In a previous article, we reviewed a paper that investigated the microbial communities living in showerhead biofilms coming from the municipal water supply. This week, we’ll take another look at what’s living in municipal drinking water and this time in Phoenix, Arizona.  This new 2010 paper from the Journal of Environmental Quality takes a close look at the presence of a pathogenic amoeba, Naegleria fowleri, in drinking water from various wells and at different times of year . The authors find a new link between the bacterial community of the water in relation to the presence or absence of N. fowleri amoeba.

Introduction:

Amoebae exist in the environment in soil and environmental waters, but not in the potable ground well water. Or so we thought.  It turns out that free-living amoeba can and do get into the drinking water system and in fact were responsible for the deaths of two children in Arizona of primary amoebic meningoencephalitis (1).  This was unusual because it was assumed that amoeba do not exist in well water because of the lack of a carbon source and because most wells have mesh casings that prevent passage of species larger than 5 microns in size. N. fowleri is 7-20 microns in size and so are well above the cut off for the well water barrier.

With this knowledge, Ian Laseke et al. (2) investigated the extent of the presence of the pathogenic amoeba N. fowleri in the warm ground water aquifers in the Phoenix area over a range of seasonal collection times and water temperatures. They also looked at different water pumps to see if the oil based pumps influence bacterial and amoeba growth.

Using molecular analysis methods, including an N. fowleri  specific PCR assay (specific for the  Mp2Cl5 gene)  and sequencing of 16S rRNA gene libraries made from the total bacterial DNA in water samples, they were able to identify amoeba positive wells and then make specific and clear correlations between the microbial communities in positive and negative wells.  To see what they discovered, keep reading.

Methods:

Well Selection: Six public water supply wells in Phoenix were sampled between winter of 2004 and autumn of 2005. To determine if presence of N. fowleri was sensitive to water temperature, the selection of sites were based on both the time of year and the water temperature at individual wells . In addition, the pump type (submersible or oil-lubed turbine) was taken into consideration to examine if  contamination of the water with oil lubricant was playing a role as a food source for bacteria that were serving as a food source for the amoeba. Control water negative for amoeba came from the Mason Drinking Water Treatment Plant in Cincinnati, Ohio.

Sampling and Filtration: Each water sample (collected in December, August, and September) were filtered through serially arranged filters (Micro-Wynd II D-PPPY, Cuno Inc.) with pore sizes of 0.5 and 1.0 micron, respectively. Volumes ranged from 236 to 4402 liters. Filters were transported to the lab at -80^oC for processing.

The negative control water was filtered through pore size 0.5 micron catridges and three 100 liter control samples were spiked with 10³, 10⁴, and 10⁵ N. fowleri cells and then filtered through 0.5 micron pore size filters similarly to the uninfected water.

DNA Extraction: A portion of the filter (~1.61 cm² ) was removed using a sterile razor and processed using the PowerSoil DNA Extraction Kit to isolate DNA. DNA was isolated from pure cultures of N. fowleri using the same method and templates were used for PCR.

Molecular Assays:  The PCR assay was a nested PCR with the first round of PCR targeting the Mp2Cl5 gene unique to the genus Naegleria and the second round of PCR was performed using primers targeting a conserved region of the Mp2Cl5 gene specific to N. fowleri (3). PCR reations were analyzed using 2% agarose gels.

To identify the predominant bacteria in the well water, 16S rRNA gene clone libraries were generated from selected samples. Clones were sequenced and phlyogenetic trees were constructed and analyzed.

Results and Discussion:

The nested PCR assay for detection of N. fowleri  was sensitive down to 10 cells/ml of water and using this method, approximately 27% of all samples tested positive N. fowleri.  While all of the samples that tested positive were from the summer and fall months, water temperature ranged from between 29 to 47^oC. So while infection of the water may be seasonal, it is not related to water temperature. It was also not related to the type of pump, indicating that the contamination of oil in the water does not enhance the level of infection of amoeba.

To look for linkages between the bacterial populations in water, which are a carbon source for amoeba, and the presence or absence of N. fowleri, the 16s rRNA gene libraries were analyzed and compared. Wells that were negative for N. fowleri consisted only of β- proteobacteria (primarily clones were within the Caldimonas and Leptothrix clades).  Clones from the positive wells, however, contained a much higher level of bacterial diversity.  N. fowleri positive wells contained members of the Bacteroides and Firmacutes phyla, as well as Deinococcus- Thermus, Nitropira, and Acintobacter.  

Summary:

Interestingly, the presence of a high level of iron and manganese oxidizing bacteria (such as the genera Caldimonas and Leptothrix)  in negative wells correlates with previous findings that amoeba require these metals to be present in environmental waters to thrive (4, 5). It has been noted that amoeba generally prefer gram-negative nonpigmented bacteria as a food source and pigments are known to possess iron chelating activity (6).  All of the negative wells were reported to have very low levels of iron and magnesium. 

To summarize, municipal warm ground water wells in the Phoenix area tested positive for N. fowleri and their presence did not correlate with water temperature, pH,  or heterotropic plate count.  Presence of the amoeba did correlate with a more diverse bacterial community, specifically an increase in some of the enteric bacterial groups and a reduction in metal oxidizing β- proteobacteria. This leads to some exciting future studies on the role of metals and metal-oxidizing bacteria on the amoeba diet.

Final note:

Personally, I would be interested in more studies on what caused some of the ground well water microbial communities to change and how to restore them to an amoeba inhibitory state. The questions that come to my mind are- what happened first; did the amoeba arrive and then the ecology changed? Or did the microbial community change and the amoeba flourished?  In addition, it might be interesting to isolate, if possible, live N. fowleri from positive water wells to determine if their size has diminished to allow them access to water wells or whether the mesh casing meant to keep large organisms out of the water wells needs to be replaced.

Thanks for reading and if you have any questions or comments, please leave us a note below or email us anytime at technical@mobio.com.

For more advice on choosing and handling filter membranes, look at this previous article on water filters. Included is a video on how to handle the water filter without damaging the membrane suface.

Have a great week,
Suzanne

References:

1- Marciano-Cabral, F. et al. 2003
Identification of Naegleria fowleri in domestic water sources by nested PCR
Appl. Environ. Microbiol. 69:5864-5869

2- Laseke, I. et al. 2010
Identification of Naegleria fowleri in Warm Water Ground Water Aquifers
Journal of Environmental Quality, vol. 39, January-February 2010, page 147-153

3- Reveiller, F.L., et al. 2002
Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri.
Parasitol. Res. 88:443-450

4- Duma, R.J. 1981.
Study of pathogenic free-living amoebas in freshwater lakes in Virginia.
Environmental Health Effects Research Series. EPA no. 600/1-800-037

5- Kyle, D.E., and G.P. Noblet. 1985.
Vertical distribution of potentially pathogenic free-living amoebae in freshwater lakes.
J. Eukaryot. Microbiol. 32:99-105

6-Singh, B.N., and G.D. Dutta. 1984.
Small free-living aerobic amoebae: Soil as a suitable habitat, isolation, culture, classification, pathogenicity, epidemiology and chemotherapy.
Indian J. Parasitol. 8:1-23