Mohamed, Al-Abbass

Shkodenko L.A., Mohamed A., Ateiah M., Rubel M.S., Koshel E.I.

The rapid and accurate diagnosis of meningitis is critical for preventing severe complications and fatalities. This study addresses the need for accessible diagnostics in the absence of specialized equipment by developing a novel diagnostic assay. The assay utilizes dual-priming isothermal amplification (DAMP) with unique internal primers to significantly reduce non-specificity. For fluorescence detection, the dye was selected among Brilliant Green, Thioflavin T, and dsGreen. Brilliant Green is preferred for this assay due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay was developed for the detection of the primary causative agents of meningitis (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae), and tested on clinical samples. The developed method demonstrated high specificity, no false positives, sensitivity comparable to that of loop-mediated isothermal amplification (LAMP), and a high S/B ratio. This versatile assay can be utilized as a standalone test or an integrated assay into point-of-care systems for rapid and reliable pathogen detection.

Deeb N., Otinov G.D., Mohamed A., Gandalipov E.R., Kovtunov E.A., Koshel E.I.

Objective: In recent years, bactofection—bacteria-mediated DNA transfer into mammalian cells is considered a promising approach to cancer gene therapy. Results and Discussion: In this study, we first attempted to develop a probiotic strain Escherichia coli Nissle 1917 for transferring shuttle plasmid into colorectal carcinoma cells HCT116. For this purpose, the strain was modified by inv and hly genes from Yersinia enterocolitica and Listeria monocytogenes. Conclusions: Although the efficiency of HCT116 transfection by GFP-plasmid was exceptionally low, the results demonstrated the potential use the modified probiotic strain for bactofection.

Pilipenko I., Murzova A., Savin A., Mohamed A., Vladimirova E., Koshel E., Shamova O., Kumacheva E.

Vidal A.G., Francis M., Chitanvis M., Takeshita K., Frame I.J., Sharma P., Vidal P., Lanata C.F., Grijalva C., Daley W., Vidal J.E.

Abstract Background Pneumococcal pneumonia continues to be a significant global health burden, affecting both children and adults. Traditional diagnostic methods for sputum analysis remain challenging. The objective of this study was twofold: to develop a rapid and easy-to-perform assay for the identification of Streptococcus pneumoniae (Spn) directly in sputum specimens using fluorescence microscopy, and to characterize with high-resolution confocal microscopy the ultrastructure of pneumococci residing in human sputum. Methods We fluorescently labeled antibodies against the pneumococcal capsule (Spn-FLUO). The specificity and sensitivity of Spn-FLUO for detecting Spn was evaluated in vitro and in vivo using mouse models of carriage and disease, human nasopharyngeal specimens, and sputum from patients with pneumococcal pneumonia. Spn was confirmed in the specimens using culture and a species-specific qPCR assay. Spn strains were serotyped by Quellung. Confocal microscopy and Imaris software analysis were utilized to resolve the ultrastructure of pneumococci in human sputum. Results Compared with cultures and qPCR, Spn-FLUO demonstrated high sensitivity (78–96%) in nasopharyngeal samples from mice and humans. The limit of detection (LOD) in nasopharyngeal samples was ≥ 1.6 × 10⁴ GenEq/ml. The specificity in human nasopharyngeal specimens was 100%. In lung specimens from mice infected with pneumococci, Spn-FLUO reached 100% sensitivity with a LOD of ≥ 1.39 × 10⁴ GenEq/ml. In human sputum, the sensitivity for detecting Spn was 92.7% with a LOD of 3.6 × 10³ GenEq/ml. Ultrastructural studies revealed that pneumococci are expectorated as large aggregates with a median size of 1336 μm². Conclusions Spn-FLUO is a rapid and sensitive assay for detecting Spn in human sputum within 30 min, encompassing a range of both vaccine and non-vaccine serotypes associated with pneumococcal pneumonia. The study highlights that most pneumococci form aggregates in human sputum.

Dai Y., Zhang Q., Gu R., Chen J., Ye P., Zhu H., Tang M., Nie X.

Vidal A.G., Francis M., Chitanvis M., Sharma P., Frame I.J., Vidal P., Lanata C., Grijalva C., Daley W., Vidal J.E.

