Environmental isolation of thermotolerant Ameobae from Wolf Creek Generating Station cooling waters
Naegleria fowleri is a free-living, thermophilic, amoeboflagellate that is the causative agent of a rare, yet severe disease of the central nervous system called Primary Amoebic Meningoencephalitis (PAM), which is nearly always fatal. PAM has yet to be diagnosed in Kansas. It has, however, been reported in two neighboring states of Kansas, Oklahoma and Missouri. The goal of this research is to demonstrate the presence of N. fowleri in Kansas through both classical morphological analysis and genetic screening using the polymerase chain reaction (PCR). Water samples were collected under special circumstances with a desire to learn whether M. fowleri could be easily extracted from the environment in the middle of winter. To test this, seven samples were taken from the cooling waters at Wolf Creek Generating Station in Burlington, KS and plated onto a low nutrient agar with a lawn of UV killed Enterobacter aerogenes as a food source. Of those samples, six yielded 38 separate amoeba samples to be further analyzed. Amoeba isolates are currently being analyzed for flagella formation, thermotolerance at 45 degrees C, and PCR using Naegleria and N. fowleri-specific primers. Microscopic examination of many of the amoebae show morphology characteristic of Naegleria sp.
Environmental Isolation of Thermotolerant Ameoba from Wolf Creek Generating Station Cooling Waters Abstract. Naegleria fowleri is a free-living, thermophilic, amoeboflagellate that is the causative agent of rare, yet severe disease of the central nervous system called Primary Amoebic Meningoencephalitis (PAM), which is nearly always fatal. PAM has yet to be diagnosed in Kansas. It has however, been reported in two neighboring states of Kansas, Oklahoma and Missouri. The goal of this research is to demonstrate the presence of N. fowleri in Kansas through both classical morphological analysis and genetic screening using the polymerase chain reaction (PCR). Water samples were collected under special circumstances with a desire to learn whether N. fowleri could be easily extracted from the environment in the middle of winter. To test this, seven samples were taken from the cooling waters at Wolf Creek Generating Station in Burlington, KS and plated onto a low nutrient agar with a lawn of UV killed Enterobacter aerogenes as a food source. Of those samples, six yielded 38 separate amoeba samples to be further analyzed. Amoeba isolates are currently being analyzed for flagella formation, thermotolerance at 45˚C, and PCR using Naegleria and N. fowleri-specific primers. Microscopic examination of many of the amoebae show morphology characteristic of Naegleria sp. Future testing in this area will involve determining if any Kansas strains of N. fowleri show genetic diversity from other neighboring strains, as well as investigating whether any undiagnosed deaths in Kansas from meningitis-like symptoms were in fact cases of PAM. The project described was, in part, supported by grant number P20 RR016475 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) Results • Over 40 amoebae isolates were obtained from 7 water samples at Wolf Creek • Of those examined to date, 6 have the transformation phenotype • Of those examined to date, 13 are thermophilic (growth at 42˚C) • Two of the 13 thermophilic amoebae are capable of forming into flagellates • See Table 1 for summary of results to date Trent Snyder and John Mullican Department of Biology Washburn University, Topeka, KS Introduction In 1965, the first case of primary amoebic meningoencephalitis (PAM) was described in Australia by M Fowler and R.F. Carter (1). The causative agent of this rare and almost always fatal disease of the central nervous system was described by Carter in 1970 as the amoeboflagellate Naegleria fowleri (2). While ubiquitous in the environment, very few incidences are recorded each year. Furthermore, out of the two hundred or so known cases of PAM, only two patients are known to have successfully recovered from the infection, following vigorous treatment regimens (3). Interestingly, the majority of PAM cases occur in individuals who are otherwise healthy (immuno-competent), but have a recent history of association with freshwater sources. N. fowleri is a thermo-tolerant species and is able to thrive in freshwater temperatures of up to 45°C (3). It has an interesting tri-cycle lifecycle, existing in three known morphological stages: the vegetative trophozoite, cyst and biflagellate forms (Figure 1). Water samples were obtained in the winter of 2011 from Wolf Creek Generating Stationʼs cooling waters in Burlington, Kansas. After processing and culturing these samples, the previously described morphological characteristics were used to select for isolates likely to be N. fowleri. Genetic detection of thermotolerant amoebae using the polymerase chain reaction (PCR) will be done to understand how Naegleria from Kansas differ from those isolated