Ceftriaxone resistant Salmonella enterica serovar Paratyphi A identified in a case of enteric fever: first case report from Pakistan.

BACKGROUND: Enteric fever is an acute systemic infectious disease associated with substantial morbidity and mortality in low- and middle-income countries (LMIC), with a global burden of 14.3 million cases. Cases of enteric fever or paratyphoid fever, caused by Salmonella enterica serovar Paratyphi A (S. Para A) have been found to rise in many endemic and non-endemic countries. Drug resistance is relatively uncommon in S. Para A. Here we report a case of paratyphoid fever caused by ceftriaxone resistant S. Para A from Pakistan. CASE PRESENTATION: A 29-year-old female presented with a history of fever, headache, and shivering. Her blood culture revealed a S. Para A isolate (S7), which was resistant to ceftriaxone, cefixime, ampicillin and ciprofloxacin. She was prescribed oral Azithromycin for 10 days, which resulted in resolution of her symptoms. Two other isolates of S. Para A (S1 and S4), resistant to fluoroquinolone were also selected for comparison. DST and whole genome sequencing was performed for all three isolates. Sequence analysis was performed for identification of drug resistance and phylogeny. Whole Genome Sequencing (WGS) of S7 revealed the presence of plasmids, IncX4 and IncFIB(K). blaCTX-M-15 and qnrS1 genes were found on IncFIB(K). The gyrA S83F mutation conferring fluoroquinolone resistance was also found present. Multi-locus sequence typing (MLST) showed the S7 isolate to belong to ST129. S1 and S4 had the gyrA S83Y and S83F mutations respectively. CONCLUSIONS: We highlight the occurrence of plasmid-mediated ceftriaxone resistant strain of S. Para A. This is of significance as ceftriaxone is commonly used to treat paratyphoid fever and resistance in S. Para A is not known. Continuous epidemiological surveillance is required to monitor the transmission and spread of antimicrobial resistance (AMR) among Typhoidal Salmonellae. This will guide treatment options and preventive measures including the need for vaccination against S. Para A in the region.

Anti quorum sensing and anti virulence activity of tannic acid and it's potential to breach resistance in Salmonella enterica Typhi / Paratyphi A clinical isolates.

Salmonella enterica Typhi and Paratyphi A are food borne pathogens causing typhoid, which is one of the most important food borne disease in the developing world. S. Typhi and S. Paratyphi A are of much concern as multi drug resistance has been on the rise. The current study is aimed to screen phytochemicals for anti quorum sensing (QS) activity against S. Typhi and S. Paratyphi A. Upon screening with swarming assay, tannic acid (TA) showed highest anti-QS activity with minimal concentration of 400µg/ml. The anti-QS activity of TA was confirmed with C. violaceum ATCC 12,472. TA showed 38-43% and 35-50% of inhibition in cell surface hydrophobicity and EPS production respectively. Through FTIR analysis, it has been observed that EPS of treated cells has a considerable change in protein and peptide. TA has also exhibited drastic reduction in the surfactant production as high as 85-90%. Blood sensitivity and antibiotic sensitivity assay revealed that TA significantly sensitizes the S. Typhi and S. Paratyphi A cells to immune components in human blood and antibiotics. It has reduced the resistance of S. Typhi and S. Paratyphi A cells against amikacin, ampicillin, ciprofloxacin, azithromycin, chloramphenicol and gentamycin, thus revitalized the usage of these antibiotics against drug resistant S. Typhi and S. Paratyphi A infections. The consistency of anti-QS potential of TA was further evaluated and established with another eight clinical isolates of S. Typhi and S. Paratyphi A. Thus TA has been proved as a promising anti QS agent that can be developed as a therapeutic combination against S. Typhi and S. Paratyphi A.

Cell mediated immune responses elicited in volunteers following immunization with candidate live oral Salmonella enterica serovar Paratyphi A attenuated vaccine strain CVD 1902.

The incidence of Salmonella enterica serovar Paratyphi A (PA) infection is on the rise and no licensed vaccines are available. We evaluated cell mediated immune (CMI) responses elicited in volunteers following immunization with a single dose (109 or 1010 cfu) of a novel attenuated live oral PA-vaccine strain (CVD 1902). Results showed increases in PA-lipopolysaccharide-specific IgG- and/or IgA B-memory cells and production of IFN-γ, TNF-α, IL-10, IL-23 and RANTES following stimulation with PA-antigens by peripheral blood mononuclear cells obtained 28 days post immunization. Flow cytometry assays revealed that vaccine elicited PA-specific CD8+ and/or CD4+ T effector/memory cells were predominantly multifunctional concomitantly expressing CD107a and/or producing IFN-γ, TNF-α and/or IL-2. Similar proportions of these MF cells expressed, or not, the gut homing marker integrin α4ß7. The results suggest that immunization with CVD 1902 elicits CMI responses against PA supporting its further evaluation as a potential vaccine candidate against paratyphoid A fever.

