Although culturability has been the focus of many investigations of H. pylori
, none have examined culturability in a potential natural reservoir, such as a freshwater environment. Furthermore, no investigations have been reported that differentiate between culturability and viability in a population. Using a LIVE/DEAD Bac
Light viability assay, culturability and viability were examined in cells incubated in both the laboratory and a natural freshwater stream. Although the use of multiple viability assays is preferable (see, e.g., reference 19
), other assays we investigated, such as the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay of Rodriguez et al. (28
) and the substrate responsiveness assay of Kogure et al. (16
), did not perform well when applied to H. pylori
. However, Boulos et al. (4
) reported Bac
Light viable counts to be comparable to those of CTC assays for bacteria present in drinking water. However, viable counts determined using the Bac
Light stain in chlorine-stressed cells were reported to be higher than those seen with CTC assays. The authors do acknowledge that the CTC-formazan granules were very small in stressed cells, suggesting that this accounts for the differences in the stains. Furthermore, reverse transcription-PCR has become an accepted method of determining cell viability, as the half-life of bacterial mRNA is on the order of minutes (10
). Our studies of H. pylori
in the environment suggest that in addition to giving positive results in Bac
Light assays, VBNC cells continue to transcribe several genes, including those known to be virulence determinants (unpublished data).
In our laboratory studies, the culturability of the cells was found to decline to <10 CFU/ml after ca. 10 days, although a large population of viable cells continued to be present (Fig. ). This indicates that in liquid culture in the laboratory, H. pylori
enters the VBNC state as the cells age. These results showing loss of culturability and morphological conversions are consistent with data published by Benïssia et al. (3
) and Catrenich and Makin (6
) for H. pylori
and by Alonso et al. (1
) for Campylobacter coli
cells. However, ours is the first investigation to show that viability is maintained in H. pylori
despite this loss of culturability. In addition to maintaining viability, the cells also maintained a constant optical density (Fig. ), indicating that cell lysis did not occur.
FIG. 1. (A) The viability (in cells per milliliter) (•), optical density (○), and culturability (in CFU per milliliter) () of cells aged in laboratory culture. Error bars represent the standard errors of the means (SEM) of duplicate samples; (more ...)
The morphology of the cells as culturability was lost was also examined. Again, the findings were consistent with those of previously published studies (3
) in that as culturability was lost in the laboratory, the percentage of rods declined and the percentage of cocci increased (Fig. ). These data suggest that as culturability declines, H. pylori
cells transition from culturable rods into nonculturable cocci, possibly passing through the O-U form as an intermediate stage. Because viability is maintained, this suggests that cells undergo a morphological conversion from rods to cocci as H. pylori
enters the VBNC state. This change in morphology has also been observed in both the laboratory and environment for other bacterial species entering the VBNC state (25
). However, when the percentage of rods was small compared to that of cocci, the culturability remained at ca. 107
for approximately 6 days. This large culturable coccus population suggests that cocci can be both culturable and nonculturable. It is also important that ca. 33% of the cocci observed in the nonculturable population in our various studies were shown to give positive results in Bac
Light assays. While it has been suggested by some investigators that the cocci are a death form of H. pylori
), our studies demonstrate the viability of nonculturable cocci.
Studies conducted in a laboratory are valuable but cannot reproduce the environment bacterial cells may be exposed to in nature. As epidemiological evidence supports the fecal-oral route for the transmission of H. pylori
), the natural reservoir for this organism could be a freshwater body. Using membrane diffusion chambers, H. pylori
cells were exposed to a natural stream environment while being monitored for culturability, viability, and morphological changes. In these environmental waters, the culturability of H. pylori
cells decreased over time, reaching nonculturability in from less than 6 to ca. 70 h. Despite the loss of culturable cells in all environmental conditions, a large number of cells remained viable, as indicated by the results of the Bac
Light assay. For example, cells suspended in 9°C creek water lost culturability by 42 h and yet approximately 1.6 × 106
cells/ml remained viable (Fig. ). This suggests that in similarity to the results observed in our laboratory studies, H. pylori
enters the VBNC state in a natural environment. It is this rapid entrance into the VBNC state that may account for the general inability to isolate H. pylori
cells from environmental sources. Lu et al. (20
) recently reported the isolation of H. pylori
cells from raw municipal wastewater taken from November to December in Mexico, employing immunomagnetic separation prior to culture. Their success may have been due to the high H. pylori
prevalence rate in their study area (74%), the likelihood that the cells were introduced into the wastewater only shortly before the isolation was made, and the low temperature during that season. Our results (Fig. ) suggest that H. pylori
is able to remain culturable in natural waters for 2 to 3 days when the waters are at a low temperature.
