Antitumour treatments are evolving, including bacteria-mediated cancer therapy which is concurrently an ancient and cutting-edge approach. Salmonella typhimurium is a widely studied bacterial species that colonizes tumor tissues, showing oncolytic and immune system-regulating properties. It can be used as a delivery vector for genes and drugs, supporting conventional treatments that lack tumor-targeting abilities. This article summarizes recent evidence on the anticancer mechanisms of S. typhimurium alone and in combination with other anticancer treatments, suggesting that it may be a suitable approach to disease management.

E. coli mediated gene delivery faces a major drawback of low efficiency despite of being a safer alternative to viral vectors. This study showed a novel, simple and effective strategy to enhance invasive E. coli DH10B vector\'s efficiency in human epithelial cells. The bactofection efficiency of invasive E .coli vector was analyzed in nine cell lines. It demonstrated highest (16%) reporter gene (GFP) expression in cervical cells. Methods were employed to further enhance its efficiency by adding transfection reagents (trans-bactofection method) to promote entry into host cells, lysosomotropic reagents for escape from lysosomal degradation or antibiotics to lyse internalized bacteria. Increased bacterial entry, as elucidated from nil to 3% expression in liver cells, was obtained upon complexing bacteria with PULSin. Chloroquine mediated endosomal escape resulted in 7.2 folds increase whereas tetracycline addition to lyse internalized bacteria caused ≈90% of GFP in HeLa. Eventually, the combined effect of these three methods exhibited close to 100% GFP in cervical and remarkable increase of 138 folds in breast cells. This is the first study showing comparative study of vector\'s gene delivery ability in various epithelial cells of the human body with improving its delivery efficiency. These data demonstrated the potential of developed bactofection method to boost up the efficiency of other bacterial vectors also, which could further be used for effectual therapeutic gene delivery in human cells.

Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting β1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.

Bactofection, a bacterial-mediated form of genetic transfer, is highlighted as an alternative mechanism for gene therapy. A key advantage of this system for immune-reactivity purposes stems from the nature of the bacterial host capable of initiating an immune response by attracting recognition and cellular uptake by antigen-presenting cells (APCs). The approach is also a suitable technique to deliver larger genetic constructs more efficiently as it can transfer plasmids of varying sizes into target mammalian cells. Given these advantages, bacterial vectors are being studied as potential carriers for the delivery of plasmid DNA into target cells to enable expression of heterologous proteins. The bacteria used for bactofection are generally nonpathogenic; however, concerns arise due to the use of a biological agent. To overcome such concerns, enhanced bacterial degradation has been engineered as an attenuation and safety feature for bactofection vectors. In particular, the ΦX174 lysis E (LyE) gene can be repurposed to both minimize bacterial survival within mammalian hosts while also improving overall gene delivery. More specifically, an engineered bacterial vector carrying the LyE gene showed improved gene delivery and safety profiles when tested with murine RAW264.7 macrophage APCs. This chapter outlines steps taken to engineer E. coli for LyE expression as a safer and more effective genetic antigen delivery bactofection vehicle in the context of vaccine utility.

Use of bacteria in cancer therapy, despite being considered as a potent strategy, has not really picked up the way other methods of cancer therapies have evolved. However, in recent years, the interest on use of bacteria to kill cancer cells has renewed considerably. The standard and widely followed strategies of cancer treatment often fail either due to the complexity of tumour biology or because of the accompanying side effects. In contrast, these limitations can be easily overcome in a bacteria-mediated approach. Salmonella is a bacterium, which is known for its ability to colonize solid or semisolid tumours more efficiently than any other bacteria. Among more than 2500 serovars of Salmonella, S. Typhimurium has been widely studied for its antagonistic effects on cancer cells. Here in, we review the current status of the preclinical and the clinical studies with a focus on the mechanisms that attribute the anticancer properties to nontyphoidal Salmonella.

