Role of disease-associated tolerance in infectious superspreaders

Abstract

Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

Keywords: acute phase response; carriers; host-to-host transmission; pathogenesis.

Fig. 1.

Superspreaders are tolerant to oral antibiotic treatment. Mice were infected for 30 d, and their transmission status was identified. (A–F) Superspreaders (SS; black circles) and nonsuperspreaders (non-SS; red squares) were treated with 5 mg of streptomycin (A–D) or neomycin (E and F) via oral gavage. Weight and fecal Salmonella burden were recorded for the indicated period. Data shown is representative of a minimum of two experiments with a total of 10–15 mice per condition. (A and B) Each data point represents an individual mouse. (C) Mice from two independent experiments were pooled with a total of 10–15 mice per condition, and weight data are shown from 5 d after streptomycin treatment. (D) Salmonella bacterial burden of indicated organs (left y axis) and whole blood (right y axis) in streptomycin-treated superspreaders and nonsuperspreaders. (E and F) Fecal Salmonella burden was quantified in neomycin treated superspreader and nonsuperspreader hosts (E), and weight data were recorded at indicated times (F). (G) Mice were infected for 4 d (shedding status cannot be confirmed at this time point) and treated with 5 mg of streptomycin (red squares) or PBS (black squares), and weight was recorded for the time period indicated. *P < 0.05; **P < 0.005; ****P < 0.0001. ns, not significant.

Fig. 2.

Antibiotic-mediated weight loss is correlated with activation of the acute-phase response. Mice were infected for 30 d, and their transmission status was identified. Superspreaders (black circles) and nonsuperspreaders (red squares) were treated with 5 mg of antibiotic via oral gavage. Superspreaders and nonsuperspreaders were treated with streptomycin and sacrificed 6 d after antibiotic treatment. Data shown are representative of two experiments with a total of 8–10 mice in each condition. (A and B) Serum (A) and intestinal cytokine (B) levels were measured by ELISA. Each point represents an individual mouse, and data are representative of two experiments with 10–15 mice per group. Nonsuperspreader hosts that lost >20% of their total body weight are represented by filled squares (Inset). (C) Fecal samples from streptomycin-treated superspreaders and nonsuperspreaders were processed, and host-derived proteins were identified by using mass spectrometry. Data show normalized spectral counts from the 17 identified proteins in the acute-phase response pathway depicted in a heat map where intensity was normalized within each protein and warmer colors represent increased spectral counts as depicted in the key. Host weight was normalized from red to blue with warmer colors depicting increased host weight. All proteins were correlated to host weight loss using Spearman’s correlation. *Significant correlations. (D) Host weight data from C is quantified, and significance is calculated by using the two-tailed Mann–Whitney test. **P < 0.005.

Fig. 3.

Antibiotic-treated nonsuperspreaders have increased inflammatory myeloid cells in the spleen, but not the gut. Mice were infected for 30 d, and their shedding status was identified. Superspreaders (black) and nonsuperspreaders (red) were treated with 5 mg of antibiotic via oral gavage. (A and B) Splenic cells were collected from streptomycin-treated superspreaders and nonsuperspreaders 6 d after antibiotic treatment. (A) Representative FACS plot shows frequency of CD11b⁺ Ly6C⁺ inflammatory myeloid cells gated on nonlymphocytes. (B) Total frequency of myeloid inflammatory cells expressed as a percentage of nonlymphocytes. (C and D) Ceca were fixed, and hematoxylin/eosin-stained sections were scored for histopathological changes. A representative figure of a cecal section from each group is displayed in C, and histopathological scores are represented in D. (E) Bars represent colonic neutrophils measured as a frequency of CD11b⁺ cells 6 d after streptomycin treatment. *P < 0.05.

Fig. 4.

CD4 T cells contribute to antibiotic-induced morbidity but not superspreader tolerance. Mice were infected for 30 d, and their shedding status was identified. Superspreaders (black circles) and nonsuperspreaders (red squares) were treated with 5 mg of streptomycin via oral gavage. Mice were treated with CD4-neutralizing antibodies or the isotype control (n = 4 or 5 in each group) 1 d before antibiotic treatment and at 2 and 4 d after antibiotic treatment. (A) Each point represents an individual mouse, and the weight data were collected daily. (B) Splenocytes were collected and representative cell populations were enumerated. In the isotype group, a single mouse was excluded due to morbidity. (C) Splenic Salmonella burden was quantified. *P < 0.05; **P < 0.005.

Fig. 5.

Neutralization of acute-phase cytokines ablates weight loss in antibiotic-treated nonsuperspreaders. Mice were infected for 30 d, and their transmission status was identified. Superspreaders (SS; black circles) and nonsuperspreaders (Non-SS; red squares) were treated with 5 mg of neomycin (A and C–F) or streptomycin (B and G) via oral gavage. Data shown are representative of two experiments with a total of 8–10 mice in each condition. Along with the antibiotic, mice were injected intraperitoneally with neutralizing antibodies against IL-1β and TNF-α (filled shapes) and the isotype control (open shapes). (A and B) Each point represents an individual mouse, and the weight data were measured at 12 d after neomycin treatment (A) and 6 d after streptomycin treatment (B). Splenocytes were collected from mice in A, and inflammatory myeloid cells (C) and neutrophils (D) were quantified as a frequency of nonlymphocytes. Cell populations were enumerated in E. Bacterial burden of selected tissues was quantified at 12 d after neomycin treatment (F) and 6 d after streptomycin treatment (G) *P < 0.05; **P < 0.005. ns, not significant.