Limits...
Transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment.

Nelson EJ, Chowdhury A, Flynn J, Schild S, Bourassa L, Shao Y, LaRocque RC, Calderwood SB, Qadri F, Camilli A - PLoS Pathog. (2008)

Bottom Line: Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low.Phage had an undetectable impact on this adaptation.Taken together, the rise of ABNC cells and lytic phage blocked transmission.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, USA.

ABSTRACT
Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water-the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.

Show MeSH

Related in: MedlinePlus

ID50 experiments of V. cholerae incubated in pond water.V. cholerae were incubated in pond water for 0 (A), 5 (B, C) and 24 h (D, E). Each symbol represents the average of the fraction of infant mice infected with V. cholerae from three phage positive patient samples (solid diamond/solid line), or the same three isogenic strains from in vitro culture (clear diamond/dashed line). The data are plotted on the X axis by the log of the culturable inputs (CFU) on the left or direct count inputs (DC) on the right; the log (ID50) is depicted with the grey dashed vertical line. The inset table summarizes the fold changes in the ID50 over 24 h for the in vitro derived samples. CI = 95% confidence interval for the ID50 determined from the fitted curve (red) modeled with the Hill Equation. Plating efficiency at 0 h neared 100% (see text). Each symbol represents the average of the 3 biological replicates (EN159, EN182, EN191) which pools data from at least 15 mice total.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2563029&req=5

ppat-1000187-g004: ID50 experiments of V. cholerae incubated in pond water.V. cholerae were incubated in pond water for 0 (A), 5 (B, C) and 24 h (D, E). Each symbol represents the average of the fraction of infant mice infected with V. cholerae from three phage positive patient samples (solid diamond/solid line), or the same three isogenic strains from in vitro culture (clear diamond/dashed line). The data are plotted on the X axis by the log of the culturable inputs (CFU) on the left or direct count inputs (DC) on the right; the log (ID50) is depicted with the grey dashed vertical line. The inset table summarizes the fold changes in the ID50 over 24 h for the in vitro derived samples. CI = 95% confidence interval for the ID50 determined from the fitted curve (red) modeled with the Hill Equation. Plating efficiency at 0 h neared 100% (see text). Each symbol represents the average of the 3 biological replicates (EN159, EN182, EN191) which pools data from at least 15 mice total.

Mentions: The ID50 for V. cholerae freshly shed from the patients (113 CFU; 95% confidence interval [CI] = 65–196 CFU) was lower compared to the in vitro grown reference (596 CFU; 95% CI = 193–1834 CFU; Fig. 4A). Hyperinfectivity was also observed after 5 h of dialysis between the patient (51 CFU; 95% CI = 13–202 CFU) and in vitro culture (680 CFU; 95% CI = 276–1673 CFU; Fig. 4B). These findings are consistent with competition experiments previously published that suggest V. cholerae maintains hyperinfectivity for at least 5 h after exit from the patient [23]. We tested if hyperinfectivity could be induced by the medium alone (stool-supernatant), and we found that hyperinfectivity could not be induced in vitro by incubation in stool supernatant (pH 9) or minimal media (M9 pH 9) (Fig. S1). Unique to the present study was that the single strain infection experiments revealed that the fraction of mice infected with high doses of patient derived V. cholerae was reduced at 5 h and 24 h compared to the in vitro reference (Fig. 4B–E). Indeed, the ID50 was not able to be calculated for the patient derived samples at 24 h because less than 50% of the animals were infected (Fig. 4D–E). The 24 h time point corresponds with the point when the titer of PFU was highest and the titer of culturable cells was lowest (Fig. 2); note again that the no phage control for this experiment are in vitro derived cells. We hypothesized, and show below, that the incomplete colonization observed is due to the presence of lytic phage in the inocula.


Transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment.

Nelson EJ, Chowdhury A, Flynn J, Schild S, Bourassa L, Shao Y, LaRocque RC, Calderwood SB, Qadri F, Camilli A - PLoS Pathog. (2008)

ID50 experiments of V. cholerae incubated in pond water.V. cholerae were incubated in pond water for 0 (A), 5 (B, C) and 24 h (D, E). Each symbol represents the average of the fraction of infant mice infected with V. cholerae from three phage positive patient samples (solid diamond/solid line), or the same three isogenic strains from in vitro culture (clear diamond/dashed line). The data are plotted on the X axis by the log of the culturable inputs (CFU) on the left or direct count inputs (DC) on the right; the log (ID50) is depicted with the grey dashed vertical line. The inset table summarizes the fold changes in the ID50 over 24 h for the in vitro derived samples. CI = 95% confidence interval for the ID50 determined from the fitted curve (red) modeled with the Hill Equation. Plating efficiency at 0 h neared 100% (see text). Each symbol represents the average of the 3 biological replicates (EN159, EN182, EN191) which pools data from at least 15 mice total.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563029&req=5

ppat-1000187-g004: ID50 experiments of V. cholerae incubated in pond water.V. cholerae were incubated in pond water for 0 (A), 5 (B, C) and 24 h (D, E). Each symbol represents the average of the fraction of infant mice infected with V. cholerae from three phage positive patient samples (solid diamond/solid line), or the same three isogenic strains from in vitro culture (clear diamond/dashed line). The data are plotted on the X axis by the log of the culturable inputs (CFU) on the left or direct count inputs (DC) on the right; the log (ID50) is depicted with the grey dashed vertical line. The inset table summarizes the fold changes in the ID50 over 24 h for the in vitro derived samples. CI = 95% confidence interval for the ID50 determined from the fitted curve (red) modeled with the Hill Equation. Plating efficiency at 0 h neared 100% (see text). Each symbol represents the average of the 3 biological replicates (EN159, EN182, EN191) which pools data from at least 15 mice total.
Mentions: The ID50 for V. cholerae freshly shed from the patients (113 CFU; 95% confidence interval [CI] = 65–196 CFU) was lower compared to the in vitro grown reference (596 CFU; 95% CI = 193–1834 CFU; Fig. 4A). Hyperinfectivity was also observed after 5 h of dialysis between the patient (51 CFU; 95% CI = 13–202 CFU) and in vitro culture (680 CFU; 95% CI = 276–1673 CFU; Fig. 4B). These findings are consistent with competition experiments previously published that suggest V. cholerae maintains hyperinfectivity for at least 5 h after exit from the patient [23]. We tested if hyperinfectivity could be induced by the medium alone (stool-supernatant), and we found that hyperinfectivity could not be induced in vitro by incubation in stool supernatant (pH 9) or minimal media (M9 pH 9) (Fig. S1). Unique to the present study was that the single strain infection experiments revealed that the fraction of mice infected with high doses of patient derived V. cholerae was reduced at 5 h and 24 h compared to the in vitro reference (Fig. 4B–E). Indeed, the ID50 was not able to be calculated for the patient derived samples at 24 h because less than 50% of the animals were infected (Fig. 4D–E). The 24 h time point corresponds with the point when the titer of PFU was highest and the titer of culturable cells was lowest (Fig. 2); note again that the no phage control for this experiment are in vitro derived cells. We hypothesized, and show below, that the incomplete colonization observed is due to the presence of lytic phage in the inocula.

Bottom Line: Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low.Phage had an undetectable impact on this adaptation.Taken together, the rise of ABNC cells and lytic phage blocked transmission.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, USA.

ABSTRACT
Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water-the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.

Show MeSH
Related in: MedlinePlus