Evaluation of Trimethoprim-sulphamethoxazole Compound in Treatment of Salmonella Infections

Fifty patients suffering from infections caused by various salmonella species were treated with trimethoprim-sulphamethoxazole compound. Twenty-three had enteric fever and two were biliary carriers of Salmonella typhi. The other 25 suffered from infections caused by salmonella species other than S. typhi or S. paratyphi B. Twenty-one of the patients with enteric fever responded clinically to the drug, one failed treatment, and one died. Two patients suffering from typhoid fever relapsed and three temporarily excreted S. typhi in stools following treatment. One of the typhoid carriers was successfully treated. All patients with infections caused by salmonella species other than S. typhi or S. paratyphi B responded to treatment but 17 continued to excrete the organism in their stools after the course of trimethoprim-sulphamethoxazole compound. Four patients developed rashes during therapy and two became anaemic.     

High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata

Background

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem especially in developing countries. Vaccines against typhoid are commonly used by travelers but less so by residents of endemic areas.

Methodology

We used single nucleotide polymorphism (SNP) typing to investigate the population structure of 372 S. Typhi isolated during a typhoid disease burden study and Vi vaccine trial in Kolkata, India. Approximately sixty thousand people were enrolled for fever surveillance for 19 months prior to, and 24 months following, Vi vaccination of one third of the study population (May 2003–December 2006, vaccinations given December 2004).

Principal Findings

A diverse S. Typhi population was detected, including 21 haplotypes. The most common were of the H58 haplogroup (69%), which included all multidrug resistant isolates (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole). Quinolone resistance was particularly high among H58-G isolates (97% Nalidixic acid resistant, 30% with reduced susceptibility to ciprofloxacin). Multiple typhoid fever episodes were detected in 22 households, however household clustering was not associated with specific S. Typhi haplotypes.

Conclusions

Typhoid fever in Kolkata is caused by a diverse population of S. Typhi, however H58 haplotypes dominate and are associated with multidrug and quinolone resistance. Vi vaccination did not obviously impact on the haplotype population structure of the S. Typhi circulating during the study period.     

Virulence and vaccine potential of Salmonella typhlmurium mutants deficient in the expression of the RpoS (σs) regulon

The alternative sigma factor RpoS (σ^s) is required for Salmonella virulence in mice. We report the immunizing capacity of Salmonella typhimurlum rpoS and rpoS aroA mutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S. typhimurium. When administered orally or intraperitoneally, rpoS derivatives of the mouse-virulent S. typhimurium strains, C52 and SL1344, were highly attenuated and were efficient single-dose live vaccines. rpoS aroA mutants were more attenuated than corresponding single aroA or rpoS mutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis. Salmonella rpoS and rpoS aroA mutants therefore deserve serious consideration for rational vaccine design. Consistent with this, Salmonella typhi Ty2, a ‘wild-type’ strain used widely for the development of human live-vaccine candidates against typhoid fever, was shown to be defective for rpoS. In addition, our results demonstrate that rpoS not only controls the growth and persistence of S. typhimurium in deep lymphoid organs, but also plays a role during the initial stages of oral infection.     

Characteristics of lipopolysaccharides ofSalmonella typhi isolated from carriers and patients suffering from typhoid fever

Lipopolysaccharides (LPS) ofSalmonella typhi strains, isolated from carriers and patients suffering from typhoid fever, were characterised according to their biochemical properties, morphological structure and degree of aggregation of complexes. All preparations of LPS, regardless of their origin, were morphologically heterogeneous. Free electrophoresis and immunoelectrophoresis demonstrated that LPS preparations were composed of components possessing different mobilities in electric fields. LPS of bacterial strains isolated from both carriers and patients, split upon reaction in immunoelectrophoresis with specific antiserum 73, rabbit antiserum toSalmonella typhi Vi Bhatnagar and 0–901 split into anodic and cathodio fractions. The anodic fraction reacted similarly as Vi antigen. LPS fromSalmonella typhi Ty-2 yielded only the cathodic fraction, typical for O antigen. LPS from strains whioh were passaged twice in nutritional medium possessed identical properties as LPS from fresh cultures ofSalmonella typhi. Electron microscopy revealed that LPS appears as long bands, rods, ellipsoid forms and amorphous material. Contrary to amorphous material, the bands, rods and ellipsoid forms possessed three-layer structure.     