Chemical composition, antibacterial activity and related mechanism of valonia and shell from Quercus variabilis Blume (Fagaceae) against Salmonella paratyphi a and Staphylococcus aureus.

BACKGROUND: Plant secondary metabolites and phytochemicals that exhibit strong bioactivities have potential to be developed as safe and efficient natural antimicrobials against food contamination and addressing antimicrobial resistance caused by the overuse of chemical synthetic preservative. In this study, the chemical composition, antibacterial activities and related mechanism of the extracts of the valonia and the shell of Quercus variabilis Blume were studied to determine its potential as a safe and efficient natural antimicrobial. METHODS: The phenolic compositions of valonia and shell extracts were determined by folin-ciocalteau colourimetric method, sodium borohydride/chloranil-based assay and the aluminium chloride method and then further identified by the reverse-phase HPLC analysis. The antibacterial activities of valonia and shell extracts were evaluated by the agar disk diffusion method and agar dilution method. The related antibacterial mechanism was explored successively by the membrane of pathogens effect, phosphorous metabolism, whole-cell proteins and the microbial morphology under scanning electron microscopy. RESULTS: The n-butanol fraction and water fraction of valonia along with n-butanol fraction of the shell contains enrich phenolics including ellagic acid, theophylline, caffeic acid and tannin acid. The n-butanol fraction and ethanol crude extracts of valonia exhibited strong antibacterial activities against Salmonella paratyphi A (S. paratyphi A) and Staphylococcus aureus (S. aureus) with the DIZ values ranged from 10.89 ± 0.12 to 15.92 ± 0.44, which were greater than that of the Punica granatum (DIZ: 10.22 ± 0.18 and 10.30 ± 0.21). The MIC values of the n-butanol fraction and ethanol crude extracts of valonia against S. paratyphi A and S. aureus were 1.25 mg/ml and 0.625 mg/ml. The related antibacterial mechanism of n-butanol fraction and ethanol crude extracts of valonia may be attributed to their strong impact on membrane permeability and cellular metabolism. Those extracts exhibited strong antibacterial activity according to inhibit the synthesis of bacterial proteins and seriously change morphological structure of bacterial cells. CONCLUSIONS: The n-butanol fraction and ethanol crude extracts of valonia had reasonably good antibacterial activities against S. paratyphi A and S. aureus. This study suggests possible application of valonia and shell as natural antimicrobials or preservatives for food and medical application.

Modeling 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) Chemistry to Design Glycoconjugate Vaccines with Desired Structural and Immunological Characteristics.

Glycoconjugation is a well-established technology for vaccine development: linkage of the polysaccharide (PS) antigen to an appropriate carrier protein overcomes the limitations of PS T-independent antigens, making them effective in infants and providing immunological memory. Glycoconjugate vaccines have been successful in reducing the burden of different diseases globally. However, many pathogens still require a vaccine, and many of them display a variety of glycans on their surface that have been proposed as key antigens for the development of high-valency glycoconjugate vaccines. CDAP chemistry represents a generic conjugation strategy that is easily applied to PS with different structures. This chemistry utilizes common groups to a large range of PS and proteins, e.g., hydroxyl groups on the PS and amino groups on the protein. Here, new fast analytical tools to study CDAP reaction have been developed, and reaction conditions for PS activation and conjugation have been extensively investigated. Mathematical models have been built to identify reaction conditions to generate conjugates with wanted characteristics and successfully applied to a large number of bacterial PSs from different pathogens, e.g., Klebsiella pneumoniae, Salmonella Paratyphi A, Salmonella Enteritidis, Salmonella Typhimurium, Shighella sonnei and Shigella flexneri. Furthermore, using Salmonella Paratyphi A O-antigen and CRM197 as models, a design of experiment approach has been used to study the impact of conjugation conditions and conjugate features on immunogenicity in rabbits. The approach used can be rapidly extended to other PSs and accelerate the development of high-valency glycoconjugate vaccines.