AbstractBackgroundPneumococcal pneumonia continues to be a significant global health burden, affecting both children and adults. Traditional diagnostic methods for sputum analysis remain challenging. The objective of this study was twofold: to develop a rapid and easy-to-perform assay for the identification ofStreptococcus pneumoniae(Spn) directly in sputum specimens using fluorescence microscopy, and to characterize with high-resolution confocal microscopy the ultrastructure of pneumococci residing in human sputum.MethodsWe fluorescently labeled antibodies against the pneumococcal capsule (Spn-FLUO). The specificity and sensitivity of Spn-FLUO for detecting Spn was evaluatedin vitroandin vivousing mouse models of carriage and disease, human nasopharyngeal specimens, and sputum from patients with pneumococcal pneumonia. Spn was confirmed in the specimens using culture and a species-specific qPCR assays. Confocal microscopy and Imaris software analysis were utilized to resolve the ultrastructure of pneumococci in human sputum.ResultsCompared with cultures and qPCR, Spn-FLUO demonstrated high sensitivity (78-96%) in nasopharyngeal samples from mice and humans. The limit of detection (LOD) in nasopharyngeal samples was ≥1.6×10⁴ GenEq/ml. The specificity in human nasopharyngeal specimens was 100%. In lung specimens from mice infected with pneumococci, Spn-FLUO reached 100% sensitivity with a LOD of ≥1.39×10⁴ GenEq/ml. In human sputum, the sensitivity for detecting Spn was 92.7% with a LOD of 3.6×10³ GenEq/ml. Ultrastructural studies revealed that pneumococci are expectorated as large aggregates with a median size of 1336 µm².ConclusionsSpn-FLUO is a rapid and sensitive assay for detecting Spn in human sputum within 30 min. The study highlights that most pneumococci form aggregates in human sputum.

Huang Y., Zia N., Ma Y., Li T., Walker G.C., Naguib H.E., Kumacheva E.

Bhowmik S., Baral B., Rit T., Jha H.C., Das A.K.

Over the past several years, a significant increase in the expanding field of biomaterial sciences has been observed due to the development of biocompatible materials based on peptide derivatives that have intrinsic therapeutic potential.

Ding Q., Mo Z., Wang X., Chen M., Zhou F., Liu Z., Long Y., Xia X., Zhao P.

The infected wounds pose one of the major threats to human health today. To address this issue, it is necessary to develop innovative wound dressings with superior antibacterial activity and other properties. Due to its potent antibacterial, antioxidant, and immune-boosting properties, epigallocatechin gallate (EGCG) has been widely utilized. In this study, a multifunctional curdlan hydrogel loading EGCG (Cur-EGCGH3) was designed. Cur-EGCGH3 exhibited excellent physicochemical properties, good biocompatibility, hemostatic, antibacterial, and antioxidant activities. Also, ELISA data showed that Cur-EGCGH3 stimulated macrophages to secrete pro-inflammatory and pro-regenerative cytokines. Cell scratch results indicated that Cur-EGCGH3 promoted the migration of NIH3T3 and HUVECs. In vivo experiments confirmed that Cur-EGCGH3 could inhibit bacterial infection of the infected wounds, accelerate hemostasis, and promote epithelial regeneration and collagen deposition. These results demonstrated that Cur-EGCGH3 holds promise for promoting healing of the infected wounds.

Patel D.K., Jung E., Priya S., Won S., Han S.S.

Hydrogels are three-dimensional networks of polymer chains containing large amounts of water in their structure. Hydrogels have received significant attention in biomedical applications owing to their attractive physicochemical properties, including flexibility, softness, biodegradability, and biocompatibility. Different natural and synthetic polymers have been intensely explored in developing hydrogels for the desired applications. Biopolymers-based hydrogels have advantages over synthetic polymers regarding improved cellular activity and weak immune response. These properties can be further improved by grafting with other polymers or adding nanomaterials, and they structurally mimic the living tissue environments, which opens their broad applicability. The hydrogels can be physically or chemically cross-linked depending on the structure. The use of different biopolymers-based hydrogels in biomedical applications has been reviewed and discussed earlier. However, no report is still available to comprehensively introduce the synthesis, advantages, disadvantages, and biomedical applications of biopolymers-based hydrogels from the material point of view. Herein, we systematically overview different synthesis methods of hydrogels and provide a holistic approach to biopolymers-based hydrogels for biomedical applications, especially in bone regeneration, wound healing, drug delivery, bioimaging, and therapy. The current challenges and prospects of biopolymers-based hydrogels are highlighted rationally, giving an insight into the progress of these hydrogels and their practical applications.

