Surviving the acid barrier: responses of pathogenic Vibrio cholerae to simulated gastric fluid

Abstract

When bacteria are subjected to low acidic pHs of the gastric environment, they may enter the viable but nonculturable (VBNC) state of survival. In this state, bacteria cannot be cultured on solid media, still exhibit signs of metabolic activity (viability). In this study, the response of pathogenic Vibrio cholerae O1 and O139 to low pH-simulated environments of the human stomach was evaluated for their survival by culturability (plate count) and viability (flow cytometry-FC) assays. Bacteria were acid challenged with simulated gastric fluid (SGF) at pH 1.5, 2.5, 3.5 and 4.5 over a period of 180 min. Exposure to SGF up to 120 min increased acid tolerance of the Vibrios up to pH 3.5 with acid challenge occurring at pH 4.5. Bacteria were culturable from pH 2.5 to 4.5 up to 60 min SGF exposure. The stationary-phase cultures of Vibrio were able to survive SGF at all pHs in an 'injured' state with FC. This could possibly mean that the bacteria have entered the VBNC stage of survival. This is a worrying public health concern due to the fact that once favourable conditions arise (intestines), these Vibrios can change back to an infectious state and cause disease.

A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid

“…These bacteria were grown on culturable agar specific for Vibrio sp. to obtain pure colonies as described previously by Singh and Barnard [ 1 ]. From the pure colonies, the V. cholerae were then grown in nutrient broth (pH 7) (BioLab) to the late stationary phase to an optical density of ~0.9 OD 600nm .…”

“…Intestinal (enteric) pathogenic bacteria, especially Vibrio cholerae ( V. cholerae ), are present in water environments of low temperature and almost neutral pH. In contrast, when these pathogenic bacteria enter a warm-blooded host, through the consumption of contaminated food or water, they will encounter an unfavorable environment such as acidic gastric fluid in the stomach before they reach the intestine to cause an infection [ 1 ]. Once ingested, in order to survive and adapt to the stressful environment, the V. cholerae alters its morphology to accommodate the in vivo cellular requirements [ 2 ].…”

“…The effectiveness of low pH gastric fluid as a bactericidal agent has been quantified using the typical cultivation methods, which is correct under standard procedures. However, this method assumes that only bacterial colonies which are present are alive [ 1 , 4 , 5 ], thereby underestimating the possibility of any viable but nonculturable bacteria.…”

“…Bacterial cells must be further investigated to distinguish between subpopulations of viable, vital, dormant, and injured cells. Fluorescent probe staining is largely centred on different cellular physiological characteristics, such as: propidium iodide for cytoplasmic membrane permeability [ 10 , 16 ], cyanine dyes for membrane potential [ 12 ], carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for esterase enzyme activity [ 17 ], and thiazole orange or SYBR Green I for relative DNA and nucleic acid content [ 1 , 10 ]. Fluorescence probes can differentiate between “live and dead” bacterial cells as well as between “vigorous, frail, and injured microorganisms” [ 18 ].…”

“…In a previous small-scale exploratory study [ 1 ], we showed that pathogenic nonacid adapted V. cholerae strains did have the capability to endure the simulated gastric acid fluid at low pHs in an “injured state” (due to the leaky membrane) which is presumably the VBNC state for survival. In this study, flow cytometry was able to show that non-acid-adapted Vibrio cholerae O1 and O139 are still metabolically active after exposure to simulated gastric fluid.…”