Selective amplification of tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A

The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of Salmonella serovars Typhi and Paratyphi A were accurately identified by this assay.     

Live Oral Salmonella enterica Serovar Typhi Vaccines Ty21a and CVD 909 Induce Opsonophagocytic Functional Antibodies in Humans That Cross-React with S. Paratyphi A and S. Paratyphi B

Live oral Salmonella enterica serovar Typhi vaccine Ty21a induces specific antibodies that cross-react against Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Paratyphi B, although their functional role in clearance remains unknown. We utilized an in vitro assay with THP-1 macrophages to compare the phagocytosis and survival of Salmonella opsonized with heat-inactivated human sera obtained before and after vaccination with Ty21a or a live oral S. Typhi vaccine, CVD 909. Opsonization with postvaccination sera predominantly increased the phagocytosis of S. Typhi relative to the corresponding prevaccination sera, and increases were also observed with S. Paratyphi A and S. Paratyphi B, albeit of lower magnitudes. Relative to prevaccination sera, opsonization with the postvaccination sera reduced the survival inside macrophages of S. Typhi but not of S. Paratyphi A or S. Paratyphi B. Higher anti-S. Typhi O antigen (lipopolysaccharide [LPS]) IgG, but not IgA, antibody titers correlated significantly with postvaccination increases in opsonophagocytosis. No differences were observed between immunization with four doses of Ty21a or one dose of CVD 909. Ty21a and CVD 909 induced cross-reactive functional antibodies, predominantly against S. Typhi. IgG anti-LPS antibodies may be important in phagocytic clearance of these organisms. Therefore, measurement of functional antibodies might be important in assessing the immunogenicity of a new generation of typhoid and paratyphoid A vaccines. (The CVD 909 study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00326443","term_id":"NCT00326443"}}NCT00326443.)     

The discrimination of d-tartrate positive and d-tartrate negative S. enterica subsp. enterica serovar Paratyphi B isolated in Malaysia by phenotypic and genotypic methods

Serotyping is not sufficient to differentiate between Salmonella species that cause paratyphoid fever from the strains that cause milder gastroenteritis as these organisms share the same serotype Salmonella Paratyphi B (S. Paratyphi B). Strains causing paratyphoid fever do not ferment d-tartrate and this key feature was used in this study to determine the prevalence of these strains among the collection of S. Paratyphi B strains isolated from patients in Malaysia. A total of 105 isolates of S. Paratyphi B were discriminated into d-tartrate positive (dT+) and d-tartrate negative (dT) variants by two lead acetate test protocols and multiplex PCR. The lead acetate test protocol 1 differed from protocol 2 by a lower inoculum size and different incubation conditions while the multiplex PCR utilized 2 sets of primers targeting the ATG start codon of the gene STM3356. Lead acetate protocol 1 discriminated 97.1% of the isolates as S. Paratyphi B dT+ and 2.9% as dT while test protocol 2 discriminated all the isolates as S. Paratyphi B dT+. The multiplex PCR test identified all 105 isolates as S. Paratyphi B dT+ strains. The concordance of the lead acetate test relative to that of multiplex PCR was 97.7% and 100% for protocol 1 and 2 respectively. This study showed that S. Paratyphi B dT+ is a common causative agent of gastroenteritis in Malaysia while paratyphoid fever appears to be relatively uncommon. Multiplex PCR was shown to be a simpler, more rapid and reliable method to discriminate S. Paratyphi B than the phenotypic lead acetate test.     

Emerging Salmonella Paratyphi A enteric fever and changing trends in antimicrobial resistance pattern of salmonella in Shimla

This retrospective study incorporates a six years, six months (January 2000-June 2006) laboratory data comprising 258 isolates of Salmonella. Cultures were identified by standard methods. Salmonella enterica serotype Typhi (S.Typhi) was the more frequent serotype isolated i.e., 61.62% with the remaining 38.37% being Salmonella enterica serotype Paratyphi A (S. Paratyphi A). There was emergence of S. Paratyphi A as the predominant serotype in 2003-2004 with resurgence of serotype Typhi thereon. A total of 66.27% isolates were resistant to one or more antibiotics. MDR S. Typhi was 10.69% and while 13.13% were MDR S. Paratyphi A. There was decrease in resistance to ampicillin, cotrimoxazole in 2004 and nalidixic acid beyond 2005 and increase in resistance to cefuroxime. We also documented a decrease in resistance to ciprofloxacin after 2005.     

Rapid multiplex PCR and real-time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C

Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.     

