Potential of 2, 2'-dipyridyl diselane as an adjunct to antibiotics to manage cadmium-induced antibiotic resistance in Salmonella enterica serovar Typhi Ty2 strain.

One of the reasons for increased antibiotic resistance in Salmonella enterica serovar Typhi Ty2 is the influx of heavy metal ions in the sewage, from where the infection is transmitted. Therefore, curbing these selective agents could be one of the strategies to manage the emergence of multidrug resistance in the pathogen. As observed in our earlier study, the present study also confirmed the links between cadmium accumulation and antibiotic resistance in Salmonella. Therefore, the potential of a chemically-synthesised compound 2, 2'-dipyridyl diselane (DPDS) was explored to combat the metal-induced antibiotic resistance. Its metal chelating and antimicrobial properties were evidenced by fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), and microbroth dilution method. Owing to these properties of DPDS, further, this compound was evaluated for its potential to be used in combination with conventional antibiotics. The data revealed effective synergism at much lower concentrations of both the agents. Thus, it is indicated from the study that the combination of these two agents at their lower effective doses might reduce the chances of emergence of antibiotic resistance, which can be ascribed to the multi-pronged action of the agents.

A novel method to generate Salmonella Typhi Ty21a ghosts exploiting the λ phage holin-endolysin system.

Human typhoid fever caused by Salmonella Typhi still poses a severe global disease burden in developing countries despite the availability of commercial vaccines. In this study, we constructed a non-living S. Typhi Ty21a vaccine candidate by employing a lambda (λ) phage-derived holin-endolysin system to efficiently construct bacterial ghosts. The lysis plasmid pJHL464 harbors an R lysis cassette that is stringently regulated by dual promoters containing cI857/λPR and ParaBAD/araC components. The plasmid was introduced into an asd gene-deleted S. Typhi Ty21a strain designated JOL1675. The in vitro expression of endolysin (~17.76 kDa) in the subsequent JOL1675 vaccine construct when grown under lysis inducible conditions was validated by immunoblotting. In scanning electron microscopy analysis, surface transmembrane tunnels and a collapsed body were visualized in the ghosts. Following 48 h of lysis, no viable JOL1675 cells remained, indicating that lysis of all cells was achieved. Subcutaneous immunizations of mice with the JOL1675 ghosts produced significantly increasing titers of serum IgG and vaginal wash secretory IgA antibodies against JOL1675 outer membrane proteins during the observational period. Further, serum collected at 6 weeks post-immunization of rabbits exhibited effective bactericidal activity against wild type S. Typhi in the presence of complement. These data showed that JOL1675 ghosts are highly immunogenic and elicit humoral and mucosal responses expected to correlate with protective immunity against S. typhi. Collectively, our findings support the conclusion that incorporating a λ phage holin-endolysin-mediated lysis construct into S. Typhi is an efficient strategy for developing a novel and safe non-living typhoid vaccine candidate.

Inhibition of Salmonella typhi growth using extremely low frequency electromagnetic (ELF-EM) waves at resonance frequency.

AIMS: Typhoid is a serious disease difficult to be treated with conventional drugs. The aim of this study was to demonstrate a new method for the control of Salmonella typhi growth, through the interference with the bioelectric signals generated from the microbe during cell division by extremely low frequency electromagnetic waves (ELF-EMW-ELF-EM) at resonance frequency. METHODS AND RESULTS: Isolated Salmonella typhi was subjected to square amplitude modulated waves (QAMW) with different modulation frequencies from two generators with constant carrier frequency of 10 MHz, amplitude of 10 Vpp, modulating depth ± 2 Vpp and constant field strength of 200 V m(-1) at 37°C. Both the control and exposed samples were incubated at the same conditions during the experiment. The results showed that there was highly significant inhibition effect for Salm. typhi exposed to 0·8 Hz QAMW for a single exposure for 75 min. Dielectric relaxation, TEM and DNA results indicated highly significant changes in the molecular structure of the DNA and cellular membrane resulting from the exposure to the inhibiting EM waves. CONCLUSIONS: It was concluded that finding out the inhibiting resonance frequency of ELF-EM waves that deteriorates Salm. typhi growth will be promising method for the treatment of Salm. typhi infection either in vivo or in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This new non-invasive technique for treatment of bacterial infections is of considerable interest for the use in medical and biotechnological applications.

A Salmonella enterica serovar Typhi plasmid induces rapid and massive apoptosis in infected macrophages.

pR(ST98) is a chimeric plasmid isolated from Salmonella enterica serovar Typhi (S. typhi) that mediates the functions of drug resistance and virulence. Previously, we reported that Salmonella plasmid virulence (spv) genes were present in S. typhi. In our current study, we investigated whether plasmid pR(ST98) exhibits significant cytotoxicity in macrophages. pR(ST98) was transferred into the avirulent Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create the transconjugant pR(ST98)/RIA. The standard S. typhimurium virulent strain SR-11, which carries a 100-kb virulence plasmid, was used as a positive control. The bacterial strains were incubated with a murine macrophage-like cell line (J774A.1) in vitro. Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling, and the survival of Salmonella strains in J774A.1 cells was determined. Results showed that macrophages infected with strain pR(ST98)/RIA displayed greater levels of apoptosis than those infected with RIA and that pR(ST98 )may increase bacterial survival in macrophages. Further studies showed that the pR(ST98)-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pR(ST98 )may activate caspase-9 and then caspase-3. The research data indicate that the virulence of bacteria that contain the pR(ST98) plasmid is enhanced; the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.