elsewhere (4). This study was undertaken to determine the presence of N. fowleri in local waters and to help understand why cases of PAM have not been previously reported in Kansas. Discussion and Conclusion This is part of an ongoing study that began in 2006 to demonstrate the prevalence of N. fowleri in the state of Kansas, which may suggest a potential health risk in contaminated waters. Numerous amoeba isolates were recovered with both surface water and sediment samples from Wolf Creek Generating Station in Burlington, KS. We are planning to cover more freshwater sources, especially bodies of water where there is a high recreational use. Previous samples in this study have yielded PCR-based confirmation of N. fowleri in Potter Lake in Lawrence, Ks; thus, the potential for one or several of the Wolf Creek Generating Station samples to contain N. fowleri is high. Since the cooling waters of the plant stay at a warm 80˚F (~27˚C) in the middle of winter and climb to temperatures of around 110°F (~43˚C) in summer, the warmer temperatures select for thermotolerant and thermophilic organisms, making the presence of Naegleria spp. in the water even more likely. Though the Wolf Creek amoeba samples in the current study are still being analyzed, there are several that are good candidates as Naegleria. To further test these samples, PCR analysis will be utilized in the very near future to examine the genotype of the amoeba isolates. Purified DNA samples from all amoebae will be analyzed. This procedure will amplify sequences specific to the N. fowleri genome and PCR fragments will be assessed via agarose gel electrophoresis. This specific amplification is accomplished by using primers that are specific for either the genus Naegleria or for N. fowleri. To our knowledge there have been no reports of PAM in Kansas to date, yet N. fowleri is most likely present in freshwater sources in the State. This finding is not surprising as Naegleria species have been found on every continent, while PAM is extremely rare. Future, planned mice studies in collaboration with Dr. Francine Marciano-Cabral (Virginia Commonwealth University, Richmond) will help determine the pathogenicity of any Kansas N. fowleri isolates. The early results of this study demonstrate the diversity of thermotolerant/ thermophilic amoebae in freshwater sources in Kansas, some of which are most likely to be Naegleria fowleri, a known pathogen. The presence of other (non- Naegleria) thermotolerant amoeba species in waters frequented by recreation seekers like water skiers, swimmers, etc. is informative as a few of these species may also be human pathogens. Further tests are needed to discover whether these other samples contain amoebae species that are known to be present in Kansas waters, and if these species are pathogenic to humans. The presence of N. fowleri in Kansas also opens the possibility that previous cases of fatal meningitis with unknown etiology may potentially be PAM cases attributed to N. fowleri infection. Upon confirmation, we will investigate possible collaborations with medical centers to examine tissue samples from patients dying from undiagnosed meningitis-like illnesses. References 1. Fowler, M., and R. F. Carter. (1965) Acute pyogenic meningitis probably due to Acanthamoeba sp. A preliminary report. BMJ 2(5464): 740-742. 2. Carter R. F. (1970) Description of Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and the experimental pathological changes induced by it. J. Pathol. 100:217-244. 3. De Jonckheere J.F. (2002) A century of research on the amoeboflagellate genus Naegleria. Acta Protozool 41:309-342. 4. Reveiller, F. L., P. A. Cabanes, and F. Marciano-Cabral. (2002) Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri. Parasitol. Res. 88:443-450. 5. Page, F. C. (1988) A new key to freshwater and soil gymnamoeba with instructions for culture. Freshwater Biological Association, Ambleside, United Kingdom. 6. Fulton, C. (1970) Ameboflagellates as Research Partners. In Methods in Cell Physiology, ed. Prescott, D.M., Vol 4, pp. 341-476. 7. Weik, R. R., and D. T. John. (1977) Agitated Mass Cultivation of Naegleria fowleri. J. Parasitol. 63:868-871. 8. Sheehan, K.B., … J. Henson. (2003) PCR Detection and Analysis of the Free-Living Amoeba Naegleria in Hot Springs in Yellowstone and Grand Teton National Parks. Appl. Env. Micro. 