A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

[Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi].

OBJECTIVE: Establishment and application of Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi. Primers specific to Salmonella paratyphi A( SPAP), Salmonella paratyphi B( SPBP), Salmonella paratyphi C( SPCP), and Salmonella typhi( STP) were designed. METHODS: A method of Taq Man probe-based quadruple real-time PCR was established according to the distinction of the 5'end of the probe mark of TET, ROX, FAM and HEX. 5 strains of SPA, 4 strains of SPB, 7 strains of SPC and 11 strains of ST were identified by amplification from SPAP, SPBP, SPCP and STP. RESULTS: While other serotypes of salmonella and17 strains of non-salmonella got negative results of amplification. Amplification rate of SPAP, SPBP, SPCP, and STP were 84. 5%, 101. 8%, 92. 4% and 90. 9%, respectively. The linear correlation coefficient( R~2) were 0. 996, 0. 975, 0. 996 and 0. 984, respectively. CONCLUSION: The PCR system is specific and sensitive for the identification of SPA, SPB, SPC and ST.

[Distribution in different Salmonella serovars and integration sites of Salmonella paratyphi C phage SPC-P1].

OBJECTIVE: To determine the prevalence of Salmonella paratyphi C phage (SPC-P1) in different Salmonella serovars and to identify the integration sites in host genome. METHODS: Based on the complete genome of SPC-P1 in S. paratyphi C RKS4594, 6 pairs of primers were designed and used to amplify the fragments of SPC-P1 in 11 S. typhi, 11 S. paratyphi A, 12 S. paratyphi B and 23 S. paratyphi C strains. At the same time, 100 complete genomes of Salmonella including 20 serovars available in National Center for Biotechnology Information (NCBI) database were downloaded and aligned by Mauve 2.3.1 to determine the prevalence of SPC-P1 in these serovars. Primers were designed according to the integration sites of SPC-P1 in the genome of RKS4594, and used to amplify ten strains having SPC-P1 in the genome. The PCR products were sequenced to investigate the integration sites of SPC-P1. RESULTS: SPC-P1 was widely distributed in S.paratyphi C genome. In the study, 14 strains had all 6 fragments and 2 strains had 3-5 fragments. All the amplified fragments showed expected sizes. In contrast, in the genomes of S. typhi, S. paratyphi A and S. paratyphi B, no or only 1-2 fragments could be amplified, and the sizes were smaller than expected. The results from Mauve showed that only in the genome of S.choleraesuis, which was a close relative of S. paratyphi C, there existed an almost complete genome of SPC-P1. The insertion site of SPC-P1 in all the ten S. paratyphi C strains tested was between pgtE and yfdC genes. CONCLUSION: SPC-P1 is a unique virulence factor of S. paratyphi C. It may play roles in the host range and pathogenicity of S.paratyphi C.

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.

A rare case of Salmonella Paratyphi C osteomyelitis: A genetic analysis and review of Salmonella osteomyelitis in England.

Salmonella osteomyelitis is rare in patients without sickling hemoglobinopathies. Invasive disease caused by Salmonella Paratyphi C is rarer still with only one case reported in the United Kingdom in the last 15 years. We report a case of relapsing S. Paratyphi C osteomyelitis in a newly diagnosed diabetic patient from Ghana. Our patient was initially treated successfully with surgical debridement followed by 6 weeks of IV ceftriaxone before recrudescence 9 months later. Due to the rarity of S. Paratyphi C and the lack of recent travel, genomic analysis was undertaken to assess possible sources with the closest related strain being from Cote d'Ivoire. The patient had likley picked up the strain several years before presentation. We review current Salmonella osteomyelitis literature and audit all cases referred to the England and Wales Salmonella national reference laboratory over the last 15 years.

[Development of modified molecular beacon based real-time PCR assay for the rapid detection of Salmonella choleraesuis and Salmonella paratyphi C].

