Typhimurium in the initial phase of plant colonization, i.e., the binding to the plant surface. We used the previously established reductionist, synthetic approach to identify factors that contribute to the surface binding of S. enterica serovar Typhimurium to leaves of L. sativa by expressing all known adhesive structures by remote control expression system.

318. A Pain in the Neck: Cervical Pyomyositis, a Rare Case of Extraintestinal Nontyphoidal Salmonellosis Further Characterized by Whole-Genome Sequencing.

Abstract Background Nontyphoidal salmonellosis (NTS) is the second-most common foodborne illness in the US, but extraintestinal manifestations are rare. We describe a case of sternocleidomastoid (SCM) pyomyositis, a rare entity, caused by Salmonella enterica, further delineated by whole-genome sequencing (WGS). A 55 year old male with liver cirrhosis and uncontrolled type-II diabetes mellitus presented with a six-day history of an enlarging left-sided neck mass. He had no fevers, chills, night sweats, nausea, vomiting or diarrhea. He had recently returned from Saudia Arabia with exposures to camels, bats and lemurs. He did not consume raw foods or dairy products. Physical exam revealed normal vital signs and a large firm neck mass. Labs were notable for leukocytosis and hyperglycemia. Neck computed tomography revealed a 6 cm heterogeneous mass inseparable from the left SCM. He required repeated drainage procedures; histopathology revealed skeletal muscle with inflammation, but no malignancy. Blood and procedural cultures grew Salmonella group B. He was treated with ceftriaxone then trimethoprim-sulfamethoxazole. Follow-up 4 weeks after presentation revealed only residual induration. Methods WGS was performed using Illumina MiSeq. Genotype and antimicrobial resistance markers were identified using MLST, KmerFinder and ResFinder on Center of Genomic Epidemiology. Virulence factors were identified using VFanalyzer. Results The bacteria was identified as Salmonella enterica subspecies enterica serovar Typhimurium and typed as ST19, the major phylogroup of Typhimurium globally. Characteristics associated with invasive NTS (iNTS) identified by WGS included genes encoding Type III secretion system and Invasin A. Other virulence factors included fimbrial adherence determinants, macrophage inducible genes, and an intracellular toxin SpvB. Resistance genes included aac(6')-Iaa, which confers resistance to tobramycin and amikacin, and qnrB19, which predicts resistance to ciprofloxacin. However, no quinolone resistance-related mutations were detected in gyrA, gyrB, parC and parE genes. AMR Genes Antimicrobial resistance genes identified by whole-genome sequencing of the Salmonella isolate. Conclusion Patients with uncontrolled diabetes are at increased risk of iNTS with or without associated diarrhea. WGS can identify additional factors that may influence morbidity and resistance. Disclosures All Authors: No reported disclosures.

Phage Therapy vs. the Use of Antibiotics in the Treatment of Salmonella-Infected Chickens: Comparison of Effects on Hematological Parameters and Selected Biochemical Markers.

Previous studies indicated that the use of a phage cocktail, composed of bacteriophages vB_SenM-2 and vB_Sen-TO17, is effective in killing cells of Salmonella enterica serovars Typhimurium and Enteritidis in vitro and in the Galleria mellonella animal model as efficiently as antibiotics (enrofloxacin or colistin) and induced fewer deleterious changes in immune responses. Here, we investigated the effects of this phage cocktail on the hematological parameters and selected biochemical markers in chickens infected with S. enterica serovar Typhimurium, in comparison to those caused by enrofloxacin or colistin. We found that treatment with antibiotics (especially with enrofloxacin) caused nonbeneficial effects on red blood cell parameters, including hematocrit, MCV, MCH, and MCHC. However, Salmonella-induced changes in the aforementioned parameters were normalized by the use of the phage cocktail. Importantly, hepatotoxicity was suggested to be induced by both antibiotics on the basis of increased alanine transaminase (ALT) and aspartate aminotransferase (AST) activities, in contrast to the phage cocktail, which did not influence these enzymes. We conclude that phage therapy with the cocktail of vB_SenM-2 and vB_Sen-TO17 in Salmonella-infected chickens is not only as effective as antibiotics but also significantly safer for the birds than enrofloxacin and colistin.

A Familiar Outbreak of Monophasic Salmonella serovar Typhimurium (ST34) Involving Three Dogs and Their Owner's Children.

Salmonella is a Gram-negative enteric bacterium responsible for the foodborne and waterborne disease salmonellosis, which is the second most reported bacterial zoonosis in humans. Many animals are potential sources of salmonellosis, including dogs, cats, and other pets. We report the case of an outbreak of salmonellosis in a family in central Italy, affecting two children and involving their three dogs as carriers. One of the children needed medical care and hospitalisation. Isolation and analysis of stool samples from the sibling and the animals present in the house were carried out. Serotyping allowed the identification of S. enterica subsp. enterica serovar Typhimurium in its monophasic variant for all the isolates. The results of whole-genome sequencing confirmed that the strains were tightly related. The minimum inhibitory concentration (MIC) test documented the resistance to ampicillin, sulfamethoxazole, and tetracycline. The origin of the zoonotic outbreak could not be assessed; however, the case study showed a clear passage of the pathogen between the human and non-human members of the family. The possibility of a transmission from a dog to a human suggests the need for further studies on the potential ways of transmission of salmonellosis through standard and alternative feed.