This study investigated the occurrence of free-living amoebae (FLA) in a public hospital in South Africa. A total of 97 water and biofilm samples from the municipal water inlet of the hospital, theatres, theatre sterilization service unit, central sterilization service unit, endoscopy/gastroscopy unit, intensive care unit and the renal unit were collected and examined for the presence of FLA using an amoebal co-culture and molecular techniques. Of the 97 samples, 77 (79.4%), 40 (52%) water and 37 (48.1%) biofilm, contained FLA. The genera Acanthamoeba, Vermamoeba (formerly Hartmanella) and Naegleria were detected by morphology, 18S rRNA PCR (polymerase chain reaction) and sequence analyses. Further sequence analysis of the five Acanthamoeba-positive isolates revealed a close resemblance with the potentially pathogenic T20 genotype. These results show a potential health risk to immuno-compromised patients and health care workers as some of the species detected are pathogenic and may harbor potential intracellular bacteria responsible for nosocomial infections. To date, this is the first report on the detection of potentially pathogenic amoebae from South African hospital water systems.

INTRODUCTION

Free-living amoebae (FLA) are unicellular eukaryotes that are ubiquitous in the environment, mainly in water and soil, where they feed on bacteria and other small microorganisms and play a role in the maintenance of soil fertility and the recycling of nutrients. FLA are present in most natural aquatic environments (rivers, streams, hot springs) as well as in man-made water systems such as domestic tap water, swimming pools and hospital water distribution networks (Thomas & Ashbolt 2011; Ovrutsky et al. 2013). Although mostly beneficial in their natural habitat, Acanthamoeba, Naegleria fowleri, Balamuthia mandrillaris and Sappinia pedata are known human pathogens responsible for opportunistic and non-opportunistic infections (Visvesvara et al. 2007; Thomas & Ashbolt 2011). These FLA can cause central nervous system (CNS) infections, amoebic encephalitis, as well as lung and skin infections (Visvesvara 2013). Acanthamoeba, in particular, is also associated with Acanthamoeba keratitis (AK), a vision threatening corneal infection that may lead to blindness in healthy individuals following corneal abrasions or corneal trauma due to injury. This infection is common in contact lens wearers, often due to poor care like rinsing of lenses with tap water and swimming in contaminated water (Trabelsi et al. 2012). These infections commonly occur in immuno-compromised and immune-suppressed individuals such as cancer patients, HIV/AIDS patients, diabetics and individuals undergoing organ transplants (Visvesvara et al. 2007, 2013). Balamuthia CNS infections have also been reported in children and older individuals with no known history of immunodeficiency. Naegleria is responsible for primary amoebic meningoencephalitis, another rare but potentially fatal CNS infection that occurs in healthy children and young adults (Lorenzo-Morales et al. 2011).

Certain pathogenic and non-pathogenic FLA also serve as a platform for the survival, multiplication and transmission of other bacterial pathogens such as Legionella pneumophila, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) (Greub & Raoult 2004; Lone et al. 2009; Ben Salah & Drancourt 2010) and numerous other gastro-intestinal and pulmonary pathogens. These amoeba-resistant bacteria (ARB) are able to infect, resist the digestive process of FLA, survive, multiply and exit FLA. The association of these pathogens with FLA protects them from common disinfection measures such as chlorination when amoebae are in cyst form. The resistant nature of FLA cysts provides involuntary protection to the pathogenic intracellular bacteria that are packaged in expelled vesicles or released during amoebae lysis (Greub & Raoult 2004; Denoncourt et al. 2014).

FLA can colonise most water systems as amoebal cysts are resistant to extremes of temperature, desiccation, pH and even disinfectants such as chlorine and glutaraldehyde (Storey et al. 2004; Dillon et al. 2014). Therefore, FLA may act as reservoirs and vectors of pathogenic bacteria that are difficult to eradicate, especially in water distribution systems where concentration of disinfectants is low (Evstigneeva et al. 2009). Furthermore, these intracellular bacteria can undergo genetic modifications within FLA, becoming more virulent and resistant to antibiotics and biocides like chlorine used in water treatment (Schmitz-Esser et al. 2010). This presents a potential public health concern, especially in hospital water distribution systems where a large population of immuno-compromised patients and medical personnel may be exposed to contaminated water. The purpose of the present study was therefore to investigate the occurrence of FLA and potential intracellular bacteria in wards and sterilization areas of a hospital in Johannesburg, South Africa.

MATERIALS AND METHODS

Sample collection

A total of 97 samples (51 water and 46 biofilm) were collected from a Johannesburg tertiary hospital. Samples were collected from the inlet of the hospital (four), theatres (41), theatre sterilization service unit (TSSD) (four), central sterilization service unit (CSSD) (eight) and endoscopy/gastroscopy unit (18), intensive care unit (ICU) (10) and the renal unit (12) after consultation with the infection control staff and management of the hospital and a walkthrough investigation of high risk patient areas. Biofilm samples were collected by swabbing 20 cm2 of surfaces prior to opening taps. Water samples were collected after running the taps/shower for 1–2 min in 1 L sampling bottles containing 5 mg/L sodium thiosulfate (Merck, SA) to neutralize the chlorine disinfectant.

Physico-chemical characteristics

At each sampling site, the water temperature, pH, turbidity, conductivity and total organic carbon (TOC) were measured with a portable COMBO TESTER® (Hanna, SA). Free residual chlorine was measured with a chlorine photometer (Hanna, SA). The samples were then collected and transported at ambient temperature in cooler boxes to the laboratory and processed on the day of receipt.