Salmonella enterica serovar Paratyphi C carries an inactive shufflon

Salmonella enterica serovar Typhi uses type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. This pilus-mediated event may be important in typhoid fever pathogenesis. We initially show that S. enterica serovar Paratyphi C strains harbor a pil operon very similar to that of serovar Typhi. An important difference, however, is located in the shufflon which concludes the pil operon. In serovar Typhi, the Rci recombinase acts upon two 19-bp inverted repeats to invert the terminal region of the pilV gene, thereby disrupting PilV synthesis and permitting bacterial self-association. In serovar Paratyphi C, however, the shufflon is essentially inactive because each of the Rci 19-bp substrates has acquired a single base pair insertion. A PilV protein is thus synthesized whenever the pil operon is active, and bacterial self-association therefore does not occur in serovar Paratyphi C. The data thus suggest that serovar Typhi bacterial self-association using type IVB pili may be important in the pathogenesis of epidemic enteric fever.     

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.     

Cross‐Reactive Immune Response Induced by the Vi Capsular Polysaccharide Typhoid Vaccine Against Salmonella Paratyphi Strains

There are no vaccines in clinical use against paratyphoid fever, caused by Salmonella Paratyphi A and B or, rarely, C. Oral Salmonella Typhi Ty21a typhoid vaccine elicits a significant cross‐reactive immune response against S. Paratyphi A and B, and some reports suggest cross‐protective efficacy against the disease. These findings are ascribed to the O‐12 antigen shared between the strains. The Vi capsular polysaccharide vaccine has been shown to elicit antibodies reactive with O‐9,12. Twenty‐five volunteers immunized with the parenteral Vi vaccine (Typherix^®) were explored for plasmablasts cross‐reactive with paratyphoid strains; the responses were compared to those in 25 age‐ and gender‐matched volunteers immunized with Ty21a (Vivotif^®). Before vaccination, 48/50 vaccinees had no plasmablasts reactive with the antigens. Seven days after vaccination, 15/25 and 22/25 Vi‐ and Ty21a‐vaccinated volunteers had circulating plasmablasts producing antibodies cross‐reactive with S. Paratyphi A, 18/25 and 23/25 with S. Paratyphi B and 16/25 and 9/25 with Paratyphi C, respectively. Compared to the Ty21a group, the Vi group showed significantly lower responses to S. Paratyphi A and B and higher to S. Paratyphi C. To conclude, the Vi vaccine elicited a cross‐reactive plasmablast response to S. Paratyphi C (Vi antigen in common) and less marked responses to S. Paratyphi A and B than the Ty21a preparation. S. Paratyphi A and B both being Vi‐negative, the result can be explained by trace amounts of bacterial cell wall O‐12 antigen in the Vi preparation, despite purification. The clinical significance of this finding remains to be determined.     

Molecular and Cellular Characterization of a Salmonella enterica Serovar Paratyphi A Outbreak Strain and the Human Immune Response to Infection

Enteric fever is an invasive life-threatening systemic disease caused by the Salmonella enterica human-adapted serovars Typhi and Paratyphi. Increasing incidence of infections with Salmonella enterica serovar Paratyphi A and the spreading of its antibiotic-resistant derivates pose a significant health concern in some areas of the world. Herein, we describe a molecular and phenotypic characterization of an S. Paratyphi A strain accounted for a recent paratyphoid outbreak in Nepal that affected at least 37 travelers. Pulsed-field gel electrophoresis analysis of the outbreak isolates revealed one genetic clone (pulsotype), confirming a single infecting source. Genetic profiling of the outbreak strain demonstrated the contribution of specific bacteriophages as a prime source of genetic diversity among clinical isolates of S. Paratyphi A. Phenotypic characterization in comparison with the S. Paratyphi A ATCC 9150 reference sequenced strain showed differences in flagellar morphology and increased abilities of the outbreak strain with respect to its motility, invasion into nonphagocytic cells, intracellular multiplication, survival within macrophages, and higher induction of interleukin-8 (IL-8) secreted by host cells. Collectively, these differences suggest an enhanced virulence potential of this strain and demonstrate an interesting phenotypic variation among S. Paratyphi A isolates. In vivo profiling of 16 inflammatory cytokines in patients infected with the outbreak strain revealed a common profile of a remarkable gamma interferon (IFN-γ) induction together with elevated concentrations of tumor necrosis factor alpha (TNF-α), IL-6, IL-8, IL-10, and IL-15, but not IL-12, which was previously demonstrated as elevated in nontyphoidal Salmonella infections. This apparent profile implies a distinct immune response to paratyphoid infections.     

Enteric fever in a 6-year-old traveler caused by Salmonella enterica serotypes Typhi and Paratyphi A: laboratory detection strategies and treatment options

We report the first pediatric case of enteric fever caused by Salmonella enterica serotypes Typhi and Paratyphi A. Mixed infections are infrequently reported, potentially because detection of two different Salmonella serotypes in blood cultures is technically challenging. We suggest laboratory strategies to aid in the recognition of mixed infections.     

