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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Tap water
 ~ Swabs
 ~ Results
 ~ Discussion
 ~  References
 ~  Article Figures
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  Table of Contents  
Year : 2015  |  Volume : 33  |  Issue : 3  |  Page : 343-348

Free living amoebae in water sources of critical units in a tertiary care hospital in India

1 Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Hospital Administration, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission25-Nov-2014
Date of Acceptance18-Feb-2015
Date of Web Publication12-Jun-2015

Correspondence Address:
S Khurana
Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.158543

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 ~ Abstract 

Background: Isolation of free-living amoebae (FLA) is reported sparsely from water taps, ventilators, air conditioners, haemodialysis units and dental irrigation systems of hospitals worldwide. Their prevalence in hospital environment especially in wards having immunocompromised patients may pose a risk to this group of susceptible population as they may cause disease themselves or may carry pathogens inside them. No study from India has performed such surveillance. Objective: To evaluate extent of FLA contamination in water sources of bone marrow transplant (BMT) intensive care unit (ICU), transplant ICU, haemodialysis unit and high dependency unit in a tertiary care hospital in India. Materials and Methods: A total of hundred samples including fifty each of tap water samples and swabs from mouth of taps used for drinking, bathing and hand washing purposes in these units were collected according to standard procedure. Samples were inoculated onto non-nutrient agar plates at room temperature followed by morphological confirmation. Molecular identification including polymerase chain reaction (PCR) and sequencing was performed in culture positive samples. Results: Four tap water samples and ten swab samples showed growth of trophozoites and cyst formation. Morphologically, four amoebae resembled Acanthamoeba spp. which was further confirmed by PCR and sequencing showed them to be of T3 and T4 genotypes. Conclusion: The presence of these FLA in hospital water sources emphasises the urgent need of implementing effective preventive measures. Further studies are required to estimate the true prevalence of FLA in Indian hospitals by taking larger number of samples.

Keywords: Free living amoebae, hospital, intensive care unit, India

How to cite this article:
Khurana S, Biswal M, Kaur H, Malhotra P, Arora P, Megha K, Taneja N, Sehgal R. Free living amoebae in water sources of critical units in a tertiary care hospital in India. Indian J Med Microbiol 2015;33:343-8

How to cite this URL:
Khurana S, Biswal M, Kaur H, Malhotra P, Arora P, Megha K, Taneja N, Sehgal R. Free living amoebae in water sources of critical units in a tertiary care hospital in India. Indian J Med Microbiol [serial online] 2015 [cited 2020 Sep 27];33:343-8. Available from:

 ~ Introduction Top

Free living amoebae (FLA) is a broad term encompassing variety of agents both pathogenic and non pathogenic organisms. [1],[2] The common pathogenic agents include Acanthamoeba, Naegleria, Balamuthia and Sappinia.[2] However, other amoebae like Vahlkampfia, Paravahlkampfia and Hartmanella have also been shown to be associated with human infection. [1] The advancement in medical therapy including transplantation, haemodialysis and critical care has contributed to the increase in susceptible population, thereby increasing number of infections by these agents. [1] Cysts of these amoebae especially Acanthamoeba spp are resistant to harsh conditions and can survive for long time thereby adding to their widespread dispersal. [3] The presence of FLA has been observed in nearly all types of habitats including soil, dust, water and air. [1],[4] Acanthamoeba isolates have been recovered from ventilators, hydrotherapy bath, air conditioners, hemodialysis and dental water supply units. [4] FLA pose a serious problem for high risk patients as apart from being pathogenic themselves, they serve as Trojan horses for many other pathogens like methicillin resistant Staphylococcus aureus, Legionella pneumophila, Pseudomonas aeruginosa, Enterobacter cloacae,  Escherichia More Details coli, Klebsiella species, Streptococcus pneumoniae, Chlamydiaceae, Parachlamydiaceae Parachlamydiaceae, non-tuberculous mycobacteria (NTM), mimivirus and enteroviruses. [3],[5] Moreover, these bacteria carried by the amoebae are observed to be resistant to multiple antibiotics leading further to spread of drug resistance among hospitalised patients. FLA are opportunistic pathogens causing serious diseases like encephalitis, keratitis and dermatologic manifestations. [2],[3] Nosocomial infections may occur due to these agents due to their persistence and survival in biofilms inside the storage tanks or main water supply to hospitals. Therefore, sources of drinking water or that for routine use as bathing or hand washing are the major foci leading to nosocomial infections and should be investigated.