The Salmonella enterica serovar Typhi type IVB self-association pili are detached from the bacterial cell by the PilV minor pilus proteins.

Salmonella enterica serovar Typhi and some strains (Vi+) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have shown previously that the extent of DNA supercoiling controls the rate of Rci-catalyzed inversion of a DNA fragment which includes the C-terminal portions of the PilV proteins. This inversion therefore controls PilV synthesis as a high inversion rate prohibits transcription of pilV-encoding DNA. Here, we describe the manner in which PilV protein expression inhibits bacterial self-association and present data which suggest that incorporation of one or a few PilV protein molecules into a growing pilus, comprised of PilS subunits, causes the pilus to detach at the bacterial membrane. The bacteria are then unable to self-associate. We suggest that this phenomenon may be relevant to the pathogenesis of typhoid fever.

Reacción de Widal: interpretación clínica / Widal test: clinical interpretation

La reacción de Widal es un test basado en el principio de aglutinación antígeno-anticuerpo, donde se determina la presencia de anticuerpos contra el antígeno O y H de la Salmonella typhi para el serodiagnóstico de fiebre tifoidea, sin embargo debido a su falta de especificidad, debe ser interpretado en el contexto clínico del paciente. Para considerar el diagnóstico de fiebre tifoidea con un titulo Anti-O y Anti-H aislado, se debe conocer su prevalencia en una determinada comunidad, en términos generales, se acepta títulos anti-O y anti-H > o = 1: 160-200 y > o = 1: 50-100 en zonas endémicas y no endémicas, respectivamente.

Complete amino acid sequence and location of Omp-28, an important immunogenic protein from Salmonella enterica serovar typhi.

Omp-28 isolated from Salmonella enterica serovar typhi presented a subunit molecular mass of 9,632 Da by MALDI-TOF MS. It was denatured, S-alkylated, and 1) directly submitted to Edman sequencing, 2) cleaved with CNBr, and 3) hydrolyzed either with endoproteinase Glu-C or Asp-N. The major CNBr peptide containing the C-terminal portion of Omp-28 was isolated by tricine-SDS-PAGE and electroblotted whereas Omp-28 enzymatic peptides were isolated by C18-RP-HPLC. All peptides were sequenced. This approach allowed the elucidation of the complete primary structure of Omp-28. Its amino acid sequence is identical to that deduced from part of the DNA of the "putative periplasmic transport protein" of either S. enterica serovar typhimurium and a multiple drug resistant S. enterica serovar typhi. Omp-28 homologous protein sequences were also deduced from Escherichia coli and Yersinia pestis genomic DNA. All proteins had their secondary structures predicted. Immunogold cytochemistry indicated that Omp-28 is found on the bacterium outer membrane.

Salmonella typhi uses CFTR to enter intestinal epithelial cells.

Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (delta508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous deltaF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in deltaF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in deltaF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.

Physical limitations on Salmonella typhi entry into cultured human intestinal epithelial cells.

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >/=40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of approximately 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta1 integrin superfamily.

Purification and characterization of type 1 fimbriae of Salmonella typhi.

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose-inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS-PAGE analysis of the fimbrial preparation showed the presence of a 20-kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino-terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti-fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.

Mucin allows survival of Salmonella typhi within mouse peritoneal macrophages.

Salmonella typhi is a facultative intracellular human specific pathogen. Both immunocompetent and immunodeficient mice are resistant to S. typhi. However, when they are infected with S. typhi suspended in mucin, the bacteria become pathogenic and infect peritoneal phagocytic cells. The LD50 for mice was 10(5) bacteria suspended in 5% mucin; mouse survival was approximately 48 hours after injection. A high number of bacteria was recovered from peritoneal cells; transmission electron microscopy disclosed a large number of vesicles filled with S. typhi cells in peritoneal cells from infected animals. The addition of mucin to cultures of the reticuloendothelial cell line J774.3 also allowed invasion of the mammalian cells with S. typhi. These data indicate that mucin allows intracellular survival of S. typhi.

Mucin allows survival of Salmonella typhi within mouse peritoneal macrophages

Salmonella typhi is a facultative intracellular human specific pathogen. Both immunocompetent and immunodeficient mice are resistant to S. typhi. However, when they are infected with S. typhi suspended in mucin, the bacteria become pathogenic and infect peritoneal phagocytic cells. The LD50 for mice was 10(5) bacteria suspended in 5 percent mucin; mouse survival was approximately 48 hours after injection. A high number of bacteria was recovered from peritoneal cells; transmission electron microscopy disclosed a large number of vesicles filled with S. typhi cells in peritoneal cells from infected animals. The addition of mucin to cultures of the reticuloendothelial cell line J774.3 also allowed invasion of the mammalian cells with S. typhi. These data indicate that mucin allows intracellular survival of S. typhi

Cell wall structures which may be important for successful immunization with Salmonella-Shigella hybrid vaccines.