Lee C., Huang H., Wang Y., Zhang Y., Chakravarthy R.D., Yeh M., Lin H., Wei J.

Hydrogels’ exceptional mechanical strength and skin-adhesion characteristics offer significant advantages for various applications, particularly in the fields of tissue adhesion and wearable sensors. Herein, we incorporated a combination of metal-coordination and hydrogen-bonding forces in the design of stretchable and adhesive hydrogels. We synthesized four hydrogels, namely PAID-0, PAID-1, PAID-2, and PAID-3, consisting of acrylamide (AAM), N,N′-methylene-bis-acrylamide (MBA), and methacrylic-modified dopamine (DA). The impact of different ratios of iron (III) ions to DA on each hydrogel’s performance was investigated. Our results demonstrate that the incorporation of iron–dopamine complexes significantly enhances the mechanical strength of the hydrogel. Interestingly, as the DA content increased, we observed a continuous and substantial improvement in both the stretchability and skin adhesiveness of the hydrogel. Among the hydrogels tested, PAID-3, which exhibited optimal mechanical properties, was selected for adhesion testing on various materials. Impressively, PAID-3 demonstrated excellent adhesion to diverse materials and, combined with the low cytotoxicity of PAID hydrogel, holds great promise as an innovative option for biomedical engineering applications.

Shkodenko L.A., Laushkina V.O., Rubel M.S., Sergeeva E.

Objective: The 2020 pandemic showed a need for quick, accessible, and user-friendly diagnostic tests to detect various pathogens applicable at the point-of-care (POC) or at home. Such diagnostics should require a minimum of steps and equipment. Results and Discussion: In this study, we have manufactured and modified a one-way valve system for the microfluidic platform and integrate it to the device for DNA extraction and loop-mediated isothermal amplification (LAMP) with a color readout which allowed rapid and effective diagnosis of Neisseria meningitidis (N. meningitidis) and herpes simplex virus (HSV) without the use of expensive equipment. The article describes the aspects of the manufacturing process and a possible presentation of the platform. Conclusions: This article presents a solution of integrating of embedded flexible films with ion-dependent colorimetric visualization.

Ivaska L., Herberg J., Sadarangani M.

Diagnostic tools to differentiate between community-acquired bacterial and viral meningitis are essential to target the potentially lifesaving antibiotic treatment to those at greatest risk and concurrently spare patients with viral meningitis from the disadvantages of antibiotics. In addition, excluding bacterial meningitis and thus decreasing antibiotic consumption would be important to help reduce antimicrobial resistance and healthcare expenses. The available diagnostic laboratory tests for differentiating bacterial and viral meningitis can be divided microbiological pathogen-focussed methods and biomarkers of the host response. Bacterial culture-independent microbiological methods, such as highly multiplexed nucleic acid amplification tests, are rapidly making their way into the clinical practice. At the same time, more conventional host protein biomarkers, such as procalcitonin and C-reactive protein, are supplemented by newer proteomic and transcriptomic signatures. This review aims to summarise the current state and the recent advances in diagnostic methods to differentiate bacterial from viral meningitis.

Samatova E., Komar A.A., Rodnina M.V.

During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome.

Garafutdinov R.R., Kupova O.Y., Sakhabutdinova A.R.

Isothermal nucleic acids amplification that requires DNA polymerases with strand-displacement activity gained more attention in the last two decades. Among the DNA polymerases with strand-displacement activity, Bst exo– is the most widely used. However, it tends to carry out nonspecific DNA synthesis through multimerization. In this study, the effect of nucleotide sequence on the Bst exo– binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the “closed” form of Bst exo– to the purine-rich DNA sequences, especially those containing dG at the 3′-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtained in silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3′- and 5′-terminal motifs. It has been shown that templates with the oligopurine 3′-terminal fragment and oligopyrimidine 5′-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplification. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

Kim S., Lee S., Kim U., Oh S.