A multiplex polymerase chain reaction method for rapid identification of Citrobacter freundii and Salmonella species, including Salmonella Typhi.

BACKGROUND AND PURPOSE: Salmonella enterica is one of the most common enteric pathogens worldwide. Conventional methods of isolation of Salmonella strains take 4-7 days to complete, are laborious and require substantial manpower. We devised a polymerase chain reaction (PCR) method that simultaneously uses three pairs of specific primers to detect inv, spv, and via genes of Salmonella. METHODS: Three primer pairs were designed, including: SPVC-1 and SPVC-2, based on the nucleotide sequences of the spvC gene; INVA-1 and INVA-2, based on the invA gene; and VIAB-1 and VIAB-2, based on the viaB gene. PCR was performed using these three primers to identify 14 clinically important bacterial organisms, including Citrobacter freundii, S. enterica serovars Typhi and Paratyphi C, Dublin, and other non-typhoidal Salmonella that harbor a virulence plasmid. RESULTS: The following strains were readily identified using the PCR: (1) C. freundii; (2) S. Typhi; and S. Paratyphi C; (3) S. Dublin (virulence antigen-positive); and (4) Salmonella serovars that harbor an spv-type virulence plasmid. S. enterica could also be identified, but required further testing to determine serovar. CONCLUSIONS: This PCR method allows S. Typhi to be identified immediately so that appropriate antibiotic treatment can be initiated without delay.     

Molecular characterization of multiresistant d-tartrate-positive Salmonella enterica serovar paratyphi B isolates

Since 1996, the National Salmonella Reference Laboratory of Germany has received an increasing number of Salmonella enterica subsp. enterica serovar Paratyphi B isolates. Nearly all of these belonged to the dextrorotatory tartrate-positive variant (S. enterica subsp. enterica serovar Paratyphi B dT(+)), formerly called S. enterica subsp. enterica serovar Java. A total of 55 selected contemporary and older S. enterica subsp. enterica serovar Paratyphi B dT(+) isolates were analyzed by plasmid profiling, antimicrobial resistance testing, pulsed-field gel electrophoresis, IS200 profiling, and PCR-based detection of integrons. The results showed a high genetic heterogeneity among 10 old strains obtained from 1960 to 1993. In the following years, however, new distinct multiresistant S. enterica subsp. enterica serovar Paratyphi B dT(+) clones emerged, and one clonal lineage successfully displaced the older ones. Since 1994, 88% of the isolates investigated were multiple drug resistant. Today, a particular clone predominates in some German poultry production lines, poultry products, and various other sources. It was also detected in contemporary isolates from two neighboring countries as well.     

Immunization with Ty21a live oral typhoid vaccine elicits crossreactive multifunctional CD8+ T-cell responses against Salmonella enterica serovar Typhi,S. Paratyphi A,and S. Paratyphi B in humans

Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune (CMI) responses in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T-cell responses to further investigate whether Ty21a elicits crossreactive CMI against S. Paratyphi A and S. Paratyphi B that also cause enteric fever. Ty21a-elicited crossreactive CMI responses against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (T_EM) and, to a lesser extent, in CD8+CD45RA+ T_EM (T_EMRA) subsets. These CD8+ T-cell responses were largely mediated by MF cells coproducing interferon-γ and macrophage inflammatory protein-1β and expressing CD107a with or without tumor necrosis factor-α. Significant proportions of Salmonella-specific MF cells expressed the gut-homing molecule integrin α₄β₇. In most subjects, similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI responses against Salmonella that could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers.     

Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A

In this study, we developed a multiplex polymerase chain reaction (mPCR) assay for pan-Salmonella detection as well as for specific detection of serovars Typhi and Paratyphi A. The assay detects members of the Salmonella genus by amplifying the outer membrane protein C (ompC). The presence of either Salmonella serovar Typhi or Paratyphi A is indicated by amplification of the putative regulatory protein gene STY4220, which is common to both serovars. Further differentiation of the serovars was achieved by targeting the intergenic region (SSPAI) between SSPA1723a and SSPA1724 in serovar Paratyphi A, and stgA, a fimbrial subunit protein, in serovar Typhi. mPCR was evaluated using 124 clinical and reference Salmonella serovars. S. enterica serovars Typhi and Paratyphi A were detected at 100% specificity and sensitivity. Each PCR reaction detected approximately 1 pg of Salmonella genomic DNA. Sensitivity of the PCR when tested on 8-h-enriched spiked blood samples of serovars Typhi and Paratyphi A was estimated at 4.5 × 10⁴–5.5 × 10⁴ CFU/ml, with similar detection levels observed for spiked fecal samples. This mPCR could prove to be a useful diagnostic tool for the detection and differentiation of serovars Typhi and Paratyphi A.     