Epidemiology of Multidrug Resistance among Salmonella enterica serovars typhi and paratyphi A at a Tertiary Pediatric Hospital in India Over a Decade; In-silico Approach to Elucidate the Molecular Mechanism of Quinolone Resistance.

BACKGROUND: Enteric fever caused by Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A remains one of the most common causes of community-acquired bloodstream infection among children in India. Multidrug resistance is emerging and is a cause of concern as it affects the choice of treatment in enteric fever. METHOD: In this study, a 10-year analysis of resistance patterns was done along with in-silico homology modeling and molecular docking to understand the commonly occurring quinolone resistance. RESULTS: A total of 1010 cases of blood culture-confirmed enteric fevers (S. typhi n=849; S. paratyphi A n=161) were recorded at the study hospital during the period from 2011-2020. Multidrug resistance among cases of S. typhi was 2.12 %, whereas it was completely absent among cases of S. paratyphi A. Fluoroquinolone resistance was high (>95%) throughout the study period. Resistance to ampicillin, chloramphenicol and co-trimoxacole was low (<3%) among S. typhi cases. No deaths were observed among study participants. Molecular docking analysis showed that quinolone had less binding affinity to mutated gyrase A than to its wild type for both S. typhi and S. paratyphi A. CONCLUSION: Quinolone resistance was high among cases of enteric fever, whereas no resistance was observed among third-generation cephalosporins. In-silico studies indicated that a mutation in gyrase A might be the cause of the gradual increase in ciprofloxacin resistance over the study period.

SopA inactivation or reduced expression is selected in intracellular Salmonella and contributes to systemic Salmonella infection.

Pseudogenes are accumulated in host-restricted Salmonella enterica serovars, while pseudogenization is primarily regarded as a process that purges unnecessary genes from the genome. Here we showed that the inactivation of sopA, which encodes an effector of Salmonella Pathogenicity Island 1, in human-restricted S. enterica serovar Typhi (S. Ty) and Paratyphi A (S. PA) is under positive selection and aimed to reduce bacterial cytotoxicity toward host macrophages. Moreover, we found that the expression of sopA in Salmonella Typhimurium (S. Tm), a broad-host-range serovar which causes systemic disease in mice, was negatively regulated during mice infection and survival in murine macrophages. The sopA repression in S. Tm is mediated by IsrM, a small RNA absent from the genome of S. Ty and S. PA. Due to the lack of IsrM, sopA expression was unregulated in S. Ty and S. PA, which might have facilitated the convergent inactivation of sopA in these two serovars. In conclusion, our findings demonstrate that sopA inactivation or intracellular repression is the target of positive selection during the systemic infection caused by S. enterica serovars.

Changes in antimicrobial resistance and molecular attributes of Salmonellae causing enteric fever in Kolkata, India, 2014-2018.

Globally, enteric fever caused by Salmonella Typhi (S. Typhi, ST) and S. Paratyphi A (SPA) remain one of the major diseases of public health importance. In this study, a total of 457 (380 ST, 77 SPA) blood isolates were collected from three tertiary care hospitals in Kolkata during 2014-18. Additionally, 66 (3.4%) ST and 5 (0.25%) SPA were recovered from blood culture of 1962 patients attending OPD of one pediatric hospital during 2016-18. The study isolates were tested for antimicrobial resistance (AMR) profiles; AMR genes; molecular sub-types by PFGE, MLVA and CRISPR. Among the total 446 ST and 82 SPA isolates, fluoroquinolone (FQ) resistance was very common in both serovars. Ciprofloxacin resistance of 24.9% and 9.8% & ofloxacin resistance of 20.9% and 87.8% were found in ST and SPA respectively. Majority (>70%) of the isolates showed decreased susceptibility to ciprofloxacin (DCS). A single point mutation in gyrA gene (S83F) was responsible for causing DCS in 37.5% (n = 42/112) ST and 63% (n = 46/73) SPA isolates. Multidrug resistance (MDR) was found only in 3.4% ST isolates and encoded the genes blaTEM-1, catA, sul, strA-strB, class 1 integron with dfrA7. All MDR ST (n = 15) possessed non-conjugative non-IncHI1 (180 kb) plasmid except one having conjugative IncHI1 (230 kb) plasmid and one without plasmid. The MDR genes were integrated near chromosomal cyaA gene site in ST with/without the presence of plasmid (nonIncH1). Almost 65.7% resistant ST belonged to H58 haplotype. PFGE showed clonally related isolates with 81% similarity in ST and 87% in SPA. Similarly, CRISPR typing showed less diversity among the isolates. However, the isolates (ST and SPA) were found to be more diverse by MLVA typing (D value 0.987 and 0.938). The study reports decrease in MDR and increase in FQ resistance among typhoidal Salmonella isolates over the years giving interesting information for enteric fever treatment.

