Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation.

Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars are exclusively adapted to the human host, where they can cause life-long persistent infection. A distinct feature of these serovars is the presence of a relatively high number of degraded coding sequences coding for metabolic pathways, most likely a consequence of their adaptation to a single host. As a result of convergent evolution, these serovars shared many of the degraded coding sequences although often affecting different genes in the same metabolic pathway. However, there are several coding sequences that appear intact in one serovar while clearly degraded in the other, suggesting differences in their metabolic capabilities. Here, we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. Thus, the inability of S. Typhi to metabolize Glucose-6-Phosphate or 3-phosphoglyceric acid is due to the silencing of the expression of the genes encoding the transporters for these compounds due to point mutations in the transcriptional regulatory proteins. In contrast, its inability to utilize glucarate or galactarate is due to the presence of point mutations in the transporter and enzymes necessary for the metabolism of these sugars. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars and highlight a limitation of bioinformatic approaches to predict metabolic capabilities. IMPORTANCE: Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars can only infect the human host, where they can cause life-long persistent infection. Because of their adaptation to the human host, these bacterial pathogens have changed their metabolism, leading to the loss of their ability to utilize certain nutrients. In this study we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars.

Estimating the subnational prevalence of antimicrobial resistant Salmonella enterica serovars Typhi and Paratyphi A infections in 75 endemic countries, 1990-2019: a modelling study.

BACKGROUND: Enteric fever, a systemic infection caused by Salmonella enterica serovars Typhi and Paratyphi A, remains a major cause of morbidity and mortality in low-income and middle-income countries. Enteric fever is preventable through the provision of clean water and adequate sanitation and can be successfully treated with antibiotics. However, high levels of antimicrobial resistance (AMR) compromise the effectiveness of treatment. We provide estimates of the prevalence of AMR S Typhi and S Paratyphi A in 75 endemic countries, including 30 locations without data. METHODS: We used a Bayesian spatiotemporal modelling framework to estimate the percentage of multidrug resistance (MDR), fluoroquinolone non-susceptibility (FQNS), and third-generation cephalosporin resistance in S Typhi and S Paratyphi A infections for 1403 administrative level one districts in 75 endemic countries from 1990 to 2019. We incorporated data from a comprehensive systematic review, public health surveillance networks, and large multicountry studies on enteric fever. Estimates of the prevalence of AMR and the number of AMR infections (based on enteric fever incidence estimates by the Global Burden of Diseases study) were produced at the country, super-region, and total endemic area level for each year of the study. FINDINGS: We collated data from 601 sources, comprising 184 225 isolates of S Typhi and S Paratyphi A, covering 45 countries over 30 years. We identified a decline of MDR S Typhi in south Asia and southeast Asia, whereas in sub-Saharan Africa, the overall prevalence increased from 6·0% (95% uncertainty interval 4·3-8·0) in 1990 to 72·7% (67·7-77·3) in 2019. Starting from low levels in 1990, the prevalence of FQNS S Typhi increased rapidly, reaching 95·2% (91·4-97·7) in south Asia in 2019. This corresponded to 2·5 million (1·5-3·8) MDR S Typhi infections and 7·4 million (4·7-11·3) FQNS S Typhi infections in endemic countries in 2019. The prevalence of third-generation cephalosporin-resistant S Typhi remained low across the whole endemic area over the study period, except for Pakistan where prevalence of third-generation cephalosporin resistance in S Typhi reached 61·0% (58·0-63·8) in 2019. For S Paratyphi A, we estimated low prevalence of MDR and third-generation cephalosporin resistance in all endemic countries, but a drastic increase of FQNS, which reached 95·0% (93·7-96·1; 3·5 million [2·2-5·6] infections) in 2019. INTERPRETATION: This study provides a comprehensive and detailed analysis of the prevalence of MDR, FQNS, and third-generation cephalosporin resistance in S Typhi and S Paratyphi A infections in endemic countries, spanning the last 30 years. Our analysis highlights the increasing levels of AMR in this preventable infection and serves as a resource to guide urgently needed public health interventions, such as improvements in water, sanitation, and hygiene and typhoid fever vaccination campaigns. FUNDING: Fleming Fund, UK Department of Health and Social Care; Wellcome Trust; and Bill and Melinda Gates Foundation.

Sensorineural hearing loss as an atypical presentation of typhoid fever: A case report.