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive isothermal nucleic acid amplification method, is a promising alternative to other molecular amplification techniques due to its superior specificity and sensitivity. However, due to primer dimerization, LAMP results in nonspecific and nontemplate amplification. And during the amplification confirmation process, there is carry-over contamination. These factors can result in false-positive results that overestimate the amount of DNA, preventing accurate detection. This review outlined several techniques for reducing false-positive LAMP results before amplification and confirming false-positive results after amplification. Before the amplification step, DNA polymerase activity can be decreased with organic additives such as dimethyl sulfoxide, betaine, and pullulan to prevent nonspecific amplification. The enzyme uracil-DNA-glycosylase (UDG) can eliminate false-positive results caused by carry-over contamination, and the hot-start effect with gold nanoparticles can reduce nonspecific amplification. When confirming false-positive results using clustered regularly interspaced short palindromic repeats, guide RNA accurately detects LAMP amplification, allowing differentiation from nonspecific amplification. By confirming amplification, the colorimetric change in the deoxyribozyme (DNAzyme) formed by the reaction of the G-quadruplex sequence of the LAMP amplicon and hemin can distinguish false-positive results. Lateral flow immunoassay can distinguish false-positive results by accurately recognizing hybridized probes to LAMP amplicons.

Raman V., Howell L.M., Bloom S.M., Hall C.L., Wetherby V.E., Minter L.M., Kulkarni A.A., Forbes N.S.

IntroductionImmunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice.MethodsTo test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion.ResultsWe showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation.DiscussionThis response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

Rubel M.S., Shkodenko L.A., Gorbenko D.A., Solyanikova V.V., Maltzeva Y.I., Rubel A.A., Koshel E.I., Kolpashchikov D.M.

Structural RNA is a challenging target for recognition by hybridization probes. This chapter addresses the recognition problem of RNA amplicons in samples obtained by multiplex nucleic acid sequence-based amplification (NASBA). The method describes the design of G-quadruplex binary (split) DNA peroxidase sensors that produces colorimetric signal upon recognition of NASBA amplicons.

Tumas S., Meldgaard T.S., Vaaben T.H., Suarez Hernandez S., Rasmussen A.T., Vazquez-Uribe R., Hadrup S.R., Sommer M.O.

AbstractIn this study we performed a step-wise optimization of biologically active IL-2 for delivery using E. coli Nissle 1917. Engineering of the strain was coupled with an in vitro cell assay to measure the biological activity of microbially produced IL-2 (mi-IL2). Next, we assessed the immune modulatory potential of mi-IL2 using a 3D tumor spheroid model demonstrating a strong effect on immune cell activation. Finally, we evaluated the anticancer properties of the engineered strain in a murine CT26 tumor model. The engineered strain was injected intravenously and selectively colonized tumors. The treatment was well-tolerated, and tumors of treated mice showed a modest reduction in tumor growth rate, as well as significantly elevated levels of IL-2 in the tumor. This work demonstrates a workflow for researchers interested in engineering E. coli Nissle for a new class of microbial therapy against cancer.

Truong H.C., Van Phan T., Nguyen H.T., Truong K.H., Do V.C., Pham N.N., Ho T.V., Phan T.T., Hoang T.A., Soetewey A., Ho T.N., Pham Q.D., Luong Q.C., Vo D.T., Nguyen T.V., et. al.

Abstract Background This retrospective hospital-based surveillance aimed to assess the epidemiology, causative pathogens trend, and serotypes distribution of pneumococcal meningitis among children aged under 5 years with bacterial meningitis in Southern Vietnam after the introduction of pentavalent vaccine in the Expanded Program on Immunization (EPI). Methods From 2012 to 2021, cerebrospinal fluid samples were collected from children aged under 5 years with suspected bacterial meningitis at Children's Hospitals 1 and 2 in Ho Chi Minh City. Probable bacterial meningitis (PBM) cases were identified using biochemistry and cytology. Real-time polymerase chain reaction was used to confirm cases of confirmed bacterial meningitis (CBM) caused by Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis. Streptococcus pneumoniae serotyping was performed. Results Of the 2560 PBM cases, 158 (6.2%) were laboratory-confirmed. The CBM proportion decreased during the 10-year study and was associated with age, seasonality, and permanent residence. Streptococcus pneumoniae was the most common pathogen causing bacterial meningitis (86.1%), followed by H influenzae (7.6%) and N meningitidis (6.3%). The case-fatality rate was 8.2% (95% confidence interval, 4.2%–12.2%). Pneumococcal serotypes 6A/B, 19F, 14, and 23F were the most prevalent, and the proportion of pneumococcal meningitis cases caused by the 10-valent pneumococcal conjugate vaccine (PCV) serotypes decreased from 96.2% to 57.1% during the PCV eras. Conclusions Streptococcus pneumoniae is the most frequent causative agent of bacterial meningitis in children aged under 5 years in Southern Vietnam over the last decade. Policymakers may need to consider introducing PCVs into the EPI to effectively prevent and control bacterial meningitis.