Evaluation of a Rapid Point-of-Care Multiplex Immunochromatographic Assay for the Diagnosis of Enteric Fever.

There is a critical need for an improved rapid diagnostic for enteric fever. We have previously demonstrated that serum IgA responses targeting Salmonella enterica serovar Typhi hemolysin E (HlyE) and lipopolysaccharide (LPS) are able to discriminate patients with acute typhoid from healthy controls in areas where enteric fever is endemic (healthy endemic controls) and from patients with other bacterial infections. We now have data demonstrating that IgA antibody responses against these antigens also work well for identifying patients with acute S. Paratyphi A infection. To develop a test for acute enteric fever detection, we have adapted a point-of-care immunochromatographic dual-path platform technology (DPP), which improves on the traditional lateral flow technology by using separate sample and conjugate paths and a compact, portable reader, resulting in diagnostics with higher sensitivity and multiplexing abilities. In this analysis, we have compared our standard enzyme-linked immunosorbent assay (ELISA) method to the DPP method in detecting acute phase plasma/serum anti-HlyE and anti-LPS IgA antibodies in a cohort of patients with culture-confirmed S. Typhi (n = 30) and Paratyphi A infection (n = 20), healthy endemic controls (n = 25), and febrile endemic controls (n = 25). We found that the DPP measurements highly correlated with ELISA results, and both antigens had an area under the curve (AUC) of 0.98 (sensitivity of 92%, specificity of 94%) with all controls and an AUC of 0.98 (sensitivity of 90%, specificity of 96%) with febrile endemic controls. Our results suggest that the point-of-care DPP Typhoid System has high diagnostic accuracy for the rapid detection of enteric fever and warrants further evaluation.IMPORTANCE Enteric fever remains a significant global problem, and control programs are significantly limited by the lack of an optimal assay for identifying individuals with acute infection. This is especially critical considering the recently released World Health Organization (WHO) position paper endorsing the role of the typhoid conjugate vaccine in communities where enteric fever is endemic. A reliable diagnostic test is needed to assess and evaluate typhoid intervention strategies and determine which high-burden areas may benefit most from a vaccine intervention. Our collaborative team has developed and evaluated a point-of-care serodiagnostic assay based on detection of anti-HlyE and LPS IgA. Our finding of the high diagnostic accuracy of the DPP Typhoid System for the rapid detection of enteric fever has the potential to have significant public health impact by allowing for improved surveillance and for control and prevention programs in areas with limited laboratory capacity.

Attenuated Salmonella Typhimurium expressing Salmonella Paratyphoid A O-antigen induces protective immune responses against two Salmonella strains.

Salmonella enterica serovar Paratyphi A is the main causative agent of paratyphoid fever in many Asian countries. As paratyphoid is spread by the fecal-oral route, the most effective means of controlling S. Paratyphi A infection is through the availability of clean water supplies and working sanitation services. Because sanitation facilities improve slowly in these poor areas and antibiotic resistance is severe, the development of a safe and effective vaccine remains a priority for controlling the spread of paratyphoid disease. In this study, we investigated the strategy of heterologous O-antigenic O2 serotype (S. Paratyphi A characterized) conversion in S. Typhimurium to prevent paratyphoid infections. A series of S. Typhimurium mutants were constructed with replacement of abe, wzxB1 and wbaVB1 genes with respective prt-tyvA1, wzxA1 and wbaVA1, and the results showed that only three genes including prt, wbaVA1 and wzxA1 from S. Paratyphi A presence enable S. Typhimurium to sufficiently express O2 antigen polysaccharide. We also constructed a series of live attenuated S. Typhimurium vaccine candidates expressing heterologous O2 O-antigens, and a mouse model was used to evaluate the immunogenicity of live vaccines. ELISA data showed that vaccine candidates could induce a comparatively high level of S. Paratyphi A and/or S. Typhimurium LPS-specific IgG and IgA responses in murine model, and IgG2a levels were consistently higher than IgG1 levels. Moreover, the functional properties of serum antibodies were evaluated using in vitro C3 complement deposition and opsonophagocytic assays. Our work highlights the potential for developing S. Typhimurium live vaccines against S. Paratyphi A.

