Calman A. MacLennan

Epidemic multiple drug resistant Salmonella Typhimurium causing invasive disease in sub-Saharan Africa have a distinct genotype

Whereas most nontyphoidal Salmonella (NTS) are associated with gastroenteritis, there has been a dramatic increase in reports of NTS-associated invasive disease in sub-Saharan Africa. Salmonella enterica serovar Typhimurium isolates are responsible for a significant proportion of the reported invasive NTS in this region. Multilocus sequence analysis of invasive S. Typhimurium from Malawi and Kenya identified a dominant type, designated ST313, which currently is rarely reported outside of Africa. Whole-genome sequencing of a multiple drug resistant (MDR) ST313 NTS isolate, D23580, identified a distinct prophage repertoire and a composite genetic element encoding MDR genes located on a virulence-associated plasmid. Further, there was evidence of genome degradation, including pseudogene formation and chromosomal deletions, when compared with other S. Typhimurium genome sequences. Some of this genome degradation involved genes previously implicated in virulence of S. Typhimurium or genes for which the orthologs in S. Typhi are either pseudogenes or are absent. Genome analysis of other epidemic ST313 isolates from Malawi and Kenya provided evidence for microevolution and clonal replacement in the field.

Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa

A highly invasive form of non-typhoidal Salmonella (iNTS) disease has recently been documented in many countries in sub-Saharan Africa. The most common Salmonella enterica serovar causing this disease is Typhimurium (Salmonella Typhimurium). We applied whole-genome sequence–based phylogenetic methods to define the population structure of sub-Saharan African invasive Salmonella Typhimurium isolates and compared these to global Salmonella Typhimurium populations. Notably, the vast majority of sub-Saharan invasive Salmonella Typhimurium isolates fell within two closely related, highly clustered phylogenetic lineages that we estimate emerged independently ∼52 and ∼35 years ago in close temporal association with the current HIV pandemic. Clonal replacement of isolates from lineage I by those from lineage II was potentially influenced by the use of chloramphenicol for the treatment of iNTS disease. Our analysis suggests that iNTS disease is in part an epidemic in sub-Saharan Africa caused by highly related Salmonella Typhimurium lineages that may have occupied new niches associated with a compromised human population and antibiotic treatment.

The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children

Nontyphoidal strains of Salmonella (NTS) are a common cause of bacteremia among African children. Cell-mediated immune responses control intracellular infection, but they do not protect against extracellular growth of NTS in the blood. We investigated whether antibody protects against NTS bacteremia in Malawian children, because we found this condition mainly occurs before 2 years of age, with relative sparing of infants younger than 4 months old. Sera from all healthy Malawian children tested aged more than 16 months contained anti-Salmonella antibody and successfully killed NTS. Killing was mediated by complement membrane attack complex and not augmented in the presence of blood leukocytes. Sera from most healthy children less than 16 months old lacked NTS-specific antibody, and sera lacking antibody did not kill NTS despite normal complement function. Addition of Salmonella-specific antibody, but not mannose-binding lectin, enabled NTS killing. All NTS strains tested had long-chain lipopolysaccharide and the rck gene, features that resist direct complement-mediated killing. Disruption of lipopolysaccharide biosynthesis enabled killing of NTS by serum lacking Salmonella-specific antibody. We conclude that Salmonella-specific antibody that overcomes the complement resistance of NTS develops by 2 years of life in Malawian children. This finding and the age-incidence of NTS bacteremia suggest that antibody protects against NTS bacteremia and support the development of vaccines against NTS that induce protective antibody.

Interleukin (IL)-12 and IL-23 Are Key Cytokines for Immunity against Salmonella in Humans

Patients with inherited deficiency of the interleukin (IL)-12/IL-23-interferon (IFN)- gamma axis show increased susceptibility to invasive disease caused by the intramacrophage pathogens salmonellae and mycobacteria. We analyzed data on 154 patients with such deficiency. Significantly more patients with IL-12/IL-23-component deficiency had a history of salmonella disease than did those with IFN- gamma -component deficiency. Salmonella disease was typically severe, extraintestinal, and caused by nontyphoidal serovars. These findings strongly suggest that IL-12/IL-23 is a key cytokine for immunity against salmonella in humans and that IL-12/IL-23 mediates this protective effect partly through IFN- gamma -independent pathways. Investigation of the IL-12/IL-23-IFN- gamma axis should be considered in patients with invasive salmonella disease.

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

Failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man

BACKGROUND Individuals who chronically excrete neurovirulent poliovirus of vaccine-origin are of considerable concern to the Global Polio Eradication programme. Chronic infection with such polioviruses is a recognised complication of hypogammaglobulinaemia. METHODS We did a series of in-vitro and in-vivo therapeutic studies, with a view to clearing persistent neurovirulent poliovirus infection in an individual with common variable immunodeficiency, using oral immunoglobulin, breast milk (as a source of secretory IgA), ribavirin, and the anti-picornaviral agent pleconaril. We undertook viral quantitation, antibody neutralisation and drug susceptibility assays, and viral gene sequencing. FINDINGS Long-term asymptomatic excretion of vaccine-derived neurovirulent poliovirus 2 was identified in this hypogammaglobulinaemic man, and was estimated to have persisted for up to 22 years. Despite demonstrable in-vitro neutralising activity of immunoglobulin and breast milk, and in-vitro antiviral activity of ribavirin, no treatment was successful at clearing the virus, although in one trial breast milk significantly reduced excretion levels temporarily. During the course of study, the virus developed reduced susceptibility to pleconaril, precluding the in-vivo use of this drug. Sequence analysis revealed the emergence of a methionine to leucine mutation adjacent to the likely binding site of pleconaril in these isolates. INTERPRETATION Chronic vaccine-associated poliovirus infection in hypogammaglobulinaemia is a difficult condition to treat. It represents a risk to the strategy to discontinue polio vaccination once global eradication has been achieved.

Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults.

HIV and Salmonella HIV-positive individuals who are infected with nontyphoidal strains of Salmonella enterica often succumb to high morbidity and mortality. Why this is the case is unknown. MacLennan et al. (p. 508; see the Perspective by Moir and Fauci) have uncovered a dysregulated antibody response to Salmonella that is the likely culprit. Sera from HIV-infected individuals do a poor job of killing S. Typhimurium, despite surprisingly elevated antibody titers. Experiments showed that HIV-infected serum inhibited the power of normal serum to kill Salmonella. Inhibition was specific to antibodies against lipopolysaccharide (LPS), a component of the cell wall of Salmonella. Hence, HIV-infected sera was able to kill Salmonella strains lacking LPS, and removing LPS immunoglobulin G from infected sera permitted Salmonella killing. Thus, not only does HIV cause defects in cell-mediated immunity but it also seems to impair humoral immunity, with severe consequences for multiple infections. Abnormal antibody responses produced in HIV-infected individuals are ineffective at clearing food-poisoning bacteria. Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibodies protect against Salmonella bacteremia. We report that high-titer antibodies specific for Salmonella lipopolysaccharide (LPS) are associated with a lack of Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from the target Salmonella or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus, our study indicates that impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whereas serum killing of Salmonella is induced by antibodies against outer membrane proteins.

Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans

Bacteremia caused by nontyphoidal strains of Salmonella is endemic among African children. Case-fatality rates are high and antibiotic resistance increasing, but no vaccine is currently available. T cells are important for clearance of Salmonella infection within macrophages, but in Africa, invasive Salmonella disease usually manifests in the blood and affects children between 4 months and 2 y of age, when anti-Salmonella antibody is absent. We have previously found a role for complement-fixing bactericidal antibody in protecting these children. Here we show that opsonic activity of antibody and complement is required for oxidative burst and killing of Salmonella by blood cells in Africans. Induction of neutrophil oxidative burst correlated with anti-Salmonella IgG and IgM titers and C3 deposition on bacteria and was significantly lower in African children younger than 2 y compared with older children. Preopsonizing Salmonella with immune serum overcame this deficit, indicating a requirement for antibody and/or complement. Using different opsonization procedures, both antibody and complement were found to be necessary for optimal oxidative burst, phagocytosis and killing of nontyphoidal Salmonella by peripheral blood cells in Africans. Although most strains of African nontyphoidal Salmonella can be killed with antibody and complement alone, phagocytes in the presence of specific antibody and complement can kill strains resistant to killing by immune serum. These findings increase the likelihood that an antibody-inducing vaccine will protect against invasive nontyphoidal Salmonella disease in African children.

Vaccines against invasive Salmonella disease: Current status and future directions

Though primarily enteric pathogens, Salmonellae are responsible for a considerable yet under-appreciated global burden of invasive disease. In South and South-East Asia, this manifests as enteric fever caused by serovars Typhi and Paratyphi A. In sub-Saharan Africa, a similar disease burden results from invasive nontyphoidal Salmonellae, principally serovars Typhimurium and Enteritidis. The existing Ty21a live-attenuated and Vi capsular polysaccharide vaccines target S. Typhi and are not effective in young children where the burden of invasive Salmonella disease is highest. After years of lack of investment in new Salmonella vaccines, recent times have seen increased interest in the area led by emerging-market manufacturers, global health vaccine institutes and academic partners. New glycoconjugate vaccines against S. Typhi are becoming available with similar vaccines against other invasive serovars in development. With other new vaccines under investigation, including live-attenuated, protein-based and GMMA vaccines, now is an exciting time for the Salmonella vaccine field.

Identification of a common immune signature in murine and human systemic Salmonellosis

Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined. We have previously shown evidence for antibody-mediating protection against invasive Salmonellosis in mice and African children. To generate an overview of antibody targeting in systemic Salmonellosis, a Salmonella proteomic array containing over 2,700 proteins was constructed and probed with immune sera from Salmonella-infected mice and humans. Analysis of multiple inbred mouse strains identified 117 antigens recognized by systemic antibody responses in murine Salmonellosis. Importantly, many of these antigens were independently identified as target antigens using sera from Malawian children with Salmonella bacteremia, validating the study of the murine model. Furthermore, vaccination with SseB, the most prominent antigenic target in Malawian children, provided mice with significant protection against Salmonella infection. Together, these data uncover an overlapping immune signature of disseminated Salmonellosis in mice and humans and provide a foundation for the generation of a protective subunit vaccine.