FIG. 2. The total numbers of viable cells (♦), viable rods (), viable O-U forms (•), and viable cocci (□) (in cells per milliliter) and the total number of culturable cells () (in CFU/ml) in the environment at 9°C. (more ...)
The times required for cells to lose culturability at various water temperatures.
While a considerable portion of the cell population gave Bac
Light-negative results in all experimental studies, ca. 10% (105
) of cells remained viable. Surprisingly, linear regression analysis indicated that as culturability was lost there was no statistically significant change at any sampling period in the percentage of viable rods, O-U forms, or cocci. Figure is representative of all of the environmental study results and shows the constancy of this number for each morphotype. These findings are unlike our laboratory data and those of previously published investigations and suggest that as they enter the VBNC state in the environment, there is no morphological conversion observed in H. pylori
cells. Therefore, in the environment all morphological forms of this bacterium may be present in the VBNC state. Judged on the basis of investigations of the morphological conversion of V. vulnificus
in the VBNC state (25
), it would be expected that as H. pylori
enters the VBNC state the predominant morphology would be that of cocci. However, it was found that in a population of nonculturable cells, large populations of viable rods and O-U forms in addition to cocci were present (Fig. ). This finding is consistent with our laboratory data, which indicate that cells may exist in all morphologies in the culturable and nonculturable states.
A link between temperature and culturability was suggested by Soltesz et al. (33
), as H. pylori
clinical isolates and type strains were found to survive longer during transport when held at temperatures less than 15°C. The existence of such a temperature-culturability relationship was further suggested when H. pylori
cells were observed to remain culturable longer in nutrient media incubated at 4°C compared to the results seen at 25, 40, and 42°C (15
). It has also been found that 99% of cells maintained respiratory activity for at least 250 days at 4°C and that all respiratory activity was lost after 24 h at 37°C (11
). When H. pylori
cells in our studies were placed into a natural environment over a 1-year period, the length of time that cells remained culturable changed (Fig. ). These seasonal studies involved water temperatures ranging from 9 to 23°C. In general, cells remained culturable longer in cooler (<20°C) waters than in warmer (>20°) waters. Throughout each sampling period, dissolved oxygen, pH, temperature, turbidity, and ammonia and phosphate levels were measured. Table summarizes the average measurements from each study.
Average values of the environmental parameters measured during each study
To determine whether there was a correlation between the number of hours H. pylori cells were culturable in the environment and the environmental parameters measured, a Spearman's coefficient of rank correlation was used. It was found that there was a significant correlation between both temperature (rs = −0.82857; P < 0.05) and phosphate (rs = 0.94286; P < 0.05) levels and culturability in the environment. When a sequential Bonferroni adjustment was applied, however, it was found that there was no statistically significant correlation between these environmental parameters and culturability. Also, multiple regression analysis was performed to determine whether any single environmental parameter accounted for the variation in culturability. It was found that no parameters met a statistically significant level of contribution to the culturability of H. pylori in the environment. Because our studies were conducted in a small, natural freshwater stream, there were many variables present which could not be accounted for or controlled, including the wash-off of construction debris into the stream during two sampling periods as well as a sewage leak during another sampling period. Although the possibility of a relationship between temperature and/or phosphate levels and culturability is suggested in our studies, a larger-scale investigation may be required to prove such a conclusion.
Several bacterial species that enter the VBNC state have been shown to resuscitate and become culturable following the modification of routine culture conditions. V. vulnificus
cells resuscitate through a temperature upshift (25
), while Campylobacter jejuni
requires passage through embryonated eggs (8
). Currently, no resuscitation techniques have been identified for H. pylori
. Cells which had entered the VBNC state in the environment were exposed to a variety of resuscitation conditions and examined for growth. No growth was observed under any condition, and attempts to resuscitate this organism continue in our laboratory.
This is the first investigation to examine the viability of H. pylori in both the laboratory and a natural environment as culturability is lost. Our data suggest that H. pylori is able to enter the VBNC state as cells age in the laboratory or are exposed to a natural, freshwater environment. Cells underwent a transition from culturable rods to predominately nonculturable cocci as they entered the VBNC state in the laboratory; however, no such morphological conversion was observed in the natural environment. Thus, H. pylori appears to exist in all morphological forms in the environment. Furthermore, our studies suggest that exposure to the environment can induce this organism to enter the VBNC state and to persist in the environment until it enters a suitable host. For this reason, it is important to consider this survival state for H. pylori in natural environments.