Spontaneous tumors regression has been associated with microbial infection for 100s of years and inspired the use of bacteria for anticancer therapy. Dr. William B. Coley (1862-1936), a bone- sarcoma surgeon, was a pioneer in treating his patients with both live bacterial-based and mixture of heat-killed bacteria known as \"Coley\'s toxins.\" Unfortunately, Coley was forced to stop his work which interrupted this field for about half a century. Currently, several species of bacteria are being developed against cancer. The bacterial species, their genetic background and their infectious behavior within the tumor microenvironment are thought to be relevant factors in determining their anti-tumor effectiveness in vivo. In this perspective article we will update the most promising results achieved using bacterial therapy (alone or combined with other strategies) in clinically-relevant animal models of cancer and critically discuss the impact of the bacterial variants, route of administration and mechanisms of bacteria-cancer-cell interaction. We will also discuss strategies to apply this information using modern mouse models, molecular biology, genetics and imaging for future bacterial therapy of cancer patients.

BACKGROUND: Tumor cell resistance to chemotherapy agents is one of the main problems in the eradication of different neoplasias. One of the mechanisms of this process is the overexpression of anti-apoptotic proteins such as Bcl-2 and Bcl-XL; blocking the activity of these proteins may contribute to the sensitization of tumor cells and allow the adequate effects of chemotherapeutic drugs.
RESULTS: This study adressed the transfection of prostate cancer cells (PC3) with a plasmid encoding a recombinant protein with an antagonist peptide from the BH3 region of the Bax protein fused to the GFP reporter protein (BaxGFP). This protein induced apoptosis of these tumor cells; further, selective transport of this plasmid to the tumor cell with Salmonella enterica serovar Typhimurium (strain SL3261), a live-attenuated bacterial vector, can induce sensitization of the tumor cell to the action of drugs such as cisplatin, through a process known as bactofection.
CONCLUSIONS: These results suggest that Salmonella enterica can be used as a carrier vector of nucleotide sequences encoding heterologous molecules used in antitumor therapy.

This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 μg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.

BACKGROUND: Pasteurella multocida B:2 causes bovine haemorrhagic septicaemia (HS), leading to rapid fatalities in cattle and buffaloes. An attenuated derivative of P. multocida B:2 GDH7, was previously constructed through mutation of the gdhA gene and proved to be an effective live attenuated vaccine for HS. Currently, only two potential live attenuated vaccine candidates for HS are being reported; P. multocida B:2 GDH7 and P. multocida B:2 JRMT12. This study primarily aims to investigate the potential of P. multocida B:2 GDH7 strain as a delivery vehicle for DNA vaccine for future multivalent applications.
RESULTS: An investigation on the adherence, invasion and intracellular survival of bacterial strains within the bovine aortic endothelial cell line (BAEC) were carried out. The potential vaccine strain, P. multocida B:2 GDH7, was significantly better (p ≤ 0.05) at adhering to and invading BAEC compared to its parent strain and to P. multocida B:2 JRMT12 and survived intracellularly 7 h post treatment, with a steady decline over time. A dual reporter plasmid, pSRGM, which enabled tracking of bacterial movement from the extracellular environment into the intracellular compartment of the mammalian cells, was subsequently transformed into P. multocida B:2 GDH7. Intracellular trafficking of the vaccine strain, P. multocida B:2 GDH7 was subsequently visualized by tracking the reporter proteins via confocal laser scanning microscopy (CLSM).
CONCLUSIONS: The ability of P. multocida B:2 GDH7 to model bactofection represents a possibility for this vaccine strain to be used as a delivery vehicle for DNA vaccine for future multivalent protection in cattle and buffaloes.

The use of bacterial cells as agents of medical therapy has a long history. Research that was ignited over a century ago with the accidental infection of cancer patients has matured into a platform technology that offers the promise of opening up new potential frontiers in medical treatment. Bacterial cells exhibit unique characteristics that make them well-suited as smart drug delivery agents. Our ability to genetically manipulate the molecular machinery of these cells enables the customization of their therapeutic action as well as its precise tuning and spatio-temporal control, allowing for the design of unique, complex therapeutic functions, unmatched by current drug delivery systems. Early results have been promising, but there are still many important challenges that must be addressed. We present a review of promises and challenges of employing bioengineered bacteria in drug delivery systems and introduce the biohybrid design concept as a new additional paradigm in bacteria-based drug delivery.