Macrophage Cell Lines and Murine Infection by Salmonella enterica Serovar Typhi L-Form Bacteria.

Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic resistance genes is well established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as that due to treatment with bacterial cell wall-inhibiting antibiotics such as ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin during the treatment of infectious diseases. This is different from in vitro drug testing, where the organism is exposed to fixed drug concentrations from the beginning until the end. To mimic the mode of antibiotic exposure of microorganisms within host tissues, we cultured the wild-type, ampicillin-sensitive Salmonella enterica serovar Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that displayed several features of the so-called L-form of bacteria, including the absence of the cell wall, altered shape, and lower growth rate compared with the parental form. Studies of the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental form. These results suggested that L-form generation following the initiation of treatment with antibiotics could lead to drug escape of S. Typhi and cell to cell (macrophages) spread of the bacteria, which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through the fecal-oral route, highlighting the need for new approaches to decrease the reservoir of infection.

Intranasal immunization of mice with chimera of Salmonella Typhi protein elicits protective intestinal immunity.

Development of safe, highly effective and affordable enteric fever vaccines is a global health priority. Live, oral typhoid vaccines induce strong mucosal immunity and long-term protection, but safety remains a concern. In contrast, efficacy wears off rapidly for injectable, polysaccharide-based vaccines, which elicit poor mucosal response. We previously reported Salmonella Typhi outer membrane protein, T2544 as a potential candidate for bivalent (S. Typhi and S. Paratyphi A) vaccine development. Here, we show that intranasal immunization with a subunit vaccine (chimera of T2544 and cholera toxin B subunit) induced strong systemic and intestinal mucosal immunity and protection from S. Typhi challenge in a mouse model. CTB-T2544 augmented gut-homing receptor expression on lymphocytes that produced Th1 and Th17 cytokines, secretory IgA in stool that inhibited bacterial motility and epithelial attachment, antibody recall response and affinity maturation with increased number of follicular helper T cells and CD4+ central and effector memory cells.