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Background: Invasive non-typhoidal Salmonella (iNTS) serovars S. Typhimurium and S. Enteritidis are major etiologic agents of invasive bacterial disease among infants and young children in sub-Saharan Africa, including in Mali. Early studies of iNTS serovars in several countries indicated that S. Typhimurium was more prevalent than S. Enteritidis, including in Mali before 2008. We investigated genomic and associated phenotypic changes associated with an increase in the relative proportion of iNTS caused by S. Enteritidis versus S. Typhimurium in Bamako, Mali, during the period 2002–2012. Methodology/Principal findings: Comparative genomics studies identified homologs of tetracycline resistance and arsenic utilization genes that were associated with the temporal shift of serovars causing iNTS shift, along with several hypothetical proteins. These findings, validated through PCR screening and phenotypic assays, provide initial steps towards characterizing the genomic changes consequent to unknown evolutionary pressures associated with the shift in serovar prevalence. Conclusions/Significance: This work identified a shift to S. Enteritidis from the more classic S. Typhimurium, associated with iNTS in Bamako, Mali, during the period 2002–2012. This type of shift in underlying iNTS pathogens are of great importance to pediatric public health in endemic regions of sub-Saharan Africa. Additionally, this work demonstrates the utility of combining epidemiologic data, whole genome sequencing, and functional characterization in the laboratory to identify and characterize genomic changes in the isolates that may be involved with the observed shift in circulating iNTS agents. [ABSTRACT FROM AUTHOR]

In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Background: In sub-Saharan Africa, systematic surveillance of young children with suspected invasive bacterial disease (e.g., septicemia, meningitis) has revealed non-typhoidal Salmonella (NTS) to be a major pathogen exhibiting high case fatality (~20%). Where infant vaccination against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae has been introduced to prevent invasive disease caused by these pathogens, as in Bamako, Mali, their burden has decreased markedly. In parallel, NTS has become the predominant invasive bacterial pathogen in children aged <5 years. While NTS is believed to be acquired orally via contaminated food/water, epidemiologic studies have failed to identify the reservoir of infection or vehicles of transmission. This has precluded targeting food chain interventions to diminish disease transmission but conversely has fostered the development of vaccines to prevent invasive NTS (iNTS) disease. We developed a mathematical model to estimate the potential impact of NTS vaccination programs in Bamako. Methodology/Principal Findings: A Markov chain transmission model was developed utilizing age-specific Bamako demographic data and hospital surveillance data for iNTS disease in children aged <5 years and assuming vaccine coverage and efficacy similar to the existing, successfully implemented, Hib vaccine. Annual iNTS hospitalizations and deaths in children <5 years, with and without a Salmonella Enteritidis/Salmonella Typhimurium vaccine, were the model’s outcomes of interest. Per the model, high coverage/high efficacy iNTS vaccination programs would drastically diminish iNTS disease except among infants age <8 weeks. Conclusions/Significance: The public health impact of NTS vaccination shifts as disease burden, vaccine coverage, and serovar distribution vary. Our model shows that implementing an iNTS vaccine through an analogous strategy to the Hib vaccination program in Bamako would markedly reduce cases and deaths due to iNTS among the pediatric population. The model can be adjusted for use elsewhere in Africa where NTS epidemiologic patterns, serovar prevalence, and immunization schedules differ from Bamako. [ABSTRACT FROM AUTHOR]

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars. Author Summary: Non-typhoidal Salmonella, such as Salmonella Typhimurium, generally cause self-limiting gastroenteritis. However, in sub-Saharan Africa, a novel genotype called sequence type (ST) 313 is circulating and causes bloodstream infections in infants and HIV-infected individuals. In contrast, the most common genotype found throughout the rest of the world is ST19, which has been highly studied. Currently, the pathogenesis of S. Typhimurium ST313 is not well understood. In our study, we present evidence that S. Typhimurium ST313 strains from Mali, West Africa, survive and replicate better within a diverse set of primary human and murine macrophages and cell lines, inducing significantly less host-cell death as compared to S. Typhimurium ST19. Flagellin expression in the S. Typhimurium ST313 strains was found to be attenuated compared to S. Typhimurium ST19, thereby causing decreased inflammation. We conclude that S. Typhimurium ST313 have evolved phenotypically to be an intermediate between S. Typhimurium ST19 and the highly invasive typhoidal Salmonella serovars S. Typhi and S. Paratyphi A. [ABSTRACT FROM AUTHOR]

Background: In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients. Methods: We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali. Principal Findings: We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains. Conclusion: We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries. Author Summary: The genus Salmonella has more than 2500 serological variants (serovars), such as Salmonella enterica serovar Typhi and Salmonella Paratyphi A and B, that cause, respectively, typhoid and paratyphoid fevers (enteric fevers), and a large number of non-typhoidal Salmonella (NTS) serovars that cause gastroenteritis in healthy hosts. In young infants, the elderly and immunocompromised hosts, NTS can cause severe, fatal invasive disease. Multiple studies of pediatric patients in sub-Saharan Africa have documented the important role of NTS, in particular Salmonella Typhimurium and Salmonella Enteritidis (and to a lesser degree Salmonella Dublin), as invasive bacterial pathogens. Salmonella spp. are isolated from blood and identified by standard microbiological techniques and the serovar is ascertained by agglutination with commercial antisera. PCR-based typing techniques are becoming increasingly popular in developing countries, in part because high quality typing sera are difficult to obtain and expensive and H serotyping is technically difficult. We have developed a series of polymerase chain reactions (PCRs) to identify Salmonella Typhimurium and variants, Salmonella Enteritidis and Salmonella Dublin. We successfully identified 327 Salmonella isolates using our multiplex PCR. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR generally differentiated diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium variant strains from other closely related strains. The PCRs described here will enable more laboratories in developing countries to serotype NTS that have been isolated from blood. [ABSTRACT FROM AUTHOR]