Adaptive acid tolerance response in Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Typhi

The survival of bacteria in various environments depends on a number of protective responses including acid tolerance response (ATR). In this study, ATR phenomenon was compared in Salmonella enterica serovar Typhi 6 and Salmonella enterica serovar Typhimurium 98 under different culture conditions. Survival of the adapted culture (pre-acid shocked to pH 5.5) was significantly better (p < 0.05) as compared to control, unadapted culture after acid shock at pH 3.3. However, the ATR varied with the serovar, incubation temperature and the growth medium used (all p-values < 0.05). S. Typhi 6 failed to grow in pH 3.3 at 45 degrees C. The addition of tetracycline or chloramphenicol (1.0 microg ml(-1)) to adapted cultures during or after acid shock (pH 3.3) had no effect on ATR expression. In S. Typhimurium 98, growth was increased by 10% or greater in adapted culture (when grown at pH 3.3) as compared to growth observed with an unadapted culture (when grown at pH 7.3) on transfer to fresh growth medium at pH 7.3. A poor ATR observed in non-growing S. Typhimurium 98 suspensions clearly showed that ATR is an energy-consuming process. Storage of S. Typhimurium 98 cultures in pH 4.5 nutrient broth at 4 degrees C demonstrated that prolonged exposure to acidic conditions is more detrimental in comparison to the cultures stored at pH 7.3 at this temperature.     

Rhabdomyolysis induced by Salmonella enterica serovar Typhi bacteraemia

Rhabdomyolysis has been reported infrequently with salmonella infection. Since 1964, there have been at least 22 reports associated with gastroenteritis or bacteraemia. Twenty cases have been associated with non-typhoidal strains of Salmonella, with single reports of Salmonella enterica serovars Paratyphi and Typhi. A second case of typhoid fever associated with rhabdomyolysis was recently diagnosed in Ann Arbor, USA in a traveller returning from an endemic area. Prompt diagnosis and treatment resulted in a good outcome. Salmonella infection should be considered by clinicians as a possibility in the differential diagnosis of rhabdomyolysis.     

Molecular basis of resistance displayed by highly ciprofloxacin-resistant Salmonella enterica serovar Typhi in Bangladesh

Highly ciprofloxacin-resistant (MIC, 512 microg/ml) strains of Salmonella enterica serovar Typhi were isolated from the blood of typhoid patients in Dhaka, Bangladesh. The strains were indistinguishable by their antibiograms, biotypes, and variable-number tandem repeat types and had matching point mutations at positions 83 and 87 of the gyrA gene. The isolation of these strains in an area of high endemicity indicates the need for continuous surveillance of antibiotic resistance of S. enterica serovar Typhi and for the rationalized use of ciprofloxacin.     

Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with Salmonella enterica serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in S. enterica serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute- versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses.     

Morphine withdrawal lowers host defense to enteric bacteria: spontaneous sepsis and increased sensitivity to oral Salmonella enterica serovar Typhimurium infection

Understanding the consequences of drug withdrawal on immune function and host defense to infection is important. We, and others, previously demonstrated that morphine withdrawal results in immunosuppression and sensitizes to lipopolysaccharide-induced septic shock. In the present study, the effect of morphine withdrawal on spontaneous sepsis and on oral infection with Salmonella enterica serovar Typhimurium was examined. Mice were chronically exposed to morphine for 96 h by implantation of a slow-release morphine pellet. Abrupt withdrawal was induced by removal of the pellet. In the sepsis model, bacterial colonization was examined and bacterial species were identified by necropsy of various tissues. It was found that at 48 h postwithdrawal, morphine-treated mice had enteric bacteria that were detected in the Peyer's patches (4/5), mesenteric lymph nodes (4/5), spleens (4/10), livers (6/10), and peritoneal cavities (8/10). In placebo pellet-withdrawn mice, only 2/40 cultures were positive. The most frequently detected organisms in tissues of morphine-withdrawn mice were Enterococcus faecium followed by Klebsiella pneumoniae. Both organisms are part of the normal gastrointestinal flora. In the infection model, mice were orally inoculated with S. enterica 24 h post-initiation of abrupt withdrawal from morphine. Withdrawal significantly decreased the mean survival time and significantly increased the Salmonella burden in various tissues of infected mice compared to placebo-withdrawn animals. Elevated levels of the proinflammatory cytokines were observed in spleens of morphine-withdrawn mice, compared to placebo-withdrawn mice. These findings demonstrate that morphine withdrawal sensitizes to oral infection with a bacterial pathogen and predisposes mice to bacterial sepsis.     

