Chromosomal Rearrangements in Salmonella enterica Serotype Typhi Affecting Molecular Typing in Outbreak Investigations

Salmonella enterica serotype Typhi strains belonging to eight different outbreaks of typhoid fever that occurred in Spain between 1989 and 1994 were analyzed by ribotyping and pulsed-field gel electrophoresis. For three outbreaks, two different patterns were detected for each outbreak. The partial digestion analysis by the intron-encoded endonuclease I-CeuI of the two different strains from each outbreak provided an excellent tool for examining the organization of the genomes of epidemiologically related strains. S. enterica serotype Typhi seems to be more susceptible than other serotypes to genetic rearrangements produced by homologous recombinations between rrn operons; these rearrangements do not substantially alter the stability or survival of the bacterium. We conclude that genetic rearrangements can occur during the emergence of an outbreak.     

Comparison of the cytolysin II genetic determinants of Actinobacillus pleuropneumoniae serotypes.

Cytolysins (Cly) I, II, and III are toxins secreted by Actinobacillus pleuropneumoniae. These toxins are thought to play an important role in the pathogenesis of porcine pleuropneumonia. ClyI and ClyII are RTX toxins and in general these toxins are encoded by operons consisting of four genes, C, A, B, and D. Our group recently cloned the C and A genes of the ClyII operon (clyIICA) of serotype 9. We found that this ClyII operon is truncated and lacked intact B and D genes (clyIIBD). B and D genes of the ClyI operon (clyIBD) were present however in serotype 9. In this study we analyzed the ClyII operons of the reference strains of the 12 A. pleuropneumoniae serotypes and compared them with the ClyII operon of serotype 9. We focused on (i) the presence, (ii) the sequence similarity, and (iii) the genomic environment of the clyIICA genes. The presence of the clyIICA sequences was studied by hybridization analysis of genomic DNA. The sequence similarity was studied by restriction fragment analysis on polymerase chain reaction-amplified DNA. The genomic environment was compared by analysis of the sequences that are located 3' of the clyIICA genes. We demonstrated that the clyIICA genes (i) are present in the reference strains of all serotypes, except serotype 10, (ii) have a high degree of sequence similarity, and (iii) are not contiguous with intact clyIIBD genes. We conclude that the organization and nucleotide sequence of the ClyII operons of A. pleuropneumoniae are very similar. We also studied the presence of clyIBD sequences and found them to be present in the reference strains of all serotypes, except serotypes 3 and 6. Thus, in most serotypes the clyIBD genes may complement the absent clyIIBD genes.     

Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica

The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.     

The Vi capsular antigen of Salmonella enterica serotype Typhi reduces Toll-like receptor-dependent interleukin-8 expression in the intestinal mucosa

Human infections with nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that in S. enterica serotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.     

Use of biochemical kinetic data to determine strain relatedness among Salmonella enterica subsp. enterica isolates

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:-:- strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of > or =95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.     

Genetic determinants and polymorphisms specific for human-adapted serovars of Salmonella enterica that cause enteric fever

Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever.     

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.     

Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons.

Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.     

An attempt to identify the evolutionary origin of a novel serotype of Salmonella enterica isolated from harbour porpoises

The isolation since 1991 of a new serotype of Salmonella enterica (antigenic formula 4,12:a:-) from harbour porpoises (Phocoena phocoena) at post-mortem examination raised the question of its evolutionary origin. Representative strains of S. enterica serotype 4,12:a:- and strains of eight other serotypes of serogroup 04 with phase-1 flagellar antigen H 'a' were examined by EcoRI ribotyping, IS200 fingerprinting and PCR-based profiling. Statistical analysis of results of multiple typing showed that strains of Salmonella serotype 4,12:a:- were genetically distant from those of antigenically similar salmonella serotypes, none of which seemed likely to be the progenitor of the 'porpoise' serotype.     

Salmonella fimbrial protein StcD induces cyclooxygenase-2 expression via Toll-like receptor 4

IntroductionThe genome of Salmonella enterica serovar Typhimurium contains 13 operons with homology to fimbrial genes.MethodsTo investigate the involvement of these fimbrial gene clusters in the expression of cyclooxygenase-2 (COX-2), which is an inducible enzyme involved in the synthesis of prostanoids, in J774 macrophages infected with S. enterica serovar Typhimurium, we constructed strains carrying a mutation in genes encoding the putative subunit proteins in 12 fimbrial operons.ResultsThe level of COX-2 expression was lower in macrophages infected with fimA or stcA mutant Salmonella than in those infected with wild-type Salmonella. Therefore, we focused on putative subunit protein StcA and adhesive like protein StcD encoded in the stc operon. Treatment of macrophages with purified recombinant StcD protein, but not StcA, resulted in the activation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, leading to the expression of not only COX-2 but also of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha. The expression of StcD-induced COX-2 was inhibited by treatment with the Toll-like receptor 4 (TLR4) inhibitor TAK-242, but not by treatment with the lipopolysaccharide (LPS) antagonist polymyxin B. Furthermore, StcD treatment stimulated HEK293 cells expressing TLR4 in the presence of CD14 and MD-2.ConclusionStcD is a pathogen-associated molecular pattern of S. enterica serovar Typhimurium that is recognized by TLR4 and plays a significant role in the induction of COX-2 expression in macrophages.     

Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.     

High level ciprofloxacin resistance in Salmonella enterica isolated from blood

PURPOSE: Over the last few years, resistance to ciprofloxacin in Salmonella enterica has become a global concern. The present study was undertaken to find out the susceptibility pattern of Salmonella enterica isolates in our hospital. METHODS: Blood cultures were done using BacT/ALERT 3D system. The antimicrobial susceptibility testing was carried out by the Kirby-Bauer disc diffusion method using CLSI breakpoints. Minimum inhibitory concentration was determined for ciprofloxacin-resistant strains using E-test and Vitek-1 automated system. RESULTS: A total of 25,953 samples of blood culture yielded 431 Salmonella enterica serotype Typhi and 198 serotype Paratyphi A isolates. Twenty-two isolates of serotype Typhi were resistant to ciprofloxacin, while two isolates of Typhi and two Paratyphi A were intermediately susceptible to ciprofloxacin. Ciprofloxacin resistance is 5.6% (24 isolates) among Salmonella enterica serotype Typhi. Ampicillin, chloramphenicol and co-trimoxazole resistance in Salmonella enterica serotype Typhi appears to have decreased to 14.9% (64/431) in comparison to the 27% (55/205) during 2003. All isolates were sensitive to ceftriaxone. CONCLUSIONS: Ciprofloxacin can no longer be considered as the drug of choice in treating Salmonella infections. While first-line antimicrobials may still have a role to play in the treatment of enteric fever, ceftriaxone remains the sole defence against ciprofloxacin-resistant Salmonella infections.     

Salmonella enterica serotype Typhimurium fimbrial proteins serve as antigens during infection of mice

The Salmonella enterica serotype Typhimurium genome contains 13 operons with homology to fimbrial gene sequences. Here we investigated the role of 11 serotype Typhimurium fimbrial proteins, including FimA, AgfA (CsgA), BcfA, StbA, SthA, LpfA, PefA, StdA, StcA, StiA, and StfA, as antigens during the infection of genetically resistant mice (CBA). Upon the growth of serotype Typhimurium in standard laboratory broth culture, only the expression of FimA could be detected by Western blot analysis. The infection of mice with serotype Typhimurium grown in broth culture, followed by at least one subsequent infection, resulted in seroconversion of animals to FimA, AgfA, BcfA, StbA, SthA, LpfA, PefA, StdA, StcA, StiA, and StfA positivity. Most animals seroconverted to only a subset of these fimbrial antigens. The immunization of mice with glutathione S-transferase (GST)-FimA, GST-AgfA, GST-BcfA, GST-StbA, GST-SthA, GST-LpfA, GST-PefA, GST-StdA, GST-StcA, GST-StiA, and GST-StfA fusion proteins resulted in reduced fecal shedding of serotype Typhimurium during a challenge compared to that by a control group immunized with purified GST protein. Collectively, these data suggest that the expression of serotype Typhimurium fimbrial antigens is induced during the infection of mice.     

Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an approximately 90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of lpfA(O113) were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf(O113) may function as an adhesin in LEE-negative isolates of EHEC.     

Differentiation of Salmonella phase 1 flagellar antigen types by restriction of the amplified fliC gene.

The large antigenic diversity (over 2,300 serotypes) expressed by Salmonella strains can probably be observed at the genetic level. The phase 1 flagellin gene fliC was amplified, and the amplified fragment was cleaved with a mixture of both endonucleases TaqI and ScaI. The restriction patterns observed allowed differentiation of flagellar types b, i, d, j, l,v, and z10. Flagellar group g (g,m, g,p, or g,m,s) could be differentiated from the other flagellar types. Flagellar types r and e,h could not be separated, although they could be distinguished from the other flagellar types studied. Practical applications of flagellar gene restriction are the distinction between serotype Gallinarum-Pullorum, which carries a cryptic gene for flagellar type g,m, and nonmotile Vi-negative variants of serotype Typhi, and the tentative assignation of nonmotile variants of Salmonella serotypes to a flagellar type.     

The capsule encoding the viaB locus reduces interleukin-17 expression and mucosal innate responses in the bovine intestinal mucosa during infection with Salmonella enterica serotype Typhi

The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica serotype Typhi. Wild-type serotype Typhi induces less CXC chemokine production in tissue culture models than does an isogenic viaB mutant. Here we investigated the in vivo relevance of these observations by determining whether the presence of the viaB region prevents inflammation in two animal models of gastroenteritis. Unlike S. enterica serotype Typhimurium, serotype Typhi or a serotype Typhi viaB mutant did not elicit marked inflammatory changes in the streptomycin-pretreated mouse model. In contrast, infection of bovine ligated ileal loops with a serotype Typhi viaB mutant resulted in more fluid accumulation and higher expression of the chemokine growth-related oncogene alpha (GROalpha) and interleukin-17 (IL-17) than did infection with the serotype Typhi wild type. There was a marked upregulation of IL-17 expression in both the bovine ligated ileal loop model and the streptomycin-pretreated mouse model, suggesting that this cytokine is an important component of the inflammatory response to infection with Salmonella serotypes. Introduction of the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GROalpha and IL-17 expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in reducing intestinal inflammation in vivo.     

Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S.enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.