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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.

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V. cholerae and phage counts during a 24 h incubation in pond water.A. The CFU decline between the patient derived (solid diamonds, N = 3) and in vitro derived (clear diamonds, N = 3) V. cholerae is similar. All patient samples had phage (EN159, EN182, EN191; N = 3). B. Direct counts (DC) do not change over 24 h. DC for V. cholerae from patient samples with phage (solid diamond, N = 3) and with no phage (shaded squares, N = 3). The in vitro grown strains isolated from phage positive stools (N = 3) or from phage negative stools (N = 3) are clear diamonds and clear squares, respectively. C. Phage bloom at 5 h (N = 2). The ratio of plaque forming units (PFU) to V. cholerae is plotted in two ways: PFU to CFU ratio (solid line); PFU to DC ratio (dashed line).
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ppat-1000187-g002: V. cholerae and phage counts during a 24 h incubation in pond water.A. The CFU decline between the patient derived (solid diamonds, N = 3) and in vitro derived (clear diamonds, N = 3) V. cholerae is similar. All patient samples had phage (EN159, EN182, EN191; N = 3). B. Direct counts (DC) do not change over 24 h. DC for V. cholerae from patient samples with phage (solid diamond, N = 3) and with no phage (shaded squares, N = 3). The in vitro grown strains isolated from phage positive stools (N = 3) or from phage negative stools (N = 3) are clear diamonds and clear squares, respectively. C. Phage bloom at 5 h (N = 2). The ratio of plaque forming units (PFU) to V. cholerae is plotted in two ways: PFU to CFU ratio (solid line); PFU to DC ratio (dashed line).

Mentions: The culturability of V. cholerae transferred to the pond microcosm was monitored by culture and direct microscopy counts. We define the non-culturable cells as ‘active but non-culturable’ (ABNC) because there were clear transcriptional changes between 5 and 24 h detected by both microarray and qRT-PCR analysis (below). Thus, our measure of ‘active’ was global transcriptional change. Culturability was rapidly lost upon transfer to the pond microcosm at 5 and 24 h with declines of 63% (SD+/−16%) and 98% (SD+/−1.0%), respectively (Fig. 2A). The V. cholerae isolates from the respective patients were grown in vitro (M9 pH 9) and transferred to the pond microcosm; the declines in culturability in the pond microcosm were similar for the in vitro derived samples compared to the patient derived samples (Fig. 2A). Despite the drop in culturable cells, the total cell numbers remained constant by direct counts (Fig. 2B) for all sample types; the cell number was also constant for phage negative patient samples and the paired in vitro grown strains (Fig. 2B). The culture counts are not available for the phage negative patient samples because two isolates were unexpectedly SM sensitive. The plating efficiency of starting cultures neared 100%. For example, the average concentration of V. cholerae from patients (EN159, EN182, EN191) at 0 h by culture counts and direct counts was 1.0×108 CFU/ml (+/−1.1×108 CFU/ml) and 1.65×108 CFU/ml (+/−0.35×108 CFU/ml), respectively.


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)

V. cholerae and phage counts during a 24 h incubation in pond water.A. The CFU decline between the patient derived (solid diamonds, N = 3) and in vitro derived (clear diamonds, N = 3) V. cholerae is similar. All patient samples had phage (EN159, EN182, EN191; N = 3). B. Direct counts (DC) do not change over 24 h. DC for V. cholerae from patient samples with phage (solid diamond, N = 3) and with no phage (shaded squares, N = 3). The in vitro grown strains isolated from phage positive stools (N = 3) or from phage negative stools (N = 3) are clear diamonds and clear squares, respectively. C. Phage bloom at 5 h (N = 2). The ratio of plaque forming units (PFU) to V. cholerae is plotted in two ways: PFU to CFU ratio (solid line); PFU to DC ratio (dashed line).
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Related In: Results  -  Collection

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ppat-1000187-g002: V. cholerae and phage counts during a 24 h incubation in pond water.A. The CFU decline between the patient derived (solid diamonds, N = 3) and in vitro derived (clear diamonds, N = 3) V. cholerae is similar. All patient samples had phage (EN159, EN182, EN191; N = 3). B. Direct counts (DC) do not change over 24 h. DC for V. cholerae from patient samples with phage (solid diamond, N = 3) and with no phage (shaded squares, N = 3). The in vitro grown strains isolated from phage positive stools (N = 3) or from phage negative stools (N = 3) are clear diamonds and clear squares, respectively. C. Phage bloom at 5 h (N = 2). The ratio of plaque forming units (PFU) to V. cholerae is plotted in two ways: PFU to CFU ratio (solid line); PFU to DC ratio (dashed line).
Mentions: The culturability of V. cholerae transferred to the pond microcosm was monitored by culture and direct microscopy counts. We define the non-culturable cells as ‘active but non-culturable’ (ABNC) because there were clear transcriptional changes between 5 and 24 h detected by both microarray and qRT-PCR analysis (below). Thus, our measure of ‘active’ was global transcriptional change. Culturability was rapidly lost upon transfer to the pond microcosm at 5 and 24 h with declines of 63% (SD+/−16%) and 98% (SD+/−1.0%), respectively (Fig. 2A). The V. cholerae isolates from the respective patients were grown in vitro (M9 pH 9) and transferred to the pond microcosm; the declines in culturability in the pond microcosm were similar for the in vitro derived samples compared to the patient derived samples (Fig. 2A). Despite the drop in culturable cells, the total cell numbers remained constant by direct counts (Fig. 2B) for all sample types; the cell number was also constant for phage negative patient samples and the paired in vitro grown strains (Fig. 2B). The culture counts are not available for the phage negative patient samples because two isolates were unexpectedly SM sensitive. The plating efficiency of starting cultures neared 100%. For example, the average concentration of V. cholerae from patients (EN159, EN182, EN191) at 0 h by culture counts and direct counts was 1.0×108 CFU/ml (+/−1.1×108 CFU/ml) and 1.65×108 CFU/ml (+/−0.35×108 CFU/ml), respectively.

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