Exquisite Tumor Targeting by Salmonella A1-R in Combination with Caffeine and Valproic Acid Regresses an Adult Pleomorphic Rhabdomyosarcoma Patient-Derived Orthotopic Xenograft Mouse Model

Adult pleomorphic rhabdomyosarcoma (RMS) is a rare and malignant mesenchymal tumor. Recently, we developed a patient-derived orthotopic xenograft (PDOX) model of adult pleomorphic RMS. In the present study, we evaluated the efficacy of tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R combined with caffeine (CAF) and valproic acid (VPA) on the adult RMS PDOX. An adult pleomorphic RMS cell line was established from the PDOX model. Cell survival after exposure to CAF and VPA was assessed, and the IC₅₀ value was calculated for each drug. The RMS PDOX models were randomized into five groups: untreated control; tumor treated with cyclophosphamide (CPA); tumor treated with CAF + VPA; tumor treated with S. typhimurium A1-R; and tumor treated with S. typhimurium A1-R + CAF + VPA. Tumor size and body weight was measured twice a week. VPA caused a concentration-dependent cytocidal effect. A synergistic effect of combination treatment with CAF was observed against the RMS cell line. For the in vivo study, all treatments significantly inhibited tumor growth compared with the untreated control. S. typhimurium A1-R combined with VPA and CAF was significantly more effective than CPA, VPA combined with CAF, or S. typhimurium A1-R alone and significantly regressed the tumor volume compared with day 0. These results suggest that S. typhimurium A1-R together with VPA and CAF could regresses an adult pleomorphic RMS in a PDOX model and therefore has important future clinical potential.     

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia)

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C. jejuni for hippuricase, GenBank: {"type":"entrez-nucleotide","attrs":{"text":"Z36940.1","term_id":"535805"}}Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.     

Mutagenicity of halogenated aliphatic hydrocarbons in Salmonella typhimurium,Streptomyces coelicolor and Aspergillus nidulans

Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyde (the last in the anhydrous and hydrate form), mono-, di- and trichloroethanol and allyl chloride, were tested for their ability to induce gene mutations in prokaryotic and eukaryotic microorganisms. The genetic systems employed were the Salmonella reversion test with strain TA1535 and TA100, with and without metabolic activation, a forward and a back-mutation system in S. coelicolor and two forward mutation systems in A. nidulans. Each compound was tested with the spot and plate incorporation assay techniques.Mono-, di- and trichloroacetaldehyde were mutagenic in all the microorganisms employed; all the halogenated ethanols were positive in A. nidulans, while in S. typhimurium and in S. coelicolor the only active forms were respectively the mono- and dichloroderivatives. Allyl chloride was active in S. typhimurium and S. coelicolor and negative in A. nidulans.The technical approach as well as the complex influence of different factors (toxicity, volatility and stability) on the genetic response of each of the compounds under test did not allow to obtain more than a qualitative relationship between mutagenic potency and chemical structure.     

Bacteriophage Typing of Salmonella. I. Isolation and Host Range Study of Bacteriophages

A series of bacteriophages, lytic for bacteria belonging to the genera Escherichia and Salmonella, were isolated. The phages were isolated from fecal samples, intestinal contents of turkey poults, and carrier cultures of S. typhimurium, S. typhimurium var copenhagen, S. heidelberg, and E. coli. The feasibility of using different habitats as sources of Salmonella phages was evaluated. The carrier cultures were the most promising source for phages active on the serotypes for which the phages were sought. A host range study of the isolated phages was made. Eight phages were selected to develop a phage typing scheme for S. typhimurium, S. typhimurium var copenhagen, and S. heidelberg.     

Measurement of drug-metabolizing systems in Salmonella typhimurium strains G46, TA1535, TA100, TA1538 and TA98

Salmonella typhimurium strains which are commonly used in the Ames test for screening potential carcinogens were examined for a number of drug-metabolizing systems. Neither cytochrome P-450 itself nor two activities catalyzed by the cytochrome P-450 system in mammalian cells, i.e., benzpyrene monooxygenase and ethoxycoumarin O-deethylation, could be detected. Nor do these bacterial strains demonstrate any ability to detoxify epoxides by hydrating them or to conjugate p-nitrophenol with glucuronic acid.On the other hand, S. typhimurium strains G46, TA1535, TA100, TA1538 and TA98 contain considerable amounts of acid-soluble thiols, approx. 5–10% of which is glutathione. These bacteria can also enzymatically conjugate glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) and can reduce oxidized glutathione using NADPH as cofactor.Thus, enzymatic and non-enzymatic reaction of immediate carcinogens with thiol groups in S. typhimurium may have a significant effect on the outcome of the Ames test in certain cases.     