Many studies around the world have been conducted to find out the prevalence of FLA especially Acanthamoeba in hospital water sources. However, no such study has been performed in India. We present here the first study from India with the aim to screen for the presence of FLA in water sources used for drinking, bathing and hand washing by high risk patients admitted to bone marrow transplant (BMT), intensive care unit, high dependency unit (HDU) that caters to haemato-oncology patients, renal transplant intensive care unit (TICU) and haemodialysis unit (HU).

 ~ Materials and Methods Top

The study was approved by Institute Ethics Committee vide number NK/530/Res/3516 Dt. 15.11.2012.

Sample collection

A total of 100 samples (50 tap water samples and 50 swabs form tap mouths) were collected from 50 taps used for drinking (including seven reverse osmosis filter taps), hand washing and bathing purposes in BMT ward (25 taps), HDU (9 taps), TICU (13 taps) and HU (3 taps) of our institute over a period of four months from March to June 2014. The taps also included those of nursing counters, coffee rooms and pantry. Sterile cotton swabs were used to swipe the mouth of the taps before opening them and then dipped into 1 ml of sterile distilled water. The mouth of the tap was then flamed and water was allowed to run for 5-10 minutes, after which 500 ml of water was collected in sterile glass bottles. [6]

After the isolation of free living amoebae from these sites, all the taps and water purifications systems were serviced and thoroughly cleaned and follow-up samples a week after, yielded negative results.

Filtration of water and culture of FLA

 ~ Tap water Top

Five hundred millilitre of tap water from each of 50 taps was filtered through sterile 0.45 μm membrane filter within 2 hours of collection. [4] Filters were then inverted onto 1.5% non-nutrient agar (NNA) plates with an overlay of E. coli ATCC 25922. All plates were sealed and incubated at 37°C. They were examined daily for growth of amoebae under 10 × magnification of light microscope for up to 2 weeks. The FLA were confirmed by their cyst and trophozoite morphology. A block was cut and placed onto fresh NNA plate overlaid with E. coli and incubated at 30°C for subculture to obtain growth for molecular study.

 ~ Swabs Top

The swabs dipped in 1 ml of distilled water during transportation, were vortexed for 30 seconds onds followed by centrifugation at 800 g for 10 minutes. [7] Pellet was resuspended in 100 μl of Page saline which was spread onto the 1.5% NNA plate overlaid with E. coli and was incubated and processed in a similar manner as that for water samples.

Nucleic acid extraction, amplification and sequencing

The nucleic acid was extracted from culture by phenol-chloroform-isoamyl alcohol method as previously described.[8] Extracted deoxynucleic acid (DNA) was subjected to PCR (polymerase chain reaction) amplification (Thermo Scientific Thermal Cycler) of 18S rRNA sequences using primers common for FLA as described earlier which amplify Acanthamoeba spp, H. vermiformis, N. fowleri, Vannella spp. and Vahlkampfia ovis. [8],[9],[10] Briefly, reaction mixture of 25 μl consisted of buffer, 0.2 mM dNTPs (Sigma) and 20 pmol each of primers and 1.25 units of Taq DNA polymerase (Sigma). The amplification cycle included 40 cycles of denaturation 94°C for 4 min annealing (63°C for 1 min) and extension (74°C for 3.5 min) followed by a final extension at 72°C for 10 min. The resultant amplicons were subjected to agarose gel (2%) electrophoresis. The samples were also subjected to amplification by Naegleria specific primers and Acanthamoeba specific primers (JDP1 and JDP2) as previously described. [10],[11] Briefly, Naegleria DNA amplification consisted of 50 cycles of denaturation (95°C for 15 s), annealing (58°C for 30 s), and extension (72°C for 45 s). The PCR amplification cycle for JDP primers was standardised which included initial denaturation at 95°C for 10 min followed by 39 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 7 min. Amplified DNA products were separated by 1.5% agarose electrophoresis stained with a solution of 0.5 μg/ml of ethidium bromide and visualised under UV light using an image analyser. Acanthamoeba spp were further classified into genotypes based on 18S ribosomal RNA nucleotide sequencing with the conserved primers 892C, using Big Dye Terminator Cycle Sequencing Kit, version 3.1 (Applied Biosystems, Foster city, CA, USA) and analysed by ABI 3130 Genetic Analyser (Applied Biosystems). [8],[11] [Table 1]. Nucleotide similarity search was performed by BLAST search of sequenced amplicons in GenBank database.
Table 1: Primer sequences used in the study

