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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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Phage positive patient derived V. cholerae have irregular shape at 24 h (arrow).Representative images of a lytic phage positive patient sample (EN182; A, B, C) and phage negative patient sample (EN124; D, E, F) incubated in pond water over 24 h. The isogenic strain EN182 was isolated away from the phage, grown in vitro and incubated for 24 h in the pond water (G, H, I). V. cholerae are labeled with a FITC conjugated monoclonal Ab to O1 LPS (left) and nucleic acid is labeled with DAPI (middle) which indicates non-V. cholerae; merge is shown on right. Data are representative of 3 independent experiments (data not shown). V. cholerae from the 0 h and 5 h time points for all sample types were indistinguishable by morphology and represented ‘F’ and ‘I’. Scale bar = 10 µm.
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ppat-1000187-g003: Phage positive patient derived V. cholerae have irregular shape at 24 h (arrow).Representative images of a lytic phage positive patient sample (EN182; A, B, C) and phage negative patient sample (EN124; D, E, F) incubated in pond water over 24 h. The isogenic strain EN182 was isolated away from the phage, grown in vitro and incubated for 24 h in the pond water (G, H, I). V. cholerae are labeled with a FITC conjugated monoclonal Ab to O1 LPS (left) and nucleic acid is labeled with DAPI (middle) which indicates non-V. cholerae; merge is shown on right. Data are representative of 3 independent experiments (data not shown). V. cholerae from the 0 h and 5 h time points for all sample types were indistinguishable by morphology and represented ‘F’ and ‘I’. Scale bar = 10 µm.

Mentions: The PFU titer was monitored at 0, 5 and 24 h in the pond microcosm (Fig. 2C). At 0 h, the average ratio of phage to V. cholerae for all three patient stools was 2.2×10−6 (SD+/−3.5×10−6 ). At 5 h, this ratio increased by 4 orders of magnitude to 1.0×10−2 (SD+/−1.2×10−2) by culture counts, or 3 orders of magnitude to 1.5×10−3 (SD+/−1.3×10−3) by direct counts. At 24 h, this ratio increased an additional 2 orders of magnitude to 4.0×10−1 (SD+/−3.9×10−1) by culture counts, but remained steady at 3.8×10−3 (+/−3.2×10−3) by direct counts. From 5 to 24 h, this ratio changed because the culturable counts decreased 14-fold. These findings are supported by micrographs that illustrate altered morphology of V. cholerae only in the patient derived samples from phage positive patients (Fig. 3A). Lytic and lysogenic vibriophage have been previously characterized from patients [27],[33],[35],[49],[50],[51],[52]; our phage isolates are consistent in terms of the tropism of those lytic phage previously published [27] because our phage had specificity for the Inaba or Ogawa serotype of the O1 El Tor V. cholerae biotype, and the phage were unable to form plaques on O139 V. cholerae (data not shown). These data indicate the phage receptor may be O1 LPS as has been demonstrated previously [34],[35]. Support for this hypothesis is the generation of LPS mutants in the presence of lytic phage (presented below).


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)

Phage positive patient derived V. cholerae have irregular shape at 24 h (arrow).Representative images of a lytic phage positive patient sample (EN182; A, B, C) and phage negative patient sample (EN124; D, E, F) incubated in pond water over 24 h. The isogenic strain EN182 was isolated away from the phage, grown in vitro and incubated for 24 h in the pond water (G, H, I). V. cholerae are labeled with a FITC conjugated monoclonal Ab to O1 LPS (left) and nucleic acid is labeled with DAPI (middle) which indicates non-V. cholerae; merge is shown on right. Data are representative of 3 independent experiments (data not shown). V. cholerae from the 0 h and 5 h time points for all sample types were indistinguishable by morphology and represented ‘F’ and ‘I’. Scale bar = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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ppat-1000187-g003: Phage positive patient derived V. cholerae have irregular shape at 24 h (arrow).Representative images of a lytic phage positive patient sample (EN182; A, B, C) and phage negative patient sample (EN124; D, E, F) incubated in pond water over 24 h. The isogenic strain EN182 was isolated away from the phage, grown in vitro and incubated for 24 h in the pond water (G, H, I). V. cholerae are labeled with a FITC conjugated monoclonal Ab to O1 LPS (left) and nucleic acid is labeled with DAPI (middle) which indicates non-V. cholerae; merge is shown on right. Data are representative of 3 independent experiments (data not shown). V. cholerae from the 0 h and 5 h time points for all sample types were indistinguishable by morphology and represented ‘F’ and ‘I’. Scale bar = 10 µm.
Mentions: The PFU titer was monitored at 0, 5 and 24 h in the pond microcosm (Fig. 2C). At 0 h, the average ratio of phage to V. cholerae for all three patient stools was 2.2×10−6 (SD+/−3.5×10−6 ). At 5 h, this ratio increased by 4 orders of magnitude to 1.0×10−2 (SD+/−1.2×10−2) by culture counts, or 3 orders of magnitude to 1.5×10−3 (SD+/−1.3×10−3) by direct counts. At 24 h, this ratio increased an additional 2 orders of magnitude to 4.0×10−1 (SD+/−3.9×10−1) by culture counts, but remained steady at 3.8×10−3 (+/−3.2×10−3) by direct counts. From 5 to 24 h, this ratio changed because the culturable counts decreased 14-fold. These findings are supported by micrographs that illustrate altered morphology of V. cholerae only in the patient derived samples from phage positive patients (Fig. 3A). Lytic and lysogenic vibriophage have been previously characterized from patients [27],[33],[35],[49],[50],[51],[52]; our phage isolates are consistent in terms of the tropism of those lytic phage previously published [27] because our phage had specificity for the Inaba or Ogawa serotype of the O1 El Tor V. cholerae biotype, and the phage were unable to form plaques on O139 V. cholerae (data not shown). These data indicate the phage receptor may be O1 LPS as has been demonstrated previously [34],[35]. Support for this hypothesis is the generation of LPS mutants in the presence of lytic phage (presented below).

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