Inhibition and eradication of human glioma with tumor-targeting Salmonella typhimurium in an orthotopic nude-mouse model

Malignant glioma tumors are the most common primary central nervous system tumors. Despite the multidisciplinary approach to treatment, prognosis remains poor. In this study, we demonstrated that the Salmonella typhimurium A1-R tumor-targeting strain can inhibit and eradicate human glioma in an orthotopic nude-mouse model. S. typhimurium A1-R was administered by injection through a craniotomy open-window or intravenously in nude mice. To establish the model, 2 × 10⁵ U87-RFP human glioma cells were injected stereotactically into the mouse brain through the craniotomy open window. Two weeks after glioma-cell implantation, mice were treated with S. typhimurium A1-R [2 × 10⁷ CFU/200 µl intravenous injection (i.v.) or 1 × 10⁶ CFU/1 µl intracranial injection (i.c.)] once a week for 3 weeks. Brain tumors were observed by fluorescence imaging through the craniotomy open window over time. S. typhimurium A1-R, administered i.c., inhibited brain tumor growth 7.6-fold compared with untreated mice (p = 0.009) and improved survival 73% (p = 0.001). Two of ten mice appeared to have their tumors eradicated. Intravenous administration of S. typhimurium A1-R was not effective. The craniotomy open window enabled observation of tumor growth in the brain in real time in both treated and untreated mice. The results of the present study demonstrate that bacterial therapy of brain cancer is a novel, effective and safe treatment strategy in a highly treatment-resistance cancer.Key words: Salmonella typhimurium A1-R, fluorescent proteins, brain cancer, mouse model, in vivo imaging     

Fine mechanisms of the interaction of silver nanoparticles with the cells of Salmonella typhimurium and Staphylococcus aureus

Silver nanoparticles possess antibacterial effect for various bacteria; however mechanisms of the interaction between Ag-NPs and bacterial cells remain unclear. The aim of our study was to obtain direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and Gram-positive bacterium S. aureus, and to study cell responses to Ag-NPs. The Ag-NPs (most 8–10 nm) were obtained by gas-jet method. S. typhimurium (7.81 × 10⁷ CFU), or S. aureus (8.96 × 10⁷ CFU) were treated by Ag-NPs (0.05 mg/l of silver) in orbital shaker at 190 rpm, 37 °C. Bacteria were sampled at 0.5, 1, 1.5, 2, 5 and 23 h of the incubation for transmission electron microscopy of ultrathin sections. The Ag-NPs adsorbed on outer membrane of S. typhimurium and cell wall of S. auereus; penetrated and accumulated in cells without aggregation and damaging of neighboring cytoplasm. In cells of S. aureus Ag-NPs bound with DNA fibers. Cell responses to Ag-NPs differed morphologically in S. typhimurium and S. aureus, and mainly were presented by damage of cell structures. The cytoplasm of S. aureus became amorphous, while S. typhimurium showed lumping and lysis of cytoplasm which led to formation of “empty” cells. Other difference was fast change of cell shape in S. typhimurium, and late deformation of S. aureus cells. The obtained results showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells. Evidently, Ag-NPs directly interact with macromolecular structures of living cells and are exert an active influence on their metabolism.     

A rapid multiplex DNA suspension array method for Salmonella typhimurium subtyping using prophage-related markers

In this study we developed a preliminary proof of concept of method for Salmonella typhimurium subtyping using multiplex PCR-based phage locus typing and a multiplex Luminex DNA suspension array for product detection. Thirty markers were selected from prophages ST64B, ST64T, ST104, P22, Gifsy-1, sopEΦ and mostly phage-related AFLP fragments, and organised into two multiplex PCRs of 15 markers each. A two-group DNA suspension array was developed using a combination of flow cytometry and Luminex xMAP® technology. To assess its subtyping capability the method was applied to 438 non-epidemiological related S. typhimurium isolates of 56 phage types. Eighty-one profiles were generated. Isolates were divided into sixteen main prophage marker profile types. There was a strong tendency for isolates with the same phage type to have the same or closely related profiles and for groups of phage types to share the same profile. The discriminatory power of this method expressed as the Simpson's Index of Diversity (D) was 0.954. A panel of 12 selected markers achieved almost the same D value (0.952) as the 30 markers. This new method provides an alternative typing scheme for S. typhimurium epidemiological investigations. The developed array is in a high-throughput format which could easily be semi-automated, making the test fast and economical.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification

The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10⁻². Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi⁻ R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes.     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well-tolerated by the immunocompetent host. Weekly intravenous injection with 2 × 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intrathoracic administration was performed with 10⁸ A1-R bacteria injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.Key words: Salmonella typhimurium, amino acid auxotroph, selective tumor targeting, lung, metastasis, RFP, GFP, fluorescence imaging, confocal microscopy     

A study in evolution: the histidine utilization genes of enteric bacteria.