OBJECTIVE: To develop real-time PCR assay based on modified molecular beacon for simultaneous detection of S. choleraesuis and S. paratyphi C. The established method was applied to the rapid detection of S. choleraesuis in food and stool samples of food poisoning, and then was applied to the identification of Salmonella C. METHODS: Based on the sequences (CP000857.1) published in GenBank, Two sets of primers and modified molecular beacon were designed. The Real-time PCR assay for the simultaneous detection of S. paratyphi C and S. choleraesuis was developed with optimized PCR procedures and PCR components, while other 11 different bacterial species were as the control. Then the sensitivity and specificity of the assay were tested using 77 Samonella strains. The assay was applied to the detection of 70 food samples. RESULTS: The limit of detection achieved was 10 fg/reaction or 20 CUF/reaction, Only Salmonella paratyphi C and Salmonella choleraesuis strains generated fluorescent signals. No cross-reaction was observed with other 11 bacterium, the sensitivity and specificity were both 100%. No samples among 70 food samples were found Salmonella positive by both real-time PCR assay and traditional culture method. It could be finished within 2 hours from template preparation to detection and the overall test would be finished within one day. CONCLUSION: The real-time PCR assay was rapid, sensitive and specific. It could be applied to the rapid diagnosis of S. paratyphi C and S. choleraesuis in food and stool samples of food poisoning and the identification of Salmonella C to guarantee food safety.

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C.

BACKGROUND: Salmonella paratyphi C is one of the few human-adapted pathogens along with S. typhi, S. paratyphi A and S. paratyphi B that cause typhoid, but it is not clear whether these bacteria cause the disease by the same or different pathogenic mechanisms. Notably, these typhoid agents have distinct sets of large genomic insertions, which may encode different pathogenicity factors. Previously we identified a novel prophage, SPC-P1, in S. paratyphi C RKS4594 and wondered whether it might be involved in pathogenicity of the bacteria. RESULTS: We analyzed the sequence of SPC-P1 and found that it is an inducible phage with an overall G+C content of 47.24%, similar to that of most Salmonella phages such as P22 and ST64T but significantly lower than the 52.16% average of the RKS4594 chromosome. Electron microscopy showed short-tailed phage particles very similar to the lambdoid phage CUS-3. To evaluate its roles in pathogenicity, we lysogenized S. paratyphi C strain CN13/87, which did not have this prophage, and infected mice with the lysogenized CN13/87. Compared to the phage-free wild type CN13/87, the lysogenized CN13/87 exhibited significantly increased virulence and caused multi-organ damages in mice at considerably lower infection doses. CONCLUSIONS: SPC-P1 contributes pathogenicity to S. paratyphi C in animal infection models, so it is possible that this prophage is involved in typhoid pathogenesis in humans. Genetic and functional analyses of SPC-P1 may facilitate the study of pathogenic evolution of the extant typhoid agents, providing particular help in elucidating the pathogenic determinants of the typhoid agents.

Laboratory-based surveillance of paratyphoid fever in the United States: travel and antimicrobial resistance.