Typhoid fever, also known as enteric fever, is a multisystemic infection primarily caused by Salmonella enterica serotype Typhi, and less commonly by Salmonella enterica serotypes Paratyphi A, B, and C. The classic presentation includes fever, malaise, diffuse abdominal pain, and constipation. If left untreated, typhoid fever can progress to delirium, obtundation, intestinal haemorrhage, bowel perforation, and death within a month of onset. However, the clinical course can deviate from the classic stepladder fever pattern, which now occurs in as few as 12% of cases.1 In this report, we describe an atypical presentation as sensorineural hearing loss in an otherwise healthy young male.

Flagella-mediated cytosolic motility of Salmonella enterica Paratyphi A aids in evasion of xenophagy but does not impact egress from host cells.

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal serovars like Paratyphi A (SPA) are human restricted and cause severe systemic diseases, while many serovars like Typhimurium (STM) have a broad host range, and usually lead to self-limiting gastroenteritis. There are key differences between typhoidal and non-typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Transcriptomes and phenotypes in epithelial cells revealed induction of motility, flagella and chemotaxis genes for SPA but not STM. SPA exhibited cytosolic motility mediated by flagella. In this study, we applied single-cell microscopy to analyze triggers and cellular consequences of cytosolic motility. Live-cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at invasion sites led to increased membrane damage in nascent Salmonella-containing vacuole, and subsequent cytosolic release. After release into the cytosol, motile bacteria showed the same velocity as under culture conditions in media. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Prior work showed that SPA does not use flagella-mediated motility for cell exit via the intercellular spread. However, cytosolic motile SPA was invasion-primed if released from host cells. Our results reveal flagella-mediated cytosolic motility as a possible xenophagy evasion mechanism that could drive disease progression and contributes to the dissemination of systemic infection.

The Identification of Enteric Fever-Specific Antigens for Population-Based Serosurveillance.

BACKGROUND: Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low- and middle-income countries. Moderate sensitivity and scalability of current methods likely underestimate enteric fever burden. Determining the serological responses to organism-specific antigens may improve incidence measures. METHODS: Plasma samples were collected from blood culture-confirmed enteric fever patients, blood culture-negative febrile patients over the course of 3 months, and afebrile community controls. A panel of 17 Salmonella Typhi and Paratyphi A antigens was purified and used to determine antigen-specific antibody responses by indirect ELISAs. RESULTS: The antigen-specific longitudinal antibody responses were comparable between enteric fever patients, patients with blood culture-negative febrile controls, and afebrile community controls for most antigens. However, we found that IgG responses against STY1479 (YncE), STY1886 (CdtB), STY1498 (HlyE), and the serovar-specific O2 and O9 antigens were greatly elevated over a 3-month follow up period in S. Typhi/S. Paratyphi A patients compared to controls, suggesting seroconversion. CONCLUSIONS: We identified a set of antigens as good candidates to demonstrate enteric fever exposure. These targets can be used in combination to develop more sensitive and scalable approaches to enteric fever surveillance and generate invaluable epidemiological data for informing vaccine policies. CLINICAL TRIAL REGISTRATION: ISRCTN63006567.

Vaccine value profile for Salmonella enterica serovar Paratyphi A.

In Asia, there are an estimated 12 million annual cases of enteric fever, a potentially fatal systemic bacterial infection caused by Salmonella enterica serovars Typhi (STy) and Paratyphi A (SPA). The recent availability of typhoid conjugate vaccines (TCV), an increasing incidence of disease caused by SPA and growing antimicrobial resistance (AMR) across the genus Salmonella makes a bivalent STy/SPA vaccine a useful public health proposition. The uptake of a stand-alone paratyphoid vaccine is likely low thus, there is a pipeline of bivalent STy/SPA candidate vaccines. Several candidates are close to entering clinical trials, which if successful should facilitate a more comprehensive approach for enteric fever control. Additionally, the World Health Organization (WHO) has made advancing the development of vaccines that protect young children and working aged adults against both agents of enteric fever a priority objective. This "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by SPA prevalent in Asia. Information is included on stand-alone SPA candidate vaccines and candidate vaccines targeting SPA combined with STy. Out of scope for the first version of this VVP is a wider discussion on the development of a universal Salmonella combination candidate vaccine, addressing both enteric fever and invasive non-typhoidal Salmonella disease, for use globally. This VVP is a detailed, high-level assessment of existing, publicly available information to inform and contextualize the public health, economic, and societal potential of pipeline vaccines and vaccine-like products for SPA. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and "Full Vaccine Value Assessment" that will inform the value proposition of an SPA vaccine. This VVP was developed by an expert working group from academia, non-profit organizations, public-private partnerships, and multi-lateral organizations as well as in collaboration with stakeholders from the WHO South-East Asian Region. All contributors have extensive expertise on various elements of the VVP for SPA and collectively aimed to identify current research and knowledge gaps.