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 ~ Results Top

Of the 100 samples, 14 samples (14%) showed positive culture of trophozoites and cysts of FLA [Figure 1]. BMT ward, HDU, TICU and HU yielded 7/50 (14%), 5/18 (27.77%), 2/26 (7.69%) and 0/6 culture positive samples respectively [Table 2] and [Table 3] shows the source of these culture positive samples and duration after which trophozoites and cysts were demonstrated in them. Most of the positive samples were obtained from swabs by swiping the inner mouth of taps representing biofilms. Swabs were observed to show growth earlier (<5 days) than tap water (≥5 days). Morphological examination of four plates demonstrated Acanthamoeba trophozoites and double layered cysts. These were confirmed to be Acanthamoeba species by PCR using both common primers (1080 bp) and specific primers (450-500 bp) [Figure 2] and [Figure 3]. Rest 10 samples showed trophozoites and cysts but were not amplified by any of the primers. Sequencing of four Acanthamoeba species classified them into T3 (scrub station tap 3 water and swab of HDU) and T4 genotype (swab from bathing tap in bathroom of room 2 and coffee room RO swab of BMT ICU).
Figure 1: Microscopic picture of culture of free living amoebae (trophozoites and cysts) on non nutrient agar (10×)

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Figure 2: PCR amplification with P-FLA-F/P-FLA-R primer (Tsvetkova et al., 2004) Lane1 and 8: 100bp Molecular marker Lane 2-3: Water Samples Negative for Acanthamoeba, Lane 4-5: Water Samples Positive for Acanthamoeba Lane 6: Negative Control (NTC, only milliQ water) Lane 7: Laboratory Maintained isolate of Acanthamoeba

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Figure 3: Genus specific PCR amplification with JDP1 and JDP2 (Schroeder et al., 2001) Lane1 and 8: 100bp Molecular marker Lane 2-3: Water Samples Negative for Acanthamoeba, Lane 4-5: Water Samples Positive for Acanthamoeba Lane 6: Negative Control (NTC, only milliQ water) Lane 7: Laboratory Maintained isolate of Acanthamoeba

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Table 2: Ward wise positive samples

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Table 3: Sources and duration of culture and PCR positivity in samples

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 ~ Discussion Top

The present study demonstrates for the first time, isolation of FLA from routine use water sources in critical units of a hospital in India. Most of the studies regarding isolation of FLA from water sources and biofilms of hospitals are reported from Europe, Iran, Egypt and Brazil. [4],[12],[13],[14],[15] The patients in BMT unit, HDU, TICU and HU constitute a group of high risk population who are more susceptible to diseases by any pathogen. The water sources of these units were screened for FLA which may be pathogenic themselves or may act as vehicle for carrying bugs especially drug resistant micro organisms which may cause nosocomial infections. Our study has demonstrated presence of FLA (morphological forms of trophozoites and cysts) in 14% of water and swab samples. Four of these samples were identified as Acanthamoeba species on the basis of morphological characteristics (double walled cyst wall) which were further confirmed by PCR. This is a significant finding as Acanthamoeba is a well known pathogen of immunocompromised patients causing serious sequelae like encephalitis. Its isolation from reverse osmosis filter (RO) taps of nursing coffee room (swab only) and pantry (swab and water both) of BMT ward is an additional challenge because these are the drinking water sources of both staff and the patients. Although faecal to oral route of transmission of FLA is not documented, still the risk of spread of pathogenic bacteria through FLA as vehicles of entry remains in the vulnerable population. Their isolation from bathing and hand-washing taps further increases the risk of acquiring these pathogens through skin, mucosa, nasal and ocular tracts. The remaining 10 samples could not be identified by PCR, though they showed trophozoites and cysts in culture. The reason for this could be that in spite of being FLA, primers used in the study could not amplify them thereby highlighting the fact that water may harbour a large number of FLA which may not be identified.