Maltzeva Y.I., Gorbenko D.A., Nikitina E.V., Rubel M.S., Kolpashchikov D.M.

Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population’s health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

Bloom S.M., O'Hare N., Forbes N.S.

Tan M., Liao C., Liang L., Yi X., Zhou Z., Wei G.

After the outbreak of SARS-CoV-2, nucleic acid testing quickly entered people’s lives. In addition to the polymerase chain reaction (PCR) which was commonly used in nucleic acid testing, isothermal amplification methods were also important nucleic acid testing methods. Among several common isothermal amplification methods like displaced amplification, rolling circle amplification, and so on, recombinase polymerase amplification (RPA) was recently paid more attention to. It had the advantages like a simple operation, fast amplification speed, and reaction at 37-42°C, et al. So it was very suitable for field detection. However, there were still some disadvantages to RPA. Herein, our review mainly summarized the principle, advantages, and disadvantages of RPA. The specific applications of RPA in bacterial detection, fungi detection, virus detection, parasite detection, drug resistance gene detection, genetically modified food detection, and SARS-CoV-2 detection were also described. It was hoped that the latest research progress on RPA could be better delivered to the readers who were interested in RPA.

Wang R., Sun M., Wang C., Dong A., Zhang J.

Mussel-inspired biomimetic dopamine (DA)-functionalized polymers have been widely studied and applied in many fields, such as biological adhesives, surface modification and biomedicine. However, the current preparation methods still face challenging obstacles, such as complex multistep processes, harsh reaction conditions and relatively low yield. Herein, a facile, efficient and versatile strategy was designed to synthesize DA-functionalized polymers by epoxy-amino click reaction between DA and epoxy-containing polymers derived from readily available commercial monomer glycidyl methacrylate (GMA). A series of DA-functionalized polymers were successfully prepared and their structure and composition were fully characterized. In addition, the performance for adhesion and surface modification of obtained DA-functionalized polymers was evaluated. The results indicated this novel synthetical strategy can readily prepare DA-functionalized polymers in a highly simple and controllable manner. Moreover, the DA-functionalized polymers prepared by epoxy-amino click reaction possessed an enhanced performance for adhesiveness. Considering the universality, simplicity and high controllability of this methodology as well as the readily available raw materials, this strategy provides new opportunities for scale-up production of DA-functionalized polymers with tunable DA content for various applications.

R． O'Steen M., M．Kolpashchikov D.

Multiplex assays often rely on expensive sensors incorporating covalently linked fluorescent dyes. Herein, we developed a self-assembling aptamer-based multiplex assay. This multiplex approach utilizes a previously established split aptamer sensor in conjugation with a novel split aptamer sensor based upon a malachite green DNA aptamer. This system was capable of simultaneous fluorescent detection of two SARS COVID-19-related sequences in one sample with individual sensors that possesses a limit of detection (LOD) in the low nM range. Optimization of the Split Malachite Green (SMG) sensor yielded a minimized aptamer construct, Mini-MG, capable of inducing fluorescence of malachite green in both a DNA hairpin and sensor format.

Howell L.M., Forbes N.S.

Engineered bacterial therapies that target the tumor immune landscape offer a new class of cancer immunotherapy. Salmonella enterica and Listeria monocytogenes are two species of bacteria that have been engineered to specifically target tumors and serve as delivery vessels for immunotherapies. Therapeutic bacteria have been engineered to deliver cytokines, gene silencing shRNA, and tumor associated antigens that increase immune activation. Bacterial therapies stimulate both the innate and adaptive immune system, change the immune dynamics of the tumor microenvironment, and offer unique strategies for targeting tumors. Bacteria have innate adjuvant properties, which enable both the delivered molecules and the bacteria themselves to stimulate immune responses. Bacterial immunotherapies that deliver cytokines and tumor-associated antigens have demonstrated clinical efficacy. Harnessing the diverse set of mechanisms that Salmonella and Listeria use to alter the tumor-immune landscape has the potential to generate many new and effective immunotherapies.