Trends in antimicrobial resistance amongst Salmonella Paratyphi A isolates in Bangladesh: 1999-2021.

BACKGROUND: Typhoid and paratyphoid remain common bloodstream infections in areas with suboptimal water and sanitation infrastructure. Paratyphoid, caused by Salmonella Paratyphi A, is less prevalent than typhoid and its antimicrobial resistance (AMR) trends are less documented. Empirical treatment for paratyphoid is commonly based on the knowledge of susceptibility of Salmonella Typhi, which causes typhoid. Hence, with rising drug resistance in Salmonella Typhi, last-line antibiotics like ceftriaxone and azithromycin are prescribed for both typhoid and paratyphoid. However, unlike for typhoid, there is no vaccine to prevent paratyphoid. Here, we report 23-year AMR trends of Salmonella Paratyphi A in Bangladesh. METHODS: From 1999 to 2021, we conducted enteric fever surveillance in two major pediatric hospitals and three clinics in Dhaka, Bangladesh. Blood cultures were performed at the discretion of the treating physicians; cases were confirmed by culture, serological and biochemical tests. Antimicrobial susceptibility was determined following CLSI guidelines. RESULTS: Over 23 years, we identified 2,725 blood culture-confirmed paratyphoid cases. Over 97% of the isolates were susceptible to ampicillin, chloramphenicol, and cotrimoxazole, and no isolate was resistant to all three. No resistance to ceftriaxone was recorded, and >99% of the isolates were sensitive to azithromycin. A slight increase in minimum inhibitory concentration (MIC) is noticed for ceftriaxone but the current average MIC is 32-fold lower than the resistance cut-off. Over 99% of the isolates exhibited decreased susceptibility to ciprofloxacin. CONCLUSIONS: Salmonella Paratyphi A has remained susceptible to most antibiotics, unlike Salmonella Typhi, despite widespread usage of many antibiotics in Bangladesh. The data can guide evidence-based policy decisions for empirical treatment of paratyphoid fever, especially in the post typhoid vaccine era, and with the availability of new paratyphoid diagnostics.

Environmental sampling for typhoidal Salmonellas in household and surface waters in Nepal identifies potential transmission pathways.

INTRODUCTION: Salmonella Typhi and Salmonella Paratyphi, fecal-oral transmitted bacterium, have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu, Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive for Salmonella Typhi and 70% for Salmonella Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease's dominant pathway of transmission remains unanswered. METHODS: We used environmental surveillance to detect Salmonella Typhi and Salmonella Paratyphi A DNA from potential sources of transmission. We collected 370, 1L drinking water samples from a population-based random sample of households in the Kathmandu and Kavre Districts of Nepal between February and October 2019. Between November 2019 and July 2021, we collected 380, 50mL river water samples from 19 sentinel sites on a monthly interval along the rivers leading through the Kathmandu and Kavre Districts. We processed drinking water samples using a single qPCR and processed river water samples using differential centrifugation and qPCR at 0 and after 16 hours of liquid culture enrichment. A 3-cycle threshold (Ct) decrease of Salmonella Typhi or Salmonella Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure. RESULTS: Among 370 drinking water samples, Salmonella Typhi was detected in 7 samples (1.8%) and Salmonella Paratyphi A was detected in 4 (1.0%) samples. Among 380 river water samples, Salmonella Typhi was detected in 171 (45%) and Salmonella Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive for Salmonella Typhi 12% of the time while samples from locations in and downstream were positive 58% and 67% of the time respectively. Individuals were observed bathing, washing clothes, and washing vegetables in the rivers. IMPLICATIONS: These results suggest that drinking water was not the dominant pathway of transmission of Salmonella Typhi and Salmonella Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems represent an important source of typhoid exposure in Kathmandu.

Can We Restart the First-Line Antibiotic as Empirical Choice When Enteric Fever is Suspected?

