Helicobacter pylori Dormant States Are Affected by Vitamin C

Abstract

Helicobacter pylori colonizes human gastric mucosa, overcoming stressful conditions and entering in a dormant state. This study evaluated: (i) H. pylori's physiological changes from active to viable-but-non-culturable (VBNC) and persister (AP) states, establishing times/conditions; (ii) the ability of vitamin C to interfere with dormancy generation/resuscitation. A dormant state was induced in clinical MDR H. pylori 10A/13 by: nutrient starvation (for VBNC generation), incubating in an unenriched medium (Brucella broth) or saline solution (SS), and (for AP generation) treatment with 10xMIC amoxicillin (AMX). The samples were monitored after 24, 48, and 72 h, 8-14 days by OD₆₀₀, CFUs/mL, Live/Dead staining, and an MTT viability test. Afterwards, vitamin C was added to the H. pylori suspension before/after the generation of dormant states, and monitoring took place at 24, 48, and 72 h. The VBNC state was generated after 8 days in SS, and the AP state in AMX for 48 h. Vitamin C reduced its entry into a VBNC state. In AP cells, Vitamin C delayed entry, decreasing viable coccal cells and increasing bacillary/U-shaped bacteria. Vitamin C increased resuscitation (60%) in the VBNC state and reduced the aggregates of the AP state. Vitamin C reduced the incidence of dormant states, promoting the resuscitation rate. Pretreatment with Vitamin C could favor the selection of microbial vegetative forms that are more susceptible to H. pylori therapeutical schemes.

Keywords: Helicobacter pylori; dormant state; persistent infections; viable but non culturable; vitamin C.

Figure 1

Study design and description of the aims of the study.

Figure 2

Induction of dormancy. (A) Culturable cell numbers of H. pylori incubated in a microaerobic atmosphere at 37 °C under different conditions. Control (BB + 2% FCS); BB (Brucella broth) and SS (saline solution) for the VBNC protocol; AMX (amoxicilllin 10xMIC) for the AP protocol recorded at t = 0, 24 h, 48 h, 72 h, 8 days, and 14 days. (B) Total number (OD₆₀₀) of H. pylori incubated in a microaerobic atmosphere at 37 °C under different conditions, in time. (C) MTT assay of H. pylori for the evaluation of metabolic activity during incubation in a microaerobic atmosphere at 37 °C under the different conditions over time.

Figure 3

Induction of dormancy. Representative images of Live/Dead staining and the percentages of viable/dead spiral/coccoid morphotypes (graphics on the right) of H. pylori 10A/13 incubated in different conditions (BB, SS, and AMX) compared with the control, in time. The images observed by fluorescent Leica 4000 DM microscopy (Leica Microsystems, Milan, Italy) were recorded at an emission wavelength of 500 nm for SYTO 9 and of 635 nm for Propidium iodide, and several fields of view were randomly examined. Original magnification, 1000×.

Figure 4

VitC effect on VBNC generation. The addition of VitC before (t = 0) the generation of H. pylori cells’ VBNC and monitoring after 8, 9, and 10 days of incubation. (A) Representative Live/Dead microscopy images of H. pylori 10A/13 incubated in SS in the presence of VitC for eight days, compared with the untreated sample (SS). Original magnification 1000×; (B) MTT values comparing the cell viability of VBNC cells generated in SS + VitC, with respect to the untreated sample (SS).

Figure 5

Effect of VitC on the VBNC state. Representative images of Live/Dead staining showing the effect of VitC on VBNC cells compared to the untreated samples (VBNC): VitC was added after 8 days of incubation after VBNC generation, and its effect was monitored for a further 24 h (9 days), 48 h (10 days), and 72 h (11 days); it was compared with the untreated sample (VBNC). The arrows indicate the viable bacillary forms in the presence of VitC. Original magnification, 1000×.

Figure 6

VitC effect on AP generation. Representative images of Live/Dead staining showing the effect of adding VitC to AMX on H. pylori cells during the AP generation. Monitoring occurred after 48 h, 72 h, and 96 h of incubation, and a comparison with the untreated sample was conducted (AMX). The second treatment with AMX guaranteed the complete killing of the population growing in AMX plus VitC for 48 h. Original magnification, 1000×. In the graphic, the MTT values show an increase in the viability of the H. pylori cells in AMX +VitC for 48 h, compared to those grown only in AMX (untreated sample).

Figure 7

Representative images of Live/Dead staining showing the disaggregation action of VitC on AP cells, monitored after a further 24 h (72 h) and 48 h (96 h), compared to the untreated sample. Original magnification, 1000×.