Identification of novel vaccine candidate against Salmonella enterica serovar Typhi by reverse vaccinology method and evaluation of its immunization

Salmonella enterica serovar Typhi (S. Typhi) is an essential enteric fever causing bacterium worldwide. Due to the emergence of multidrug-resistant strains, urgently attention is needed to prevent the global spread of them. Vaccination is an alternative approach to control these kinds of infections. Currently available commercial vaccines have significant limitations such as non-recommendation for children below six years of age and poor long-term efficacy. Thus, the development of a new vaccine overcoming these limitations is immediately required. Reverse Vaccinology (RV) is one of the most robust approaches for direct screening of genome sequence assemblies to identify new protein-based vaccines. The present study aimed to identify potential vaccine candidates against S. Typhi by using the RV approach. Immunogenicity of the best candidate against S. Typhi was further investigated. The proteome of S. Typhi strain Ty2 was analyzed to identify the most immunogenic, conserved, and protective surface proteins. Among the predicted vaccine candidates, steD (fimbrial subunit) was the best for qualifying all the applied criteria. The synthetic steD gene was expressed in E.coli, and the mice were immunized with purified recombinant steD protein and then challenged with a lethal dose of S. Typhi. Immunized animals generated high protein-specific antibody titers and demonstrated 70% survival following lethal dose challenge with S. Typhi. Pretreatment of the S. Typhi cells with immunized mice antisera significantly decreased their adhesion to Caco-2 cells. Altogether, steD as a protective antigen could induce a robust and long term and protective immunity in immunized mice against S. Typhi challenge.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

The ompB Operon Partially Determines Differential Expression of OmpC in Salmonella typhi and Escherichia coli

Expression of the Escherichia coli OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in Salmonella typhi is not influenced by osmolarity, while OmpF is regulated as in E. coli. To better understand the lack of osmoregulation of OmpC expression in S. typhi, we compared the expression of the ompC gene in S. typhi and E. coli, using ompC-lacZ fusions and outer membrane protein (OMP) electrophoretic profiles. S. typhi ompC expression levels in S. typhi were similar at low and high osmolarity along the growth curve, whereas osmoregulation of E. coli ompC in E. coli was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in S. typhi, while expression of both was regulated by osmolarity in E. coli. Complementation experiments with either the S. typhi or E. coli ompB operon in an S. typhi ΔompB strain carrying the ompC-lacZ fusions showed that both S. typhi and E. coli ompC were not regulated by osmolarity when they were under the control of S. typhi ompB. Interestingly, in the same strain, both genes were osmoregulated under E. coli ompB. Surprisingly, in E. coli ΔompB, they were both osmoregulated under S. typhi or E. coli ompB. Thus, the lack of osmoregulation of OmpC expression in S. typhi is determined in part by the ompB operon, as well as by other unknown trans-acting elements present in S. typhi.     

Surveillance for typhoid fever in Matsuyama city during 1974-1981 and detection of Salmonella typhi in sewage and river waters

Eighty-two cases of typhoid fever were found in Matsuyama city in the period from 1974 to 1981. Seventy-six cases were found to be infected with Salmonella typhi other three with Salmonella paratyphi A, and the remaining three were diagnosed only clinically. The strains of S. typhi isolated from these patients showed such a variety of Vi-phage types as D1, D2, E1, M1, 53 and degraded Vi-positive strain (DVS). The concurrent survey of the city sewage and river waters for typhoid bacilli was conducted with total 578 samples taken therefrom. S. typhi was isolated from 120 of those samples. The Vi-phage types of the isolates were closely related with those of the isolates from the patients. The periodical examinations of the city sewage and the draining river may serve as a useful means for the controlling typhoid fever epidemics.     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

Molecular cloning of a Salmonella typhi LT-like enterotoxin gene

Diarrhoea is a common event during typhoid fever; nevertheless, the possible participation of a diarrhoea-inducing enterotoxin has not been described (Roy et al., 1985). Recombinant bacteriophage lambda FDC1 was isolated from a genomic library of Salmonella typhi, the causal agent of typhoid fever, by screening with a probe for the B subunit gene of the heat-labile, cholera-like, Escherichia coli enterotoxin (LT). Lambda FDC1 codes for an enterotoxin that causes secretion in rat ileal loops, that elongates Chinese hamster ovary (CHO) cells, that is recognized by antibodies against LT, and does not bind in vitro to ganglioside GM1. These results should allow further studies towards elucidating a possible role for the S. typhi enterotoxin in the pathogenesis of typhoid fever.     