Enteric fever is a major health problem in Bangladesh. Antibiotic resistance especially against first-line antibiotics is a major concern in the management and thereby not practicing by physician as first choice thinking their resistance. This retrospective study was carried out in the Department of Microbiology, Bangladesh Medical College Hospital, Dhaka, Bangladesh from January of 2017 to December of 2019 to identify the year wise sensitivity pattern of first-line antibiotics like Amoxycillin, Cotrimoxazole and Chloramphenocol against Salmonella typhi and Salmonella paratyphi. All the blood samples sent for culture and sensitivity were evaluated to see the microbiom and their sensitivity pattern. Salmonella typhi and paratyphi were the major isolates in last 3 years which were 73.74% and 15.32% respectably. Sensitivity pattern of Amoxycillin, Cotrimoxazole and Chloramphenocol for Salmonella typhi is increased from 2017 to 2019 which were 66.0 to 83.0%, 80.0 to 83.0% and 84.0 to 85.0% respectively. Similar increasing pattern of sensitivity found in Salmonella paratyphi which was 82.5 to 89.2%, 76.2 to 95.4%, 98.7 to 98.5% respectively. They were also found highly sensitive (>90.0%) to 3rd generation cephalosporin. This study recommends the use of first-line antibiotics as empirical agent of choice in enteric fever as they are still highly sensitive against Salmonella typhi and Salmonella paratyphi.

A pattern of antibiotic drug resistance of Salmonella Typhi and Salmonella Paratyphi among children with enteric fever in a tertiary care hospital in Lahore, Pakistan.

AIM: To establish the pattern of antibiotic resistance and assess the frequency of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Salmonella Typhi and Salmonella Paratyphi among children with enteric fever. METHODS: This cross-sectional study was carried out in the Department of Pediatrics, Sharif Medical City Hospital, Lahore, from July 2020 to January 2021. The study involved patients aged between 0 to 15 years who attended our outpatient department or were admitted to the ward with the suspicion of typhoid fever. A convenience sample of patients with blood cultures positive for S. Typhi and S. Paratyphi was enrolled. RESULTS: Of the 105 participants, 70 (66.7%) were male. The mean age was 8.48±4.18 years, and the most affected age group was 6-10 years (n=46, 43.8%). Among the cultured organisms, 95 (90.5%) isolates were S. Typhi and 10 (9.5%) were S. Paratyphi A. Antibiotic resistance was highest against ampicillin (n=91, 86.7%), and all of the isolates were sensitive to imipenem and meropenem. Twenty-three (21.9%) cultured organisms were MDR and 54 (56.8%) were XDR. CONCLUSION: An alarming antibiotic drug resistance pattern was observed among children with enteric fever in Lahore. The lowest resistance was noted for azithromycin, meropenem, and imipenem. Our findings warrant the immediate implementation of tailored antibiotic stewardship and infection control strategies.

Bacterial ghost cell based bivalent candidate vaccine against Salmonella Typhi and Salmonella Paratyphi A: A prophylactic study in BALB/c mice.

Typhoid and emerging paratyphoid fever are a severe enteric disease worldwide with high morbidity and mortality. Licensed typhoid vaccines are in the market, but no paratyphoid vaccine is currently available. In the present study we developed a bivalent vaccine against Salmonella Typhi and Paratyphi A using a bacterial ghost platform. Bacterial ghost cells (BGs) are bacteria-derived cell membranes without cytoplasmic contents that retain their cellular morphology, including all cell surface features. Furthermore, BGs have inherent adjuvant properties that promote an enhanced humoral and cellular immune reaction to the target antigen. Sodium hydroxide was used to prepare ghost cells of Salmonella Typhi and Paratyphi A. The bacterial ghost cells were characterised using electron microscopy. Then BALB/c mice were immunized three times (0th, 14th and 28th day) with the bivalent typhoidal bacterial ghost cells. Haematological study of adult mice throughout immunization period reflected that the immunogen was safe to administer and does not affect the animals' health. After complete immunization, we found significant serum antibody titter against whole cell lysate, outer membrane protein and lipopolysaccharide of both bacteria, and cell-mediated immunity was observed in an ex-vivo experiment. CD4+, CD8a+ and CD19+ splenic cell populations were increased in immunized animals. Bivalent Typhoidal ghost cell immunized mice showed better survival, less bacterial colonization in systemic organs, and less inflammation and/or destruction of tissue in histopathological analysis than non-immunized control mice.Serum antibodies of immunized animals can significantly inhibit bacterial motility and mucin penetration ability with better killing properties against Salmonella Typhi and Paratyphi A. Furthermore, significant passive protection was observed through the adoptive transfer of serum antibody and lymphocytes of immunized animals to naïve animals after bacterial infection. In summary, Bivalent Typhoidal Bacterial Ghost cells (BTBGs) enhances immunogenic properties and serves as a safe and effective prevention strategy against Salmonella Typhi and Paratyphi A.

