Role of amoebae for survival and recovery of ‘non-culturable’ Helicobacter pylori cells in aquatic environments

Abstract

Helicobacter pylori is a fastidious Gram-negative bacterium that infects over half of the world's population, causing chronic gastritis and is a risk factor for stomach cancer. In developing and rural regions where prevalence rate exceeds 60%, persistence and waterborne transmission are often linked to poor sanitation conditions. Here we demonstrate that H. pylori not only survives but also replicates within acidified free-living amoebal phagosomes. Bacterial counts of the clinical isolate H. pylori G27 increased over 50-fold after three days in co-culture with amoebae. In contrast, a H. pylori mutant deficient in a cagPAI gene (cagE) showed little growth within amoebae, demonstrating the likely importance of a type IV secretion system in H. pylori for amoebal infection. We also demonstrate that H. pylori can be packaged by amoebae and released in extracellular vesicles. Furthermore, and for the first time, we successfully demonstrate the ability of two free-living amoebae to revert and recover viable but non-cultivable coccoid (VBNC)-H. pylori to a culturable state. Our studies provide evidence to support the hypothesis that amoebae and perhaps other free-living protozoa contribute to the replication and persistence of human-pathogenic H. pylori by providing a protected intracellular microenvironment for this pathogen to persist in natural aquatic environments and engineered water systems, thereby H. pylori potentially uses amoeba as a carrier and a vector of transmission.

Exploring the nature of interaction between shiga toxin producing Escherichia coli (STEC) and free-living amoeba - Acanthamoeba sp

“…Interestingly, the resuscitation of several species of VBNC bacteria upon being internalized by the amoebae has been documented by several authors (Steinert et al, 1997;Garcıá et al, 2007;Epalle et al, 2014;Casini et al, 2018;Dey et al, 2019;Dey et al, 2020) as it has been observed in this study (Table 2). To date, the mechanism of this shift is unknown and understanding the driving forces behind these interactions could allow developing more effective culture methods that might improve the detection of pathogens in their reservoirs and certain vehicles of infections.…”

“…The resuscitation of several species of Viable But Not Culturable (VBNC) bacteria upon being internalized by the amoebae has been documented (Steinert et al, 1997;Garcıá et al, 2007;Epalle et al, 2014;Casini et al, 2018;Dey et al, 2020). The biological reason of this shift is unknown and largely unexplored.…”

Helicobacter pyloriresults in lysis and death after exposure to water

“…This study showed that H. pylori could not survive in water by itself. However, it has been suggested that H. pylori could survive inside free‐living amoebae in environmental water, such as drinking water and waste water, 29,44,45 so, it may be possible that H. pylori can survive in water through symbiosis or interaction with amoebae. The bacteria that have endured in this way may be detected as the VBNC state in drinking water, well water, and tap water.…”

Paradigms of Protist/Bacteria Symbioses Affecting Human Health: Acanthamoeba species and Trichomonas vaginalis

“…Interestingly, amoebae are also involved in VBNC resuscitation; the shift back to the culturable, active and most importantly virulent form of the bacterium. The resuscitation of several species of VBNC bacteria upon being internalized by the amoebae has been documented ( Steinert et al, 1997 ; García et al, 2007 ; Epalle et al, 2014 ; Casini et al, 2018 ; Dey et al, 2019 , 2020 ), whereby they begin multiplying within the intracellular space of the amoeba. The exact reason for this shift is unknown and relatively unexplored, however it has been reported than the presence of extracellular pyruvate and glutamate can drive the shift to the replicative form, perhaps acting as antioxidants and facilitating cell recovery ( Ducret et al, 2014 ).…”

“…While most studies investigate the interactions of Acanthamoeba with bacteria, other free-living amoebae should also be considered. Recently, Dey and colleagues demonstrated the potential for both W. magna and Vermamoeba vermiformis to not only carry and allow multiplication of H. pylori but to resuscitate the bacteria to a culturable state ( Dey et al, 2020 ). It is becoming increasingly more evident that to minimize the risks of these pathogens to human health the whole microbial community should be considered, and further research should aim to better understand the interactions between these organisms.…”