Most of the positive cultures were recovered from the swab samples rather than tap water which suggests that these amoebae may be entrapped in the biofilms formed in the moist tap mouth. Lasjerdi et al., from Iran showed isolation of FLA in 52.9% (59.4% dust and 45.4% biofilms) of dust and biofilms formed in moist environments of hospital wards. [1] 96.9% of Acanthamoeba species isolated in the study belonged to T4 genotype. Other FLA included Hartmanella and Vahlkampfia. A study by Rohr et al. from Germany showed high isolation of FLA up to 52% and 47% in hot water taps and swabs respectively. [16] They included six hospitals of Germany and sampled 56 hot water taps and 49 swabs from drains, tap mouths, floor tiles and bathrooms and observed isolation of Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmanella (15%) and Vanella (7%). Another study from Germany by Michel et al., also showed presence of FLA including Acanthamoeba, Neagleria, Hartmanella and Echinamoeba in 54% water samples in a new hospital building. [17] We used common primers for FLA as described earlier which could identify the four Acanthamoeba isolates which were also further confirmed by using specific JDP primers. However, the common primers could not identify the remaining ten FLA which may be due to lack of such sequence in these FLA as water harbours a plethora of organisms which may have some different sequences. [9] Some other target in 18S rRNA itself may help in resolving this issue. Bagheri et al., reported presence of Acanthamoeba in 48% of tap water samples collected from different wards of hospitals in 13 cities of Iran. [12] Carlesso et al., demonstrated FLA in 35% of swab samples from dust and biofilms of which 34% were Acanthamoeba species. [18] In another study by Carlesso et al., 23% of similar samples showed presence of Acanthamoeba species of T3 and T4 genotypes in dust and T5 genotype in biofilms. [15] A study from France by Ovrutsky has shown isolation of FLA including Acanthamoeba of T3 and T4 genotype in 14.8% samples of water and biofilms formed in shower drains and sink drains. [7] The four Acanthamoeba isolates in our study also belonged to both T3 and T4 genotypes (two each). The pathogenic Acanthamoebae earlier have been observed to be of T3 and T4 genotype, thereby suggesting these isolates obtained from critical units may pose danger to immunocompromised patients. [19] Another study from France by Lasheras et al. showed a high presence of 68.9% of FLA in hot water faucets, showers, hot water tanks and cooling towers of 10 hospitals. [20] A study from Switzerland by Thomas et al. have shown isolation of FLA predominantly Hartmanella vermiformis in 7.5% in tap water, swabs from tap and showerhead of ICU, surgery ward, medicine and other wards in a hospital. [14] Haemodialysis and dental units have also shown higher isolation rate of 32.6-42.9% of Acanthamoeba spp in studies from Tunisia and Egypt. [4],[21],[22] Studies by Dendana et al., and Trabelsi et al., from Tunisia have shown 29% acanthpodial forms and 69% FLA including Acanthamoeba in water samples of haemodialysis and dental unit respectively. [21],[23] Our study identified presence of Acanthamoeba species in two samples each from BMT ward (coffee room RO swab, bathing tap swab of room 2) and HDU (scrub station 3 water and swab from tap mouth). However, samples from transplant ICU and haemodialysis unit in our study did not show isolation of Acanthamoeba species.

Acanthamoeba cysts are known to be resistant to chlorine used for disinfecting water and therefore may survive in water. [1] This may lead to widespread dispersal of cysts. Many hospitals have water storage tanks for emergency water supply. They may also act as source of contamination by FLA. [12] One sample in our study was collected from such storage tank but it turned out to be negative in culture thereby demonstrating that contamination was taking place in pipes which may be due to the persistence of these FLA in the biofilms. Taking into account, the increase in the immunosuppressed population, it is very important to take urgent steps to prevent nosocomial infections by these agents or by the drug resistant bacteria they harbour. There are no guidelines for the prevention of colonisation by amoeba in health care settings. However, going by the results of our study and others, there is an urgent need to focus on methods of prevention/removal of biofilms caused by FLA in hospital water especially those housing high risk patients. Servicing/replacement of the RO filter seemed to eradicate the focus. However the frequency of sampling and/or servicing needs to be explored in future studies.