Retrospective Review of Blood Culture-Confirmed Cases of Enteric Fever in Navi Mumbai, India: 2014-2018.

India has one of the highest estimated burdens of enteric fever globally. Prior to the implementation of Typbar-TCV typhoid conjugate vaccine (TCV) in a public sector pediatric immunization campaign in Navi Mumbai, India, we conducted a retrospective review of blood culture-confirmed cases of typhoid and paratyphoid fevers to estimate the local burden of disease. This review included all blood cultures processed at a central microbiology laboratory, serving multiple hospitals, in Navi Mumbai (January 2014-May 2018) that tested positive for either Salmonella Typhi or Salmonella Paratyphi A. Of 40,670 blood cultures analyzed, 1,309 (3.2%) were positive for S. Typhi (1,201 [92%]) or S. Paratyphi A (108 [8%]). Culture positivity was highest in the last months of the dry season (April-June). Our findings indicate a substantial burden of enteric fever in Navi Mumbai and support the importance of TCV immunization campaigns and improved water, sanitation, and hygiene.

Salmonella enterica serovar Paratyphi A-induced immune response in Caenorhabditis elegans depends on MAPK pathways and DAF-16.

Salmonella enterica serovar Paratyphi A (S. Paratyphi A) is a pathogen that can cause enteric fever. According to the recent epidemic trends of typhoid fever, S. Paratyphi A has been the major important causative factor in paratyphoid fever. An effective vaccine for S. Paratyphi A has not been developed, which made it a tricky public health concern. Until now, how S. Paratyphi A interacts with organisms remain unknown. Here using lifespan assay, we found that S. Paratyphi A could infect Caenorhabditis elegans (C. elegans) at 25°C, and attenuate thermotolerance. The immune response of C. elegans was mediated by tir-1, nsy-1, sek-1, pmk-1, mpk-1, skn-1, daf-2 and daf-16, suggesting that S. Paratyphi A could regulate the MAPK and insulin pathways. Furthermore, we observed several phenotypical changes when C. elegans were fed S. Paratyphi A, including an accelerated decline in body movement, reduced the reproductive capacity, shortened spawning cycle, strong preference for OP50, arrested pharyngeal pumping and colonization of the intestinal lumen. The virulence of S. Paratyphi A requires living bacteria and is not mediated by secreting toxin. Using hydrogen peroxide analysis and quantitative RT-PCR, we discovered that S. Paratyphi A could increase oxidative stress and regulate the immune response in C. elegans. Our results sheds light on the infection mechanisms of S. Paratyphi A and lays a foundation for drugs and vaccine development.

Accelerating clinical development of a live attenuated vaccine against Salmonella Paratyphi A (VASP): study protocol for an observer-participant-blind randomised control trial of a novel oral vaccine using a human challenge model of Salmonella Paratyphi A infection in healthy adult volunteers.

INTRODUCTION: This is the first efficacy study of an oral live attenuated vaccine against Salmonella Paratyphi A using a human challenge model of paratyphoid infection. S. Paratyphi A is responsible for 3.3 million cases of enteric fever every year, with over 19 000 deaths. Although improvements to sanitation and access to clean water are vital to reduce the burden of this condition, vaccination offers a cost-effective, medium-term solution. Efficacy trials of potential S. Paratyphi vaccine candidates in the field are unlikely to be feasible given the large number of participants required. Human challenge models therefore offer a unique, cost-effective solution to test efficacy of such vaccines. METHODS AND ANALYSIS: This is an observer-blind, randomised, placebo-controlled trial phase I/II of the oral live-attenuated vaccine against S. Paratyphi A, CVD 1902. Volunteers will be randomised 1:1 to receive two doses of CVD 1902 or placebo, 14 days apart. One month following second vaccination all volunteers will ingest S. Paratyphi A bacteria with a bicarbonate buffer solution. They will be reviewed daily in the following 14 days and diagnosed with paratyphoid infection if the predefined microbiological or clinical diagnostic criteria are met. All participants will be treated with antibiotics on diagnosis, or at day 14 postchallenge if not diagnosed. The vaccine efficacy will be determined by comparing the relative attack rate, that is, the proportion of those diagnosed with paratyphoid infection, in the vaccine and placebo groups. ETHICS AND DISSEMINATION: Ethical approval for this study has been obtained from the Berkshire Medical Research Ethics Committee (REC ref 21/SC/0330). The results will be disseminated via publication in a peer-reviewed journal and presentation at international conferences. TRIAL REGISTRATION NUMBER: ISRCTN15485902.