BACKGROUND: The incidence of paratyphoid fever, including paratyphoid fever caused by antimicrobial-resistant strains, is increasing globally. However, the epidemiologic and laboratory characteristics of paratyphoid fever in the United States have never been studied. METHODS: We attempted to interview all patients who had been infected with laboratory-confirmed Salmonella serotypes Paratyphi A, Paratyphi B, or Paratyphi C in the United States with specimens collected from 1 April 2005 through 31 March 2006. At the Centers for Disease Control and Prevention (CDC), isolates underwent serotype confirmation, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis typing. RESULTS: Of 149 patients infected with Salmonella Paratyphi A, we obtained epidemiologic information for 89 (60%); 55 (62%) of 86 were hospitalized. Eighty-five patients (96%) reported having travel internationally, and 80 (90%) had traveled to South Asia. Of the 146 isolates received at the CDC, 127 (87%) were nalidixic acid resistant; nalidixic acid resistance was associated with travel to South Asia (odds ratio, 17.0; 95% confidence interval, 3.8-75.9). All nalidixic acid-resistant isolates showed decreased susceptibility to ciprofloxacin (minimum inhibitory concentration, > or = 0.12 microg/mL). Of 49 patients infected with Salmonella Paratyphi B, only 12 (24%) were confirmed to have Paratyphi B when tested at the CDC. Four (67%) of 6 patients were hospitalized, and 5 (83%) reported travel (4 to the Andean region of South America). One case of Salmonella Paratyphi C infection was reported in a traveler to West Africa with a urinary tract infection. CONCLUSIONS: Physicians should be aware of the increasing incidence of infection due to Salmonella Paratyphi A and treatment options given its widespread antimicrobial resistance. A paratyphoid fever vaccine is urgently needed. Continued surveillance for paratyphoid fever will help guide future prevention and treatment recommendations.

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.

Diverse genome structures of Salmonella paratyphi C.

BACKGROUND: Salmonella paratyphi C, like S. typhi, is adapted to humans and causes typhoid fever. Previously we reported different genome structures between two strains of S. paratyphi C, which suggests that S. paratyphi C might have a plastic genome (large DNA segments being organized in different orders or orientations on the genome). As many but not all host-adapted Salmonella pathogens have large genomic insertions as well as the supposedly resultant genomic rearrangements, bacterial genome plasticity presents an extraordinary evolutionary phenomenon. Events contributing to genomic plasticity, especially large insertions, may be associated with the formation of particular Salmonella pathogens. RESULTS: We constructed a high resolution genome map in S. paratyphi C strain RKS4594 and located four insertions totaling 176 kb (including the 90 kb SPI7) and seven deletions totaling 165 kb relative to S. typhimurium LT2. Two rearrangements were revealed, including an inversion of 1602 kb covering the ter region and the translocation of the 43 kb I-CeuI F fragment. The 23 wild type strains analyzed in this study exhibited diverse genome structures, mostly as a result of recombination between rrn genes. In at least two cases, the rearrangements involved recombination between genomic sites other than the rrn genes, possibly homologous genes in prophages. Two strains had a 20 kb deletion between rrlA and rrlB, which is a highly conservative region and no deletion has been reported in this region in any other Salmonella lineages. CONCLUSION: S. paratyphi C has diverse genome structures among different isolates, possibly as a result of large genomic insertions, e.g., SPI7. Although the Salmonella typhoid agents may not be more closely related among them than each of them to other Salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes followed by genome structure rearrangements. Comparison of multiple Salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes.

Serodiagnosis of Salmonella enterica serovar Typhi and S. enterica serovars Paratyphi A, B and C human infections.

The aim of this study was to evaluate an immunoassay for the detection of human serum antibodies to the LPS and flagellar antigens of Salmonella Typhi and Salmonella Paratyphi A, B and C, and to the Vi capsular polysaccharide of S. Typhi and S. Paratyphi C. A total of 330 sera were used; these originated from 15 patients who were culture-positive for S. Typhi and 15 healthy controls, together with 300 sera submitted to the Laboratory of Enteric Pathogens for Salmonella serodiagnosis. By SDS-PAGE/immunoblotting, all 15 sera from culture-positive patients had serum antibodies to the 9,12 LPS antigens and 10 had antibodies to the 'd' flagellar antigens. Of the 300 reference sera, 22 had antibodies to the 9,12 LPS antigens, one to the 1,4,5,12 LPS antigens and 12 to the 6,7 LPS antigens. Only two sera had antibodies to flagellar antigens, one of which bound to the 'b' and the other to the 'd' antigen. An ELISA was developed that successfully detected serum antibodies to the Vi capsular polysaccharides, but because of the kinetics of serum antibody production to the Vi, these antibodies may be of limited value in the serodiagnosis of acute infection with S. Typhi and S. Paratyphi C. The immunoassays described here provide a sensitive means of detecting serum antibodies to the LPS, flagellar and Vi antigens of S. Typhi and S. Paratyphi, and constitute a viable replacement for the Widal assay for the screening of sera. The Salmonella serodiagnosis protocols described here are the new standard operating procedures used by the Health Protection Agency's National Salmonella Reference Centre based in the Laboratory of Enteric Pathogens, Colindale, UK.