 ~ References Top

Lasjerdi Z, Niyyati M, Haghighi A, Shahabi S, Biderouni FT, Taghipour N, et al. Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran. Parasitol Res 2011;109:575-80.  Back to cited text no. 1
Schuster FL. Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 2002;15:342-54.  Back to cited text no. 2
Bradbury RS, French LP, Blizzard L. Prevalence of Acanthamoeba spp. in Tasmanian intensive care clinical specimens. J Hosp Infect 2014;86:178-81.  Back to cited text no. 3
Hassan A, Farouk H, Hassanein F, Abdul-Ghani R, Abdelhady AH. Acanthamoeba contamination of hemodialysis and dental units in Alexandria, Egypt: A neglected potential source of infection. J Infect Public Health 2012;5:304-10.  Back to cited text no. 4
Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 2004;17:413-33.  Back to cited text no. 5
Senior BW. Examination of water, milk, food and air. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackey and McCartney Practical Medical Microbiology. 14 th ed. India: Churchill Livingstone; 2011. p. 883-921.  Back to cited text no. 6
Ovrutsky AR, Chan ED, Kartalija M, Bai X, Jackson M, Gibbs S, et al. Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata. Appl Environ Microbiol 2013;79:3185-92.  Back to cited text no. 7
Schroeder JM, Booton GC, Hay J, Niszl IA, Seal DV, Markus MB, et al. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 2001;39:1903-11.  Back to cited text no. 8
Coskun KA, Ozcelik S, Tutar L, Elaldi N, Tutar Y. Isolation and identification of free-living amoebae from tap water in Sivas, Turkey. Biomed Res Int 2013;2013:675145.  Back to cited text no. 9
Tsvetkova N, Schild M, Panaiotov S, Kurdova-Mintcheva R, Gottstein B, Walochnik J, et al. The identification of free-living environmental isolates of amoebae from Bulgaria. Parasitol Res 2004;92:405-13.  Back to cited text no. 10
Schild M, Gianinazzi C, Gottstein B, Muller N. PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections. J Clin Microbiol 2007;45:564-7.  Back to cited text no. 11
Bagheri H, Shafiei R, Shafiei F, Sajjadi S. Isolation of Acanthamoeba spp. From drinking waters in several hospitals of Iran. Iran J Parasitol 2010;5:19-25.  Back to cited text no. 12
Briancesco R, Semproni M, Della Libera S, Sdanganelli M, Bonadonna L. Non-tuberculous mycobacteria and microbial populations in drinking water distribution systems. Ann Ist Super Sanita 2010;46:254-8.  Back to cited text no. 13
Thomas V, Herrera-Rimann K, Blanc DS, Greub G. Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl Environ Microbiol 2006;72:2428-38.  Back to cited text no. 14
Carlesso AM, Artuso GL, Caumo K, Rott MB. Potentially pathogenic Acanthamoeba isolated from a hospital in Brazil. Curr Microbiol 2010;60:185-90.  Back to cited text no. 15
Rohr U, Weber S, Michel R, Selenka F, Wilhelm M. Comparison of free-living amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance. Appl Environ Microbiol 1998;64:1822-4.  Back to cited text no. 16
Michel R, Burghardt H, Bergmann H. Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa, after their isolation from a microbiologically contaminated drinking water system in a hospital. Zentralbl Hyg Umweltmed 1995;196:532-44.  Back to cited text no. 17
Carlesso AM, Simonetti AB, Artuso GL, Rott MB. Isolation and identification of potentially pathogenic free-living amoebae in samples from environments in a public hospital in the city of Porto Alegre, Rio Grande do Sul. Rev Soc Bras Med Trop 2007;40:316-20.  Back to cited text no. 18
Booton GC, Kelly DJ, Chu YW, Seal DV, Houang E, Lam DS, et al. 18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong. J Clin Microbiol 2002;40:1621-5.  Back to cited text no. 19
Lasheras A, Boulestreau H, Rogues AM, Ohayon-Courtes C, Labadie JC, Gachie JP. Influence of amoebae and physical and chemical characteristics of water on presence and proliferation of Legionella species in hospital water systems. Am J Infect Control 2006;34:520-5.  Back to cited text no. 20
Dendana F, Sellami H, Jarraya F, Sellami A, Makni F, Cheikhrouhou F, et al. Free-living amoebae (FLA): Detection, morphological and molecular identification of Acanthamoeba genus in the hydraulic system of an haemodialysis unit in Tunisia. Parasite 2008;15:137-42.  Back to cited text no. 21
Singh T, Coogan MM. Isolation of pathogenic Legionella species and legionella-laden amoebae in dental unit waterlines. J Hosp Infect 2005;61:257-62.  Back to cited text no. 22
Trabelsi H, Sellami A, Dendena F, Sellami H, Cheikh-Rouhou F, Makni F, et al. Free-living Amoebae (FLA): Morphological and molecular identification of Acanthamoeba in dental unit water. Parasite 2010;17:67-70.  Back to cited text no. 23


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]


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