69(10):5914-5918. Materials and Methods Environmental Sampling Each free water sample was collected in a 50 mL centrifuge tube in one of two ways. In the surface sample method a 50 mL centrifuge tube was placed just under the water surface to collect water circulating near the surface. In the sediment collection method the sediment was stirred up by hand and a 50 mL centrifuge tube was placed inverted into the water until is was ~1 inch from the sediment and surrounded by a cloud of stirred up sediment. It was then righted and capped. In total, 7 samples (4 surface water and 3 sediment samples) were collected. Temperature and pH data were also recorded each time a sample was obtained. Processing Samples All samples were vortexed upon return to the lab in order to thoroughly mix the contents of the centrifuge tubes. Each sample (1 mL) was then pipetted from the centrifuge tube onto its own NM agar (6) plate containing a lawn of UV-killed Enterobacter aerogenes (henceforth referred to as NMEA plates). Following this, the sample tubes were then centrifuged at 3000 rpm for 10 min in order to collect all the present solids at the bottom of the tube. The supernatant was then drained off until only 1 mL remained. This was then centrifuged in order to resuspend the solids. This was then added to a NMEA plate. Plates were then incubated at 37° C for at least 24 hours. Additional time was allowed if amoeba plaques were either not present or smaller than 1.0 cm in diameter. Plaque isolation Individual clearings of the bacterial lawn (plaques) were transferred to a second plate by cutting a cube of agar from the original plate with a sterile stainless steel spatula with the amoeba-side down on a new agar plate covered with an inactivated bacterial lawn. These plates were incubated at 37°C until the outgrowth plaques were large enough to obtain another scraping or agar cube to another plate with the intention of assessing temperature tolerance and extracting DNA. If any fungal contamination was present, amoebae were transferred to new plates until a clean sample could be isolated from the plate. Assessing Temperature Tolerance Each isolate from the previous step was transferred to two separate plates. One plate was incubated at 37°C and one at 42°C for at least 48 hours. Any plates containing plaques that continued to expand in 42°C after 48 hours (“thermotolerant samples”) were removed and stored or directly transferred to liquid culture medium. Starvation Test for Assessing Flagella Formation Each isolate from isolation procedure was also challenged with a starvation stress in order to see if the organism could form flagella. This was accomplished by using a sterile loop to transfer amoeba from the growth edge of the sample into 0.75 mL of sterile water in a chamber slide. Once placed in this nutrient-deficient environment, those amoebae capable of transforming into flagellates will do so in at minimum 60 min. Samples were checked after 2 and 3 hours for flagella formation. Collection Method Sample # # of Plaques # of Thermotolerant Isolates # of Amoeboflagellates Both Dip 1 10 3 1 0 2 3 1 0 0 Sediment 1 3 NA NA NA 2 10 4 1 1 3 13 5 5 1 *Thermotolerance and Flagella testing are ongoing Figure 3. NM agar plate with E. aerogenes lawn spread across surface. At edges streak marks in lawn are still visible. Featureless circle in the middle is the expanding colony of amoeba. Dark square in center is the plug of NM agar used to transplant the amoeba from a previous plate. Figure 4. This is a microscopic view of the amoebic cysts that make up the dormant center of the grey circle seen in Figure 3. When an amoeba encounters extended, harsh conditions it will encyst in order to preserve its life. Figure 5. This picture is taken from the very edge of the grey circle seen in Figure 3. Here there is a surplus of nutrients provided by the E. aerogenes bacteria coating the agar. The amoeba are in trophozoite form and dividing rapidly. Figure 4 Figure 5 Example of water sample collection using 50 mL conical centrifuge tubes. Table 1. Summary of Wolf Creek Amobae Isolates to Date. Wolf Creek Generating Station, Burlington, KS. Yellow arrow indicates the hot water outflow into the cooling waters. The yellow dot represents the area where the water samples were retrieved. Figure 1. Partial Restriction Maps of the Circular Ribosomal DNA Elements from Naegleria spp. Represented in Linear Format. pGRUB and pFOWL are the cloned extrachromosomal rDNA elements from N. gruberi, EGB strain and N. fowleri, LEE strain, respectively. The DNA sequence of pFOWL is incomplete as demonstrated on the lower portion of the figure. An approximately 2 kbp region within the 8 kbp EcoRI fragment of pFOWL has not been sequenced completely. This region has been very difficult to sequence, due presumably to numerous repetitive sequences that have made it difficult to subclone and/or sequence the region. The hatched region ( ) represents the un-sequenced gap in pFOWL. Recall that while these are circular molecules, we represent them in linear fashion to better illustrate similarities and differences. The green (ori?) boxes within the 2.0 kbp XhoI fragment of pGRUB are putative origin of replication sites mapped previously via 2D agarose gel electrophoresis. ori? ori? Putative origin of replication - mapped XhoI XhoI pFOWLR8 H S R R ……… pFOWLR8_del2 pFOWLR8_del1 pFOWLR8_del3 pFOWLR8_del4 pFOWLR8_del5 pFOWLR8_del6 ExoIII S1 nuclease 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 EcoRI Digest of pFOWLR8 clones Figure 2 Key R = EcoRI cut site H = HindIII cut site S = SacI cut site Strategy for the Analysis and Sequencing of Subclones Figure 3 pFOWLR8 EcoRI digest