Three separate lots of S. typhi/S. sonnei hybrid (Ty/Shig) live oral vaccine strain 5076-1C were tested for efficacy in human volunteers challenged with virulent S. sonnei. Two lots (2 and 5) protected volunteers, a third lot (8) did not. The three lots were evaluated by immunological tests and electron microscopy. Lots 2 and 5, which protected, contained bacteria that reacted with anti-flagellar serum and had observable attached flagella and pili. Lot 8, which failed to protect, did not react with anti-flagellar serum, and had no observable pili. There was no correlation between vaccine efficacy and the reaction of IgG in patient's sera in western blot analysis. Surface structures on the Ty-Shig hybrid may be important for generating a protective immune response.

Surface co-expression of Vibrio cholerae and Salmonella typhi O-antigens on Ty21a clone EX210.

In an attempt to construct a bivalent, live, oral cholera-typhoid vaccine, genes specifying the biosynthesis of Vibrio cholerae O-antigen have been transferred into a modified version of the attenuated, oral typhoid vaccine strain Salmonella typhi Ty21a. The present report investigates the production of V. cholerae and S. typhi O-antigens by one such clone, EX210. When cultured without galactose supplementation EX210 produces surface O-antigen of V. cholerae type, as detected by haemagglutination-inhibition and bactericidal assays, and by immuno-electron microscopy. However, the protective efficacy of Ty21a depends upon growth in the presence of exogenous galactose and under these conditions only S. typhi O-antigen is detectable on the surface of EX210. Subsequent experiments revealed that the proportion of polysaccharide of S. typhi type is dependent upon the level of galactose supplementation, and identified a limiting sugar concentration which results in surface co-expression of both O-antigens. Visualization of the two polysaccharides on silver-stained polyacrylamide gels indicates that S. typhi O-antigen subunits are polymerized into longer sidechains, suggesting that at higher galactose concentrations their predominance results in a masking of the shorter V. cholerae O-antigen.

[The antigen H:z66 in 1,000 strains of Salmonella typhi from the Antilles, Central America and South America]. / Recherche de l'antigène H:z66 chez 1,000 souches de Salmonella typhi provenant des Antilles, d'Amérique Centrale et d'Amérique du Sud.

The research of the flagellar antigen H:z66 of Salmonella typhi was performed in 1,000 strains from the West Indies, Central America and South America. A method based on the immobilization of motile strains in soft agar with immune serum anti-H: d was used to detect strains carrying this flagellar antigen. No strains had antigen H:z66 irrespective to their biovar, phage-type, colicinogeny, drug susceptibility and geographical origin. Future investigations are needed for a better knowledge of the worldwide distribution of Salmonella typhi strains carrying antigen H:z66.

[Ultrastructural study of intestinal bacteria grown on Endo- and Levine-type nutrient media based on nonfood raw materials]. / Izuchenie ul'trastruktury nekotorykh kishechnykh bakterii, vyrashchennykh na pitatel'nykh sredakh tipa Endo i Levina s osnovoi na nepishchevogo syr'ia.

The comparative electron-microscopic study of several test strains (Salmonella typhi H-901, Shigella flexneri la 8516, and Escherichia coli 055) grown in experimental Endo and Levine media prepared on the basis of raw materials unsuitable for human consumption and in commonly used similar media prepared on the basis of sprat hydrolysate has shown the test strains grown in media containing aminopeptide and fodder yeast hydrolysate to retain their typical ultrastructure, which confirms the possibility of using these protein bases for the preparation of Endo and Levine media.

[Ultrastructural aspects of the process of Salmonella typhi bacterial interaction with L929-line cells]. / Ul'trastrukturnye aspekty protsessa vzaimodeistviia briushnotifoznykh bakterii s kletkami linii L929.

The study of the use of scanning electron microscopy and the analysis of the initial stages of interaction between S. typhi and eukaryotic cells by the method of three-dimensional reconstruction has revealed that the infective agent penetrates into the cytoplasm on the principle of internalization. The internalization of S. typhi occurs with the active participation of the eukaryotic cells which, at the beginning, envelopes the bacteria with its processes, and the infective agents firmly adhere to the glycocalyx of the host cell by means of special fimbria-like formations differing from fimbriae by their lesser rigidity and thickness; then the microbes fixed to the membrane penetrate inside the cell without destroying its cytoplasmic membrane. Differences in the processes of the interaction of eukaryotic cells with S. typhi initial strain 238 and its variant free from the plasmid with a molecular weight of 6 Md, characterized by its lower capacity for association with cells of continuous cell culture L929, have been revealed. The factors stimulating the ingestion of S. typhi by eukaryotic cells are under study at present.

Cytopathogenic effect of Salmonella typhi GIFU 10007 on M cells of murine ileal Peyer's patches in ligated ileal loops: an ultrastructural study.

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice, cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.