The histidine utilization (hut) system, MIGCPUH, comprises two operons, whose promoters are M and P. The DNA segments of phage λ vectors coding for the hut genes of Salmonella typhimurium and Klebsiella aerogenes were compared by using electron microscopy to study the nature of the hut DNA homology. The hut S. typhimurium/hut K. aerogenes DNA heteroduplexes were spread and mounted for electron microscopy under more and more denaturing conditions. The homology between hut S. typhimurium and hut K. aerogenes is extensive but, as denaturing conditions make base-pairing more difficult, the regions of lower homology are melted, while the regions of higher homology remain base-paired. The denaturation map could be correlated with the genetic map by the use of λhut S. typhimurium phages carrying different portions of the hut genome. The sequences with the highest homology are in the structural genes for the four enzymes, while the regulatory regions, the promoters and the hut repressor gene, are much less homologous.     

Salmonella typhimurium A1-R targeting of a chemotherapy-resistant BRAF-V600E melanoma in a patient-derived orthotopic xenograft (PDOX) model is enhanced in combination with either vemurafenib or temozolomide

A metastatic melanoma obtained from the right chest wall of a patient was previously established orthotopically in the right chest wall of nude mice as a patient-derived orthotopic xenograft (PDOX) model. We previously showed that the combination of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and chemotherapy was highly effective against the melanoma PDOX. In the present study, we investigated the mechanism of the high efficacy of this combination. Two weeks after implantation, 40 PDOX mouse models were randomized into 4 groups of 10 mice each: untreated control (n = 10); treated with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with temozolomide (TEM) (25 mg/kg, p.o. for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with vemurafenib (VEM) (30 mg/kg, p.o., for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks) (n = 10). On day 14 from initiation, all treatments significantly inhibited tumor growth compared with untreated control (S. typhimurium A1-R: p < 0.01; TEM combined with S. typhimurium A1-R: p < 0.01; VEM combined with S. typhimurium A1-R: p < 0.01). Combination therapy with S. typhimurium A1-R was significantly more effective on tumor growth than S. typhimurium A1-R alone (with TEM: p < 0.01; with VEM: p < 0.01). Combination therapy significantly increased S. typhimurium A1-R tumor targeting alone (S. typhimurium A1-R + TEM: p < 0.01, S. typhimurium A1-R + VEM: p < 0.01), relative to S. typhimurium A1-R alone, respectively. In conclusion, chemotherapy drugs promoted targeting of S. typhimurium A1-R of melanoma, thereby enhancing efficacy against the melanoma PDOX.     

Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface

Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.     

Macrophages recognize and adhere to an OmpD-like protein of Salmonella typhimurium

Murine peritoneal macrophages bind to Salmonella typhimurium in vitro in the absence of exogenous opsonins. We have identified an outer membrane protein of S. typhimurium that mediates this adhesion. Biotin-labeled macrophages were used to probe electroblotted envelope proteins of S. typhimurium that had been previously resolved by polyacrylamide electrophoresis under denaturing and reducing conditions. Macrophages bound to an outer membrane protein with an apparent molecular mass of 44 kDa. The protein was purified to homogeneity and free of detectable lipopolysaccharide. Limited microsequencing of this protein resulted in a 15-amino acid query sequence of A-E-V-Y-N-K-D-G-N-K-L-D-L-Y-G, which shares complete identity with a 15-mer of both the OmpD of S. typhimurium SH 7454 and the OmpC polypeptide of Escherichia coli K-12. Picomolar concentrations of this purified protein significantly inhibited the subsequent adherence of ³⁵S-labeled S. typhimurium to macrophages in monolayers. We propose that this 44-kDa protein is involved in the recognition of S. typhimurium by macrophage during the initial stages of infection.     

Gene Transfer between Related Bacteria by Electrotransformation: Mapping Salmonella typhi Genes in Salmonella typhimurium

Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction. Gene transfer is hampered in Salmonella typhi and in other pathogenic bacteria by the lack of a generalized transduction system. We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering S. typhi DNA into suitable S. typhi or Salmonella typhimurium recipients. Transferred DNA can recombine with the homologous region in the host chromosome. In one application of the method, mutations isolated in S. typhi were genetically mapped in S. typhimurium.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

The araC gene of Citrobacter freundii

The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.     

Interactions Between Macrophages of Guinea Pigs and Salmonellae I. Fate of Salmonella typhimurium Within Macrophages of Normal Guinea Pigs

A suspended cell culture procedure was described for the cultivation of guinea pig macrophages infected with Salmonella typhimurium. The fate of the intracellular bacteria was assessed by quantitative recovery of viable bacteria with 0.5% solution of sodium desoxycholate. Two strains of S. typhimurium with different degrees of virulence for mice were compared. There was an initial destruction of intracellular bacteria of both strains; however, the extent of this destruction differed. Approximately 1% of the avirulent bacteria initially phagocytized survived at the end of 4 hr, whereas approximately 8% of the virulent bacteria survived at the end of 3 hr. After this initial killing, the intracellular bacteria began to multiply at a logarithmic rate between 3 and 21 hr after phagocytosis, and then a stationary phase was attained. The rate of this multiplication was comparable for both strains.