[Retrospective study on the isolated strains of Salmonella in an Iranian hospital in Kermanshah]. / Etude rétrospective des souches de Salmonella isolées à l'hôpital iranien de Kermanchah.

A survey of occurrence of Salmonella in blood and bone marrow cultures was conducted in 1989-1990 and 1999-2000 (Sina hospital, Kermanshah). A total of 496 (12.3%) and 60 (2.4%) Salmonella strains were isolated (from 4020 and 2447 cultures). In 1989-1990, the isolated strains were: S. typhi 448 (98.5%), S. paratyphi A 40 (8%), S. paratyphi B 5 (1%) and S. paratyphi C 3 (0.5%). In 1999-2000, the isolated strains were S. typhi 59 (98%) and S. paratyphi B 1 (1.5%). There was a 60.9% reduction in the number of specimens over the 2 periods. The rate of Salmonella isolation fell from 12.3% (1989-1990) to 2.4% (1999-2000). There was a 10.2, 8.3 and 6.6 times increase in resistance of S. typhi strains to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole respectively.

Case Series of Imported Enteric Fever at a Referral Center in Tokyo, Japan: Antibiotic Susceptibility and Risk Factors for Relapse.

Owing to the increase in Salmonella strains with decreased fluoroquinolone susceptibility in the endemic areas, we have been treating enteric fever with intravenous ceftriaxone empirically since 2007. In this study, we reevaluated our treatment protocol. This retrospective cohort study was conducted at a single institute in Tokyo, Japan, between January 2006 and December 2013. Enteric fever was defined as isolation of Salmonella Typhi or Salmonella Paratyphi A, B, and C from the blood and/or stool of patients with fever. Of the 35 patients with imported enteric fever, 28 (80%) had returned from south Asia. Ciprofloxacin-susceptible strains were detected in only 12% of the cases. The isolates showed excellent susceptibility to ampicillin (91%), chloramphenicol (94%), ceftriaxone (97%), and azithromycin (97%). One case of Salmonella Paratyphi B was excluded, and of the remaining 34 patients, 56% were treated with ceftriaxone alone, 26% with ceftriaxone then fluoroquinolone, and 9% with levofloxacin alone. The overall relapse rate was 6.1%; however, among those receiving ceftriaxone monotherapy, the relapse rate was 11% (N = 2). The relapse group was characterized by longer times to treatment initiation (P = 0.035) and defervescence (> 7 days) after treatment initiation (P = 0.022). In such cases, we recommend that ceftriaxone treatment be continued for > 4 days after defervescence or be changed to fluoroquinolone if the strains are found to be susceptible to prevent relapse. Furthermore, ampicillin and chloramphenicol, which are no longer prescribed, may be reconsidered as treatment options in Asia.

Trends in antibiotic susceptibility of enteric fever isolates in East London.

