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.

Seroincidence of Enteric Fever, Juba, South Sudan.

We applied a new serosurveillance tool to estimate typhoidal Salmonella burden using samples collected during 2020 from a population in Juba, South Sudan. By using dried blood spot testing, we found an enteric fever seroincidence rate of 30/100 person-years and cumulative incidence of 74% over a 4-year period.

Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death.

Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD-mediated pyroptosis via activation of caspase-1, caspase-4 and caspase-8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island-1 (SPI-1) injectisome, HilA-mediated overexpression of the SPI-1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O-antigen length regulator, which induces the production of very long O-antigen chains. Using a ΔfepE mutant we established that the very long O-antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome-mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar-specific inflammasome modulation, which mirrors the pro- and anti-inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome-mediated detection through the elaboration of very long LPS O-polysaccharides.

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.

Salmonella Paratyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever.

Typhoid and paratyphoid fevers have a high incidence worldwide and coexist in many geographical areas, especially in low-middle-income countries (LMIC) in South and Southeast Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized modules for membrane antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment. Here, we engineered S Paratyphi A using the pDC5-viaB plasmid to generate GMMA displaying the heterologous S Typhi Vi antigen together with the homologous O:2 O antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells, and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2, and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars, which could benefit LMIC residents and travelers.

Engineering a Novel Bivalent Oral Vaccine against Enteric Fever.

Enteric fever is a major global healthcare issue caused largely by Salmonella enterica serovars Typhi and Paratyphi A. The objective of this study was to develop a novel, bivalent oral vaccine capable of protecting against both serovars. Our approach centred on genetically engineering the attenuated S. Typhi ZH9 strain, which has an excellent safety record in clinical trials, to introduce two S. Paratyphi A immunogenic elements: flagellin H:a and lipopolysaccharide (LPS) O:2. We first replaced the native S. Typhi fliC gene encoding flagellin with the highly homologous fliC gene from S. Paratyphi A using Xer-cise technology. Next, we replaced the S. Typhi rfbE gene encoding tyvelose epimerase with a spacer sequence to enable the sustained expression of O:2 LPS and prevent its conversion to O:9 through tyvelose epimerase activity. The resulting new strain, ZH9PA, incorporated these two genetic changes and exhibited comparable growth kinetics to the parental ZH9 strain. A formulation containing both ZH9 and ZH9PA strains together constitutes a new bivalent vaccine candidate that targets both S. Typhi and S. Paratyphi A antigens to address a major global healthcare gap for enteric fever prophylaxis. This vaccine is now being tested in a Phase I clinical trial (NCT04349553).

Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies.

Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

Overview of the Nontyphoidal and Paratyphoidal Salmonella Vaccine Pipeline: Current Status and Future Prospects.

Nontyphoidal Salmonella and Salmonella Paratyphi are responsible for significant morbidity and mortality worldwide. To date, no vaccine has been licensed against these organisms. The development of effective vaccines remains an urgent priority. In this review, the rationale for and current status of various vaccine candidates against S. Paratyphi and nontyphoidal Salmonella are presented, with a focus on the research findings from the 2019 International Conference on Typhoid and Other Invasive Salmonelloses. Additionally, other vaccine candidates that are currently undergoing clinical development are highlighted. Future approaches, which may include antigens that are genetically conserved across Salmonella and confer broad, non-serotype-specific protection, are also discussed.

Review of Methods Suitable for Environmental Surveillance of Salmonella Typhi and Paratyphi.

Typhoid fever is an enteric disease caused by the pathogens Salmonella Typhi and Salmonella Paratyphi. Clinical surveillance networks are lacking in many affected areas, thus presenting a need to understand transmission and population prevalence. Environmental surveillance (ES) has been suggested as a potentially effective method in the absence of (or in supplement to) clinical surveillance. This review summarizes methods identified in the literature for sampling and detection of typhoidal Salmonella from environmental samples including drinking water, wastewater, irrigation water, and surface waters. Methods described use a trap or grab sampling approach combined with various selective culture and molecular methods. The level to which the performance of identified methods is characterized for ES in the literature is variable, thus arguing for the optimization and standardization of ES techniques.

Antimicrobial Resistance in Typhoidal Salmonella: Around the World in 3 Days.

With the increasing antibacterial resistance in typhoidal Salmonella and the dearth of novel antimicrobials on the horizon, we risk losing our primary defense against widespread morbidity and mortality from enteric fever. During 26-28 March 2019, researchers from around the world came together in Hanoi, Vietnam, and shared some of their latest findings on antimicrobial resistance. From the 258 abstracts presented at the conference, at least 50 discussed phenotypic and genotypic characteristics of antimicrobial resistance in typhoidal Salmonella, covering data of at least 24 different countries, spanning 5 continents. Here, we summarize the key findings, focusing on our global journey ahead.