John Wain

Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.

Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

Quantitation of Bacteria in Bone Marrow from Patients with Typhoid Fever: Relationship between Counts and Clinical Features

ABSTRACT Enteric fever is the only bacterial infection of humans for which bone marrow examination is routinely recommended. A prospective study of the concentrations of bacteria in the bone marrow and their relationship to clinical features was conducted with 120 Vietnamese patients with suspected enteric fever, of whom 89 had confirmed typhoid fever. Ninety-three percent of the Salmonella entericaserovar Typhi samples isolated were resistant to ampicillin, chloramphenicol, and co-trimoxazole. For 81 patients with uncomplicated typhoid and satisfactory bone marrow aspirates, the number of serovar Typhi CFU in bone marrow aspirates was a median value of 9 (interquartile range [IQR], 1 to 85; range, 0.1 to 1,580) compared to 0.3 (IQR, 0.1 to 10; range, 0.1 to 399) CFU/ml in simultaneously sampled blood. The ratio of individual blood counts to bone marrow counts was 10 (IQR, 2.3 to 97.5). The number of bacteria in blood but not bone marrow was correlated inversely with the duration of preceding fever. Thus, with increasing duration of illness the ratio of bone marrow-to-blood bacterial concentrations increased; the median ratio was 4.8 (IQR, 1 to 27.5) during the first week compared with 158 (IQR, 60 to 397) during the third week. After lysing the host cells, the median ratio of viable bone marrow to blood increased, reflecting the higher concentration of intracellular serovar Typhi in the bone marrow. Effective antibiotic pretreatment had a significantly greater effect in reducing blood counts compared to bone marrow counts (P < 0.001). Thus, bacteria in the bone marrow of typhoid patients are less affected by antibiotic treatment than bacteria in the blood. The numbers of bacteria in bone marrow correlated negatively with the white blood cell (R = −0.3, P = 0.006) and platelet counts (R = −0.32, P = 0.01) and positively with fever clearance time after treatment (R = 0.4,P < 0.001). The bacterial load in bone marrow therefore may reflect the clinical course of the infection, and high levels may suppress neutrophil proliferation.

Typhoid fever: pathogenesis and disease.

Typhoid fever is an infectious disease of global distribution. Although there is a wealth of data on Salmonella typhimurium infection in the mouse and the interaction of this serovar with human cell lines in vitro, there is a relatively small amount of data on S. typhi and the pathogenesis of typhoid fever. In this review we focus on three areas: adherence to and invasion of gut epithelial cells, dissemination to systemic sites, and survival and replication within host cells. In addition, we attempt to put current salmonella research into the context of typhoid fever.

The molecular mechanisms of severe typhoid fever

Salmonella typhi continues to cause severe disease in many parts of the world, its most feared complication being perforation of ulcerated Peyers patches within the small intestine, leading to peritonitis with associated mortality. The pathogenesis of this process is not well understood. In this article, we present a theoretical mechanism as to how bacterial factors and host immunological mediators within infected tissue might contribute to the observed intestinal pathology, and propose that necrosis of the Peyers patches observed in typhoid is caused by a mechanism similar to the Shwartzman and Koch reactions.

A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.

Infection Biology of a Novel α-Crystallin of Mycobacterium tuberculosis: Acr2

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the α-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-γ-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.

Risk factors for typhoid fever in the Mekong delta, southern Viet Nam: a case-control study.

In order to identify risk factors for typhoid fever in a highly endemic area, we undertook a case-control study in the Mekong delta, Viet Nam. Cases were 144 consecutive patients admitted to hospital with blood culture-confirmed typhoid fever. Two controls (1 in the hospital and 1 in the community) were chosen for each case. Standardized interviews were conducted with questions regarding recent contact with a typhoid fever patient, eating habits, hygiene and socio-economic level. Cases were more likely to have been in contact with a patient with typhoid fever than hospital controls (adjusted odds ratio (OR) = 5.2, 95% confidence interval (95% CI) 1.7-15.9) or community controls (adjusted OR = 11.9, 95% CI 2.3-60.7); 11% and 14% of typhoid fever cases (compared to hospital or community controls, respectively) were attributable to recent contact with a patient with this disease. These findings suggest that strategies directed towards the persons in contact with a patient might reduce the incidence of secondary cases of typhoid fever.

Effect of Deletion or Overexpression of the 19-Kilodalton Lipoprotein Rv3763 on the Innate Response to Mycobacterium tuberculosis

ABSTRACT The 19-kDa lipoprotein of Mycobacterium tuberculosis is an important target of the innate immune response. To investigate the immune biology of this antigen in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19-kDa-lipoprotein gene (Δ19) and complemented this strain by reintroduction of the 19-kDa-lipoprotein gene on a multicopy vector to produce Δ19::pSMT181. The Δ19 strain multiplied less well than Δ19::pSMT181 in human monocyte-derived macrophages (MDM) (P = 0.039). Surface expression of major histocompatibility complex class II molecules was reduced in phagocytes infected with M. tuberculosis; this effect was not seen in cells infected with Δ19. Δ19 induced lower interleukin 1β (IL-1β) secretion from monocytes and MDM. Overexpression of the 19-kDa protein increased IL-1β, IL-12p40, and tumor necrosis factor alpha secretion irrespective of phagocyte maturity. These data support reports that the 19-kDa lipoprotein has pleiotropic effects on the interaction of M. tuberculosis with phagocytes. However, this analysis indicates that in the context of the whole bacillus, the 19-kDa lipoprotein is only one of a number of molecules that mediate the innate response to M. tuberculosis.