Research Article: Glucose- but Not Rice-Based Oral Rehydration Therapy Enhances the Production of Virulence Determinants in the Human Pathogen Vibrio cholerae

Date Published: December 4, 2014

Publisher: Public Library of Science

Author(s): Juliane Kühn, Flavio Finger, Enrico Bertuzzo, Sandrine Borgeaud, Marino Gatto, Andrea Rinaldo, Melanie Blokesch, Pamela L. C. Small.

Abstract: Despite major attempts to prevent cholera transmission, millions of people worldwide still must address this devastating disease. Cholera research has so far mainly focused on the causative agent, the bacterium Vibrio cholerae, or on disease treatment, but rarely were results from both fields interconnected. Indeed, the treatment of this severe diarrheal disease is mostly accomplished by oral rehydration therapy (ORT), whereby water and electrolytes are replenished. Commonly distributed oral rehydration salts also contain glucose. Here, we analyzed the effects of glucose and alternative carbon sources on the production of virulence determinants in the causative agent of cholera, the bacterium Vibrio cholerae during in vitro experimentation. We demonstrate that virulence gene expression and the production of cholera toxin are enhanced in the presence of glucose or similarly transported sugars in a ToxR-, TcpP- and ToxT-dependent manner. The virulence genes were significantly less expressed if alternative non-PTS carbon sources, including rice-based starch, were utilized. Notably, even though glucose-based ORT is commonly used, field studies indicated that rice-based ORT performs better. We therefore used a spatially explicit epidemiological model to demonstrate that the better performing rice-based ORT could have a significant impact on epidemic progression based on the recent outbreak of cholera in Haiti. Our results strongly support a change of carbon source for the treatment of cholera, especially in epidemic settings.

Partial Text: The diarrheal disease cholera remains a major problem in developing countries. In 2012, almost 250’000 cases were reported to the WHO; however estimated numbers, including non-reported cases, are argued to reach several million cases every year. The recent disease outbreak in Haiti demonstrated the devastating effects of cholera epidemics. Because of these dramatic consequences, the problem of how to stop an epidemic at an early stage, or at least to slow it down, was addressed and the employment of general intervention strategies was discussed. In this context, mathematical models were developed and used to predict the outcome of major interventions, such as vaccination or extended use of antibiotics and sanitation [1]–[6]. Remarkably, more general treatment strategies have not been considered. This fact appears surprising because, without treatment, the case fatality rate for severe cholera is approximately 50%; however, if handled properly, nearly all deaths can be avoided. The general treatment of cholera patients is based on a so-called oral rehydration therapy (ORT), which is a cost-effective and easily applicable method to replace lost fluids and electrolytes. For the latter purpose, the administered solution contains a mixture of several compounds that were designated oral rehydration salts (ORS), including sodium, chloride, and potassium ions as well as glucose. Indeed, glucose is the most commonly added carbohydrate because it stimulates sodium and therefore, water absorption in the small intestine [7]. However, in field studies, it was shown that ORT might be improved by the substitution of the carbon source (or through the addition of amino acids and supplementation with trace elements) [8]. The molecular mechanisms underlying these findings remain to be resolved. Moreover, a meta-analysis comparing the treatment with standard, glucose-based versus rice-based ORS illustrated the beneficial effects of the latter composition, such as reduced episodes of vomiting, a decrease of the stool volume, and a shortened recovery time [9]–[12].

Cholera remains a major social emergency in developing countries and despite major research efforts this disease is far from being eradicated. Here, we aimed at understanding why an alternative cholera treatment, which relies on rice-based instead of glucose-based ORS, has beneficial effects on disease outcome as reported in field studies (Fig. 1). For this purpose, we tested the expression of virulence-associated genes in V. cholerae using defined virulence-inducing minimal medium conditions and a variety of carbon sources. Using this approach, we demonstrated that the virulence genes were upregulated in different clinical isolates of V. cholerae as long as those strains were grown in the presence of glucose or other PTS sugars (Fig. 2A, Fig. S1 and Table 1). The amount of cholera toxin produced under these conditions reflected the expression data (Fig. 2B). Because the cholera toxin is primarily responsible for the severe symptoms that are associated with the disease, our study highlights the negative effects of glucose-based ORT. Interestingly, in 2002, the WHO announced the recommendation to reduce the osmolarity in ORS [46]. Concomitantly, the final concentration of glucose was lowered from the initial 111 mM (WHO-ORS) to 75 mM (HYPO- ORS). Nevertheless, we observed that lowering the concentration of glucose in this range (from 111 mM glucose that was previously used, toward the 75 mM current recommendation, or even down to 50 mM, such as in our assays, because this concentration might better reflect the concentration within the intestine) did not reduce virulence gene expression in V. cholerae (Table 1). To our knowledge, this study is the first to describe the PTS sugar-dependent expression of virulence genes in V. cholerae cells grown in minimal salt conditions, although a putative cAMP-CRP binding site in the tcpA promoter region has been proposed previously [47] and the repression of TCP and CT by cAMP-CRP has been described [48]. Furthermore, a link between carbon catabolite repression and QS-dependent virulence repression has been reported before [33], [35], [49]; however, our data indicate that PTS-sugars also influence virulence expression in a QS-independent manner (Fig. S2), which might be due to the binding of the cAMP-CRP complex to the putative cAMP-CRP binding site [47] mentioned above.



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