Single-nucleotide-polymorphism typing and genetic relationships of Salmonella enterica serovar Typhi isolates

Salmonella enterica serovar Typhi is a clone with a low level of variation. We developed a molecular typing method for serovar Typhi using 38 genome-wide single-nucleotide polymorphisms (SNPs) as markers detected by PCR-restriction enzyme digestion. The 73 worldwide serovar Typhi isolates studied were separated into 23 SNP profiles and four distinct genetic groups. Serovar Typhi isolates expressing the unique flagellar antigen z66 were found to cluster together and branch off from the ancestral group, suggesting that serovar Typhi was initially monophasic with only an H1 antigen and subsequently gained the z66 antigen. Typing using the 38 SNPs gave a discriminatory power of 0.87, and a minimum of 16 SNPs may be used to achieve the same level of differentiation. The SNP typing method we developed will be a valuable tool for global epidemiology studies of serovar Typhi.     

Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates from Pakistan

The accurate identification of Salmonella enterica subsp. enterica serovar Typhi variants that fail to express the capsular polysaccharide, Vi, is an important and much discussed issue for medical microbiology. We have tested a multiplex PCR method which shows the presence or absence of the genetic locus required for Vi expression. Of 2,222 Salmonella serovar Typhi clinical isolates collected from patients' blood over a 4-year period in a region of Pakistan where typhoid is endemic, 12 tested negative for Vi expression by serological agglutination. However, only 1 of these 12 was Vi negative by the multiplex PCR method. This result was confirmed by immunofluorescence, the most sensitive method for Vi characterization in Salmonella serovar Typhi. The multiplex PCR described therefore represents a simple and accurate method for surveillance for Vi-negative variants of Salmonella serovar Typhi in Pakistan. Testing of clinical isolates of Salmonella serovar Typhi, before subculture, from other regions where Vi-negative Salmonella serovar Typhi has been described should be carried out so that the impact of vaccination with purified Vi antigen on the levels of Vi-negative Salmonella serovar Typhi in bacterial populations can be assessed.     

Immune responses against Salmonella enterica serovar enteritidis infection in virally immunosuppressed chickens

To understand the role of immune mechanisms in protecting chickens from Salmonella infections, we examined the immune responses of Salmonella enterica serovar Enteritidis-infected chickens and the effect of chicken anemia virus (CAV), a T-cell-targeted virus, on S. enterica serovar Enteritidis-induced immune responses. One-day-old chicks were orally inoculated with S. enterica serovar Enteritidis with or without intramuscular injection of CAV. The bacterial infection, pathology, and immune responses of chickens were evaluated at 14, 28, and 56 days postinoculation. The infection increased the levels of S. enterica serovar Enteritidis-specific mucosal immunoglobulin A (IgA), the number of gut-associated T cells, and the titer of serum IgG specific for S. enterica serovar Enteritidis surface antigens. CAV infection depressed these immune responses, especially the mucosal immune responses, but did not increase the number of S. enterica serovar Enteritidis-infected cells in the intestine. The severity of pathological lesions appeared to be reciprocal to the level of immune responses, but the S. enterica serovar Enteritidis infection persisted. These results suggest that oral infection of S. enterica serovar Enteritidis in chickens induces both mucosal and systemic immune responses, which have a limited effect on the S. enterica serovar Enteritidis infection under conditions designed to mimic the field situation.     

Commensal bacteria increase invasion of intestinal epithelium by Salmonella enterica serovar Typhi

The intestinal microflora consists of a heterogeneous population of microorganisms and has many effects on the health status of its human host. Here, it is shown that the products of certain strains of bacteria normally present in the intestinal microflora are able to trigger redistribution of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in epithelial cells. CFTR is used by Salmonella enterica serovar Typhi as a receptor on epithelial cells which mediate the translocation of this microorganism to the gastric submucosa. Serovar Typhi-epithelial cell adhesion and CFTR-dependent invasion by serovar Typhi of epithelial cells were increased following commensal-mediated CFTR redistribution. These data suggest that commensal microorganisms present in the intestinal lumen can affect the efficiency of serovar Typhi invasion of the intestinal submucosa. This could be a key factor influencing host susceptibility to typhoid fever.     

Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to sef, fim, saf, and tcf, which had been described previously in serotype Typhi, we identified eight new putative chaperone-usher-dependent fimbrial operons, which were termed bcf, sta, stb, ste, std, stc, stg, and sth. Hybridization analysis performed with 16 strains of Salmonella reference collection C and 22 strains of Salmonella reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal Salmonella serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other Salmonella serotypes investigated in that it contained a unique combination of putative fimbrial operons.     