Chromogenic media for the detection of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Paratyphi A in human stool samples: evaluation in a reference setting

Detection of Salmonella Paratyphi A stool carriers by conventional stool culture media is hindered by the absence of hydrogen sulphide production compared to most other Salmonella serovars. This study evaluated the detection of Salmonella Paratyphi A in stool samples using Salmonella chromogenic media compared to a conventional medium. Four chromogenic media, COMPASS Salmonella agar (Biokar Diagnostics, Beauvais, France), BBL? CHROMagar? Salmonella (BD Diagnostics, Erembodegem-Aalst, Belgium), Brilliance? Salmonella agar (Oxoid Ltd., Basingstoke, UK) and Salmonella PLUS CHROMagar? (CHROMagar, Paris, France), were compared to conventional Salmonella-Shigella agar (Oxoid Ltd.). The colony morphology of 29 freshly grown stock isolates (Salmonella and competing organisms) was assessed. The limit of detection (LOD) was also determined using saline and stool suspensions. Finally, recognizability of Salmonella Paratyphi A isolates was assessed using 20 human stool samples spiked with different concentrations of Salmonella Paratyphi A. All Salmonella Paratyphi A isolates demonstrated detectable growth with typical purple-coloured colonies that could be clearly differentiated from competing organisms on all four chromogenic media. The LOD for Salmonella Paratyphi A was 10³ colony-forming units (CFU)/ml for all media, except for Brilliance agar (10⁵ CFU/ml of stool). Salmonella Paratyphi A was easy to differentiate from competing organisms in the spiked stool samples. Colony yields improved when an enrichment step (Selenite-F broth (BD Diagnostics, Erembodegem-Aalst, Belgium)) and prolonged incubation for 48 h were performed. Chromogenic media demonstrated good specificity and a low LOD for the detection of Salmonella Paratyphi A in stool samples.     

Glycoconjugate vaccine strategies for protection against invasive Salmonella infections

Salmonella enterica serovars Typhi and Paratyphi A and B and certain non-typhoidal Salmonella enterica (NTS) serovars are important causes of invasive Salmonella disease worldwide. NTS serovars Typhimurium and Enteritidis typically cause gastroenteritis in healthy children and adults in industrialized countries but in certain hosts (e.g., young infants, the elderly, immunocompromised individuals) they also cause invasive infections. These two serovars also cause invasive disease in infants and young children in sub-Saharan Africa. Whereas Salmonella surface polysaccharides are poor immunogens in animal models and do not generate immunologic memory, conjugation with carrier proteins overcomes these limitations. S. Typhi expresses a Vi polysaccharide capsule; Vi either alone or as a glycoconjugate protects humans from typhoid fever. In contrast, S. Paratyphi A and B and NTS (with rare exceptions) do not express capsular polysaccharides. Rather, their surface polysaccharides are the O polysaccharide (OPS) of lipopolysaccharide. In animal studies, immunization with Salmonella COPS (core polysaccharide-OPS) conjugated with carrier proteins generates functional immunity and protects against fatal Salmonella challenge. Conjugating to Salmonella proteins (flagellin, porins) may extend immune responses to another relevant target for antibody generation and enhance the glyconjugate's efficacy.     

Antimicrobial resistance trends in blood culture positive Salmonella Paratyphi A isolates from Pondicherry,India

Enteric fever is a public health problem with the upsurge in the occurrence of Salmonella isolates that are resistant to ciprofloxacin. In this study, a total of 284 blood culture isolates of S. Paratyphi A were investigated. Of these isolates, 281 (98.9%) were nalidixic acid resistant. A high rate (6.3%) of high-level resistance (≥4 μg/mL) was found to ciprofloxacin. The isolates with ciprofloxacin minimum inhibitory concentrations (MICs) of ≥12 μg/mL had 4 mutations, 2 mutations within the quinolone resistance-determining region of gyrA and 2 mutations also in parC. According to the Clinical Laboratory Standards Institute 2012 MIC breakpoints, 75.0% of isolates were resistant to ciprofloxacin. Finally, 3 major pulsed-field gel electrophoresis patterns were observed among the S. Paratyphi A isolates. The spread of fluoroquinolone resistant S. Paratyphi A necessitates a change toward ‘evidence-based’ treatment for enteric fever. The research provides a perspective on the increasing prevalence of antimicrobial resistant S. Paratyphi A isolates in this region of India.