Comparative genomic analysis between typhoidal and non-typhoidal Salmonella serovars reveals typhoid-specific protein families.

BACKGROUND: The genus Salmonella contains more than 2600 serovars. While most cause a self-limiting gastroenteritis, four serovars, S. Typhi, S. Paratyphi A, B and C, elicit typhoid, a potentially fatal systemic infection. Because of the prevalence in certain regions, such as South Asia, and the disease severity of typhoidal Salmonella infections, comprehensive studies are needed to elucidate the pathogenesis of diseases caused by these typhoidal serovars. RESULTS: We performed comparative genomic analyses on eight human typhoidal strains and 27 non-human typhoidal Salmonella strains to elucidate their evolutionary relationships and identify the genes specific to the four typhoidal serovars. Our results indicate that Salmonella may have an open pan-genome. A core-genome based phylogeny demonstrated that divergence between S. Paratyphi A and S. Typhi took place not long ago and S. Paratyphi B shared a recent common ancestor with S. Paratyphi C. Of great interest, the divergence between S. Paratyphi B and S. Paratyphi C was shown to be more recent than that between S. Paratyphi A and S. Typhi. Alignment and comparisons of the genomes identified unique complements of protein families to each of the typhoidal serovars. Most of these protein families are phage related and some are candidate virulence factors. Importantly, we found 88 protein families specific to two to three of the four typhoidal serovars. All but two of the 88 genes are present in S. Typhi, with a few in the three paratyphoidal serovars but none in the non-human typhoidal serovars. Most of these genes are predicted to encode hypothetical proteins and some are known to code for virulence factors such as Vi polysaccharide related proteins. CONCLUSIONS: By comprehensive genomic comparisons, we identified protein families specific to the human typhoidal serovars, which will greatly facilitate investigations on typhoid pathogenesis.

Synthesis and immunogenicity evaluation of Salmonella enterica serovar Paratyphi A O-specific polysaccharide conjugated to diphtheria toxoid.

Salmonella enterica serovar Paratyphi A (S. Paratyphi A) is a human restricted pathogen that can cause systemic infection (paratyphoid fever) with recently increased incidence particularly in developing countries. Currently there is no licensed vaccine for prevention of infection from S. Paratyphi A. In this study the O-specific polysaccharide (OSP) of S. Paratyphi A was conjugated to diphtheria toxoid (DT) with and without adipic acid dihydrazide (ADH) as a linker. Binding of the OSP to a carrier protein was intended to convert a T-cell independent OSP response to a T-cell dependent response inducing higher levels of anti-OSP antibodies and immunological memory. These conjugates (OSP-AH-DT and OSP-DT) were evaluated for their immunogenicity in mice. The S. Paratyphi A OSP-DT conjugate induced a poor anti-OSP response less than that observed with LPS while the OSP-AH-DT conjugate induced a significantly higher antibody titer compared with LPS alone. The study also demonstrated diphtheria toxoid as a potential carrier protein for conjugate vaccine candidates using S. Paratyphi A OSP.

Mix-infection of S. Typhi and ParaTyphi A in Typhoid Fever and Chronic Typhoid Carriers: A Nested PCR Based Study in North India.

INTRODUCTION: Enteric fever is a systemic disease caused by Salmonella organism such as serotypes Typhi and ParaTyphi A, B, C. Salmonella ParaTyphi A contributes more than 50% of all the enteric fever cases and it has recently been projected as an emerging pathogen. MATERIALS AND METHODS: The present study was aimed to detect Salmonella Typhi and ParaTyphi A in urine, blood and stool specimens collected from cases of enteric fever (110), chronic typhoid carriers (46) and healthy controls (75) to explore the possibility of mixed infection by nested PCR. A new nested PCR primer was designed targeting putative fimbrial protein (stkG) gene which is one of the fimbrial gene families to Salmonella ParaTyphi A and for S. Typhi already reported primers targeting flagellin (fliC) gene. RESULTS: Large volume of urine specimens (15 ml) was found to be the best for detection of Salmonella serotypes. The urine sample was found to have mixed-infection by both the serotypes in 40.9% of the cases but lower in blood (27.3%) and stool (13.6%). CONCLUSION: The present study concludes that occurrence of mixed infection may be quite frequent in typhoid and chronic typhoid carriers' individuals, although the reported recent rise in ParaTyphi A incidence may not be real.