Role of RpoS in fine-tuning the synthesis of Vi capsular polysaccharide in Salmonella enterica serotype Typhi

Regulation of the synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi, is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some serotype Typhi strains exhibit overexpression of Vi polysaccharide, which masks clinical detection of lipopolysaccharide O antigen. This variation in Vi polysaccharide and O antigen display (VW variation) has been observed since the initial studies of serotype Typhi. In this study, we report that rpoS plays a role in this increased expression in Vi polysaccharide. We constructed a variety of isogenic serotype Typhi mutants that differed in their expression levels of RpoS and examined the role of the rpoS product in synthesis of Vi polysaccharide under different osmolarity conditions. Vi polysaccharide synthesis was also examined in serotype Typhi mutants in which the native promoter of the rpoS was replaced by an araCP(BAD) cassette, so that the expression of rpoS was arabinose dependent. The RpoS(-) strains showed increased syntheses of Vi polysaccharide, which at low and medium osmolarities masked O antigen detection. In contrast, RpoS(+) strains showed lower syntheses of Vi polysaccharide, and an increased detection of O antigen was observed. During exponential growth, when rpoS is unstable or present at low levels, serotype Typhi RpoS(+) strains overexpress the Vi polysaccharide at levels comparable to those for RpoS(-) strains. Our results show that RpoS is another regulator of Vi polysaccharide synthesis and contributes to VW variation in serotype Typhi, which has implications for the development of recombinant attenuated Salmonella vaccines in humans.     

利用IVIAT技术初步筛选伤寒沙门菌的体内诱导基因

目的 利用体内诱生抗原筛选技术初步筛选伤寒沙门菌的体内诱导基因.方法 首先将3个伤寒病人恢复期的血清等体积混合,用体外培养的伤寒沙门菌全菌体、超声裂解产物及加热变性裂解产物充分吸收处理.然后采用pET-30a/b/c表达载体系统构建伤寒沙门菌Ty2菌株的基因组表达文库,并以吸收后的血清为探针,筛选伤寒沙门菌基因组表达文库.结果 从5 000个克隆中获得5个阳性克隆,对阳性克隆测序和序列检索分析表明,涉及 6 个开放阅读框.结论 本实验利用IVIAT筛选体内诱导基因的各个程序是正确的,并初步筛选到伤寒沙门菌的体内诱导基因.     

Antibodies in action: role of human opsonins in killing Salmonella enterica serovar Typhi

Although vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance of Salmonella enterica serovar Typhi infections is poorly defined. Here, we describe the first functional characterization of S. Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 ΔaroC ΔssaV). We determined that postvaccination sera increased the uptake of wild-type S. Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2- to 9.5-fold more S. Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival of S. Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killing S. Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing of S. Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever.     

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association

Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.     

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.     

Differences in resistance to Salmonella enterica serovar Gallinarum infection among indigenous local chicken ecotypes in Tanzania.

A study was conducted to evaluate the disease resistance potential in 105 chickens of six indigenous local chicken ecotypes in Tanzania by orally challenging 1-week-old chicks with 2.5 x 10(8) colony-forming units of virulent S. Gallinarum. For 14 days post infection, clinical signs, necropsy findings, antibody titres, packed cell volume, leukocyte population count, and viable bacterial cell counts in the liver and spleen were recorded. Clinical signs were recorded daily but other parameters were recorded on the day of infection, then on days 3, 6, 10 and 14 after infection. Clinical signs of fowl typhoid were evident in chickens from day 3 post infection and disappeared by day 9 post infection. Pathological lesions on sacrificed birds included enlargement of the liver and spleen with foci of necrosis on the liver, spleen and myocardium. The mean viable bacterial cell counts in the liver and spleen varied between ecotypes, although the differences were not statistically significant. There were significant differences in the leukocyte population in the peripheral blood, with one ecotype (Morogoro-medium) showing a consistent and significantly higher heterophil count compared with other ecotypes. It was concluded that there is a selectable resistance potential to S. Gallinarum among the local chicken ecotypes in Tanzania that may be attributable to non-specific host immune responses. Further studies are suggested.     

Evaluation of Salmonella enteritidis-immune lymphokines on host resistance to Salmonella enterica ser. gallinarum infection in broiler chicks

The prophylactic treatment of neonatal broiler chicks with lymphokines derived from S. enteritidis-immurazed chickens (SE-ILK) was evaluated for its effect on the birds' resistance to an experimental infection S. enterica ser. gallinarum (SG). On the day of hatch, chicks were injected intraperitoneally with either SE-ILK, control non-immune lymphokines (NILK), or were left untreated. Thirty minutes later, all chicks were orally gavaged with either 10(4) colony forming units (CFU) or 10(6) CFU SG. The chicks were observed twice daily for 10 days for morbidity and mortality. Chicks that died during the experiment had their livers cultured for SG. The survivors were killed and their livers, spleens and caecal tonsils cultured for SG. The prophylactic treatment of chickens with SE-ILK induced significant protection against extraintestinal SG infection when compared to the NILK-treated or non-treated controls as evidenced by: (1) a significant reduction (P< 0.005) in the mortality of chicks challenged with either 10(4) and 10(6) CFU SG; (2) an increased average weight gains of chicks challenged with either 10(4) and 10(6) CFU SG; and (3) a significant (P< 0.001) reduction in the number of chicks with organs culture-positive for SG. The results suggest that the prophylactic administration of SE-ILK can confer non-specific protection to chicks against a pathogenic species of Salmonella resulting in reduced morbidity, mortality, and organ infectivity caused by SG infections of broiler chicks, while enhancing performance during the first 10 days of Ufe.