Susceptibility to typhoid fever is associated with a polymorphism in the cystic fibrosis transmembrane conductance regulator (CFTR)

The cystic fibrosis transmembrane conductance regulator (CFTR) is the affected protein in cystic fibrosis (CF). The high rate of CF carriers has led to speculation that there must be, similar to the sickle cell haemoglobin advantage in malaria, a selective advantage for heterozygotes. Such a selective advantage may be conferred through reduced attachment of Salmonella typhi to intestinal mucosa, thus providing resistance to typhoid fever. We tested this hypothesis by genotyping patients and controls in a typhoid endemic area in Indonesia for two highly polymorphic markers in CFTR and the most common CF mutation. We found an association between genotypes in CFTR and susceptibility to typhoid fever (OR=2.6). These analyses suggest that the role CFTR plays in vitro in S. typhi infection is also important for infection in the human population.Electronic Supplementary Material Supplementary material is available for this article at     

Prediction and evaluation of multi epitope based sub-unit vaccine against Salmonella typhimurium

Salmonella enteric serovar Typhimurium is the most common enteric pathogen in humans and animals. Consumption of contaminated food or water triggers inflammation that allows Salmonella to spread into the gut and causes gastrointestinal diseases. The infection spreads by intestinal invasion, phagocyte internalization and subsequent dissemination in many other patients. This research used TolA, a Salmonella typhimurium membrane protein, to computationally design a multi-epitope vaccine against the pathogen. Complete consistency of the candidate vaccine was checked In silico, and molecular dynamics simulations confirmed the vaccine's stability. According to docking report, the vaccine has a good affinity with toll-like receptors. In silico cloning and codon optimization techniques improved the vaccine's efficacy in Salmonella typhimurium manifestation process. The candidate vaccine induced an efficient immune response, as determined by In silico immune simulation. Computational studies revealed that the engineered multi-epitope vaccine is structurally stable, capable of eliciting particular immunological reactions, and therefore a candidate for a latent Salmonella typhimurium vaccine. However, wet lab studies and further investigations are required to confirm the results.     

The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer

Salmonella typhi, the causative agent of typhoid fever, annually infects 16 million people and kills 600 000 world wide. Plasmid-encoded multiple drug resistance in S.typhi is always encoded by plasmids of incompatibility group H (IncH). The complete DNA sequence of the large temperature-sensitive conjugative plasmid R27, the prototype for the IncHI1 family of plasmids, has been compiled and analyzed. This 180 kb plasmid contains 210 open reading frames (ORFs), of which 14 have been previously identified and 56 exhibit similarity to other plasmid and prokaryotic ORFs. A number of insertion elements were found, including the full Tn10 transposon, which carries tetracycline resistance genes. Two transfer regions, Tra1 and Tra2, are present, which are separated by a minimum of 64 kb. Homologs of the DNA-binding proteins TlpA and H-NS that act as temperature-regulated repressors in other systems have been located in R27. Sequence analysis of transfer and replication regions supports a mosaic-like structure for R27. The genes responsible for conjugation and plasmid maintenance have been identified and mechanisms responsible for thermosensitive transfer are discussed.     

IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine

Cellular immunity against Salmonella typhi was observed by using a direct anti-bacterial in vitro assay in volunteers orally vaccinated with the live S. typhi mutant strain Ty 21a. With this experimental approach, it was demonstrated that Ty 21a vaccine also induces cellular immunity against S. paratyphi A and B. Interestingly, the mechanism involved in cellular immunity against bacteria seems to be of an antibody-dependent cellular cytotoxicity (ADCC) type, with IgA acting as the humoral arm and CD4+ T lymphocytes as the cellular one. In accordance with the increase in IgA-driven ADCC against S. typhi, a major rise in IgA against O and H antigens was observed in the serum of vaccinees in parallel to an increase in IgG of identical specificity. Furthermore, a Ty 21 vaccine induced cellular activity against flagellar antigens. These results indicate that IgA-ADCC by T lymphocytes against bacteria can originate from local stimulation of the gut mucosal immune system. This cellular defense mechanism might be at the origin of the protection induced by Ty 21a vaccine.     

Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection

Heat shock proteins (Hsps) have been reported to be dominant antigens for the host immune response to various pathogens and thus, have great potential for use in vaccination. In the present study, we evaluated the immunogenicity and protective efficacy of GroEL of Salmonella enterica serovar Typhi against lethal infection by S. typhi Ty2 in mice with or without adjuvants. Anti GroEL–IgG titers were significantly higher in mice immunized with either GroEL-alone or in combination with alum/Complete Freund’s adjuvant (CFA) as compared to the control. Analysis of antibody isotypes suggested predominance of Th2 type immune response in GroEL + alum immunized animals as revealed by higher IgG1/IgG2a ratio. Whereas, immunization of animals with GroEL + CFA or GroEL-alone shifted the immune response toward Th1 phenotype. Mice immunized with GroEL with or without adjuvants, showed a significant increase in lymphocyte proliferation and cytokine levels. The animals immunized with GroEL + CFA or GroEL-alone showed higher IFN-γ and IL-2 levels than alum group, indicating Th1 response whereas IL-4 levels (Th2 response) were found to be highest in alum group as compared to other two immunized groups. Immunization of mice with GroEL-alone, GroEL + alum, and GroEL + CFA provided 70, 50 and 80% protection, respectively, against lethal challenge by S. typhi in mice. The differences in the percentage protection among various groups were attributed to the differences in the immune responses generated by respective immunizations. The present study shows that GroEL forms an ideal candidate molecule to develop a recombinant protein based vaccine against human typhoid.     

Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine

Salmonella enterica serovar Typhi (S. typhi) strain Ty21a remains the only licensed attenuated typhoid vaccine. Despite years of research, the identity of the protective immunological mechanisms elicited by immunization with the Ty21a typhoid vaccine remains elusive. The present study was designed to characterize effector T cell responses in volunteers immunized with S. typhi strain Ty21a typhoid vaccine. We determined whether immunization with Ty21a induced specific CTL able to lyse S. typhi-infected cells and secrete IFN-gamma, a key effector molecule against intracellular pathogens. We measured the functional activity of these CTL by a (51)Cr-release assay using 8-day restimulated PBMC from Ty21a vaccinees as effector cells and S. Typhi-infected autologous PHA-activated PBMC as target cells. Most vaccinees exhibited consistently increased CD8-mediated lysis of targets by postimmunization PBMC when compared with preimmunization levels. We also developed an IFN-gamma ELISPOT assay to quantify the frequency of IFN-gamma spot-forming cells (SFC) in PBMC from Ty21a vaccinees using an ex vivo system. Significant increases in the frequency of IFN-gamma SFC following immunization (mean +/- SD, 393 +/- 172; range 185-548 SFC/10(6) PBMC; p = 0.010), as compared with preimmunization levels, were observed. IFN-gamma was secreted predominantly by CD8(+) T cells. A strong correlation was recorded between the cytolytic activity of CTL lines and the frequency of IFN-gamma SFC (r(2) = 0.910, p < 0.001). In conclusion, this work constitutes the first evidence that immunization of volunteers with Ty21a elicits specific CD8(+) CTL and provides an estimate of the frequency of CD8(+) IFN-gamma-secreting cells induced by vaccination.     

Salmonella paratyphi C: Genetic Divergence from Salmonella choleraesuis and Pathogenic Convergence with Salmonella typhi

Background

Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved.

Methodologies/Principal Findings

We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis.

Conclusions

S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man.     

Temporal fluctuation of multidrug resistant salmonella typhi haplotypes in the mekong river delta region of Vietnam

Background

Typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005.

Principal Findings

The population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene.

Significance

The H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam.     

Pathogenetic and immunological characteristics of different forms of the Salmonella typhi carrier state

Infectious granulomas with macrophages containing Salmonella typhi have been detected in the immune organs of the intestine of typhoid patients by means of morphological investigation techniques, immunofluorescent and electron microscopy. This suggests that typhoid granulomas form the basis of S. typhi primary carriership complicated by the relapses of this infection in cases of weakened cell-mediated immunity, which is proved by a decrease in the level of T-lymphocytes and by increased leukocyte migration index in relapses of typhoid fever and in S. typhi primary carriership. At the same time, the formation of S. typhi secondary carriership occurs in the process of the colonization of the altered organs and tissues of the body by S. typhi. This secondary carriership differs from the primary one by a number of pathogenetic signs. The detailed characterization of these two forms of S. typhi carriership is presented.