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.

Genomic analysis unveils genome degradation events and gene flux in the emergence and persistence of S. Paratyphi A lineages.

Paratyphoid fever caused by S. Paratyphi A is endemic in parts of South Asia and Southeast Asia. The proportion of enteric fever cases caused by S. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory of S. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study "Surveillance of Enteric Fever in India (SEFI)." In the study period (2017-2020), S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis highlights that the genome degradation events and gene acquisitions or losses are key molecular events in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 hypothetically disrupted coding sequences (HDCS) or pseudogenes-forming mutations possibly associated with the emergence of lineages were identified. The pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We have identified six characteristic missense mutations associated with lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A, however, these mutations confer only a low structural impact and possibly have minimal impact on vaccine effectiveness. Since S. Paratyphi A is human-restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and monitoring by means of genomic surveillance would be constantly needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today.

Phylogenomic investigation of an outbreak of fluoroquinolone-resistant Salmonella enterica subsp. enterica serovar Paratyphi A in Phnom Penh, Cambodia.

In early 2020, the Medical Biology Laboratory of the Pasteur Institute of Cambodia isolated an unusually high number of fluoroquinolone-resistant Salmonella enterica subspecies enterica serovar Paratyphi A strains during its routine bacteriological surveillance activities in Phnom Penh, Cambodia. A public-health investigation was supported by genome sequencing of these Paratyphi A strains to gain insights into the genetic diversity and population structure of a potential outbreak of fluoroquinolone-resistant paratyphoid fever. Comparative genomic and phylodynamic analyses revealed the 2020 strains were descended from a previously described 2013-2015 outbreak of Paratyphi A infections. Our analysis showed sub-lineage 2.3.1 had remained largely susceptible to fluoroquinolone drugs until 2015, but acquired chromosomal resistance to these drugs during six separate events between late 2012 and 2015. The emergence of fluoroquinolone resistance was rapidly followed by the replacement of the original susceptible Paratyphi A population, which led to a dramatic increase of fluoroquinolone-resistant blood-culture-confirmed cases in subsequent years (2016-2020). The rapid acquisition of resistance-conferring mutations in the Paratyphi A population over a 3 year period is suggestive of a strong selective pressure on that population, likely linked with fluoroquinolone use. In turn, emergence of fluoroquinolone resistance has led to increased use of extended-spectrum cephalosporins like ceftriaxone that are becoming the drug of choice for empirical treatment of paratyphoid fever in Cambodia.

Antimicrobial susceptibility of bacteraemic isolates of Salmonella enterica serovar typhi and paratyphi infection in Pakistan from 2017-2020.

OBJECTIVE: To determine the antibacterial susceptibility pattern of bacteraemia isolates of Salmonella enterica serovar typhi and paratyphi. METHODS: The retrospective descriptive observational study was conducted at the Microbiology section of Dow Diagnostic Research and Reference Laboratory, and comprised blood culture reports from January 1, 2017, to Dec 30, 2020, which were screened for the presence of Salmonella typhi and paratyphi growth The frequency of the isolates and their antibiotic resistance patterns were analysed. Data was analysed using SPSS 20. RESULTS: Of the 174,190 blood culture samples, 62,709(36%) were positive for bacterial growth. Salmonella were isolated in 8,689(13.8%) samples of which 8,041(92.5%) were Salmonella typhi, 529(6%) were Salmonella paratyphi A and 119(1.3%) were Salmonella paratyphi B. There was a drastic increase in resistance to third-generation cephalosporin in Salmonella typhi from 71(12.8%) in 2017 to 1,420(71%) in 2018, 2,850(74.6%) in 2019 and 1,251(77%) in 2020. All isolates were sensitive to meropenem and azithromycin. CONCLUSIONS: A high number of extensively drug-resistant typhoid cases due to Salmonella typhi were found. All isolates were sensitive to meropenem and azithromycin.