BACKGROUND: The study sought evidence for changes in the proportions of antibiotic resistant strains among isolates of Salmonella enterica serovar Typhi (S. typhi) and Salmonella enterica serovar Paratyphi (S. paratyphi) between 2005 and 2012. METHODS: Blood culture isolates of S. typhi and S. paratyphi from patients attending Newham and The Royal London Hospitals were included in the study. The organisms were cultured on selective media and identified by Maldi-ToF, API 20E and serology. Minimum inhibitory concentrations (MICs) of augmentin, chloramphenicol, co-trimoxazole, ceftriaxone, ciprofloxacin and azithromycin were determined by E tests for 194 isolates. RESULTS: Median MICs of ciprofloxacin and ceftriaxone were stable at 0.5 mg/L and 0.125 mg/L, respectively. Chloramphenicol, azithromycin, co-trimoxazole and augmentin median MICs were 4 mg/L, 8 mg/L, 0.064 mg/L and 0.5 mg/L, respectively. MIC90 values were lower than the resistant breakpoint for ceftriaxone, azithromycin and augmentin, but were >256 mg/L for chloramphenicol, 32 mg/L for co-trimoxazole and 1 mg/L for ciprofloxacin. CONCLUSIONS: Antibiotic resistance remained stable for enteric fever isolates between 2005 and 2012. The isolates remained susceptible to augmentin, ceftriaxone and azithromycin over this period, but the MIC90 was greater than the resistant breakpoint for chloramphenicol, cotrimoxazole and ciprofloxacin. The implications for clinical practice are that isolates of S. typhi and S. paratyphi from East London remain sensitive to ceftriaxone and azithromycin.

Enteric fever and its impact on returning travellers.

Enteric fever, a systemic illness, is caused by Salmonella enterica serovar Typhi or S. enterica serovar Paratyphi A, B or C. The organism is transmitted to humans by the faecal oral route and is endemic in countries with poor sanitation and lacking clean drinking water. There are around 27 million individuals infected with S. Typhi worldwide annually. Enteric fever is a particular problem in travellers to endemic areas, especially those visiting friends and relatives. Currently, the two main vaccines recommended for travellers are the Vi polysaccharide vaccine and the oral Ty21a vaccine. These internationally licensed vaccines are safe and effective against S. Typhi. However, there is currently no commercially available vaccine against S. Paratyphi, which is increasingly reported as a cause of enteric fever. Vaccine uptake and taking appropriate precautions are poor in travellers visiting friends and relatives abroad; this problem requires addressing. Ciprofloxacin is no longer recommended for empirical treatment of infection because of increasing reports of resistance, especially from South Asia. Ceftriaxone and azithromycin are currently the most commonly used antimicrobials for empirical treatment of enteric fever but resistance to both these agents is emerging.

Comparison of ViaB regions of Vi-positive organisms.

We cloned the vipR genes from Salmonella paratyphi C, S. dublin, and Citrobacter freundii strains and compared them with the S. typhi sequence to clarify the genetic relationship of the ViaB regions of Vi-positive organisms. ViaB regions were divided into two groups based on their sequences, the Salmonella and C. freundii groups. The vipR coding sequences of the Salmonella group were identical. Southern blot hybridization results using the full-length ViaB region as a probe support these findings.

Acute disseminated histoplasmosis complicated with hypercalcaemia.

A case of acute progressive disseminated histoplasmosis complicated with hypercalcemia is reported and the literature is reviewed. This and the previously reported cases imply that physicians should have a higher index of suspicion for this infection and the probable underlying diseases resulting from impaired cellular-mediated immunity when encountering patients with hypercalcaemia.

Extraintestinal infection by group C Salmonella: a case report and review of the literature.

Infected or mycotic aneurysms of the aorta are not very frequent but they are associated with high morbidity and mortality rates. Vascular infections due to Salmonella are not very frequent, but in recent years the reports of infections of this type have been on the increase. The authors report their experience with a case of aneurysm of the abdominal aorta infected by group C Salmonella and go on to review the literature on the subject.

Diffuse abdominal gallium-67 citrate uptake in salmonella infections.

Two pediatric patients with salmonella infections (one with typhoid fever and the second with salmonella C2 gastroenteritis), had a diffuse abdominal uptake of Ga-67 citrate. The possible explanation for this finding is discussed. Salmonella infection should be included as a cause in the differential diagnosis of diffuse accumulation of Ga-67 citrate.