Media preparation

Page's modified Neff's amoebae saline (PAS) was prepared by dissolving 120 mg NaCl, 4 mg MgSO4.7H2O, 4 mg CaCl2.2H2O, 142 mg Na2HPO4, and 136 mg KH2PO4 in 1 L of distilled water. Nonnutritive agar (NNA) plates were prepared by adding 15 g agar/L (Merck, SA) of PAS. All media were sterilized by autoclaving for 15 min at 121 °C.

Isolation of free-living amoebae

The amoebal enrichment technique for the detection of FLA was adapted from Thomas et al. (2006) and Lamoth & Greub (2010). Five hundred milliliters of water samples were concentrated by filtration using a 0.45 μm pore size cellulose nitrate membrane (Millipore, SA). Swabs were vortexed for 30 s in 10 mL PAS in individual sterile tubes and the suspensions were concentrated by filtration. The filter membranes were placed upside down onto NNA overlaid with a suspension of heat-killed Escherichia coli (ATCC 25922) (E. coli; 100 μL for each plate). The plate was then incubated aerobically at 32 °C and examined daily for 3 weeks under a light microscope (Olympus, Japan) with a ×10 objective for appearance of amoebal trophozoites and/or cysts. Plates were recorded as negative after 3 weeks of observation. Plates positive for amoebae were sub-cultured by cutting small agar plugs, placing them upside down onto fresh NNA-E. coli plates and incubating as before. This was done three or four times to purify amoebae as much as possible. Once purified, amoebae were harvested by gently scraping the agar surface and re-suspending in 2 mL of PAS. To further remove extracellular bacteria and debris, the suspension was centrifuged three times at 1,000 g for 20 min. The washed pellet was then suspended in 1 mL PAS. Nine hundred and fifty microlitres was inoculated in 12 well, flat bottomed microtitre wells (Nunc, USA) and incubated at 32 °C. The plates were checked daily under an inverted microscope (Leica, Germany), with a 40× objective, for appearance of FLA. Transmission electron microscopy (TEM) was used to determine the presence of intracellular bacteria.

Transmission electron microscopy

Each non-nutrient agar plate with amoebal trophozoites was flooded with fixative and left overnight. The fixative contained 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.15), osmotically adjusted by the addition of 0.01 M CaCl2, 0.01 M MgCl2, and 0.09 M sucrose. The agar surface was then scraped, and for subsequent steps in the protocol, the sample was pelleted by gentle centrifugation (300 g for 20 min). Buffer rinses (30 min each) and post fixation with 1% osmium tetroxide for 1 hour preceded graded ethanol dehydration and embedding in a low viscosity resin. Sections of 70 nm were cut on a Leica EM UC6 ultramicrotome, placed on Formvar®-coated slot grids, double stained with uranyl acetate and lead citrate, and viewed on an FEI BioTwin Spirit transmission electron microscope at 80 kV (FEI, USA). Images were captured with an Olympus Quemesa charge-coupled device (CCD) camera.

Polymerase chain reaction and sequence analysis of the positive FLA samples

Amoebal DNA was extracted from 200 μL of the prepared amoebae suspension (from each of the amoebae-positive plates) using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The nucleic acid was eluted in 100 μL elution buffer into a 1.5-mL microcentrifuge tube and stored at −20 °C for subsequent analysis by polymerase chain reaction (PCR), agarose gel electrophoresis and sequence analysis. An 18S rRNA PCR was performed using the primers Ami6F1 (5′-CCA GCT CCA ATA GCG TAT ATT-3′), Ami6F2 (5′-CCA GCT CCA AGA GTG TAT ATT-3′), and Ami9R (5′-GTT GAG TCG AAT TAA GCC GC-3′) at a concentration of 0.5 μM (Thomas et al. 2006) with PCR Master Mix No. Rox (Eurogentec, Belgium). After a first step consisting of 94 °C for 5 min, 40 cycles of amplification were performed by using denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and elongation at 72 °C for 2 min and a final cycle at 72 °C for 10 min. PCR products were visualised by 2% agarose gel electrophoresis on 2% gel (data not shown). Sample purification was performed by the Genelute Kit (Sigma-Aldrich, USA) following manufacturer's protocol. Concentration of DNA was determined by photometric method (Qubit 2.0 Fluorometer, Life Technologies, USA) and subsequently sequenced with 3130xl Genetic Analyzer (Applied Biosystems, USA) using the PCR primers. In order to determine Acanthamoeba genotype, follow-up PCR was performed with the primers set JDP1 (5′-GGC CCA GAT CGT TTA CCG TGA A-3′) and JDP2 (5′-TCT CAC AAG CTG CTA GGG GAG TCA-3′) described by Schroeder et al. (2001). Cycling conditions were as follows: 95 °C for 5 min for the initial denaturation step, followed by 35 cycles of 15 s at 95 °C for denaturation, 15 s at 62 °C for annealing, 3 s at 72 °C for extension, and a final extension at 72 °C for 10 min. PCR products were visualised and sequenced as described above (data not shown). All 18S rRNA gene sequences were deposited to the GenBank database at the National Center for Biotechnical Information (NCBI) under the accession numbers KT18374-KT183626. Obtained sequences were aligned with sequences of Acanthamoeba genotypes T1–T20 (Corsaro et al. 2015). A list of the used reference strains and their GenBank accession numbers can be found in the Supplementary Material (available in the online version of this paper). Phylogenetic trees were constructed using neighbour-joining trees with a generation of 1,500 bootstrapped replicates using the MEGA 6.06 (Mega Software, Tempe, Arizona, USA) software.

RESULTS

Physico-chemical characteristics

Water temperatures recorded ranged between 14.8 and 27.3 °C (mean 23.1 °C), pH ranged between 1.74 and 8.77 (mean 7.77), TDS ranged between 10 and 187 mg/L (mean 126 mg/L) and residual chlorine ranged between 0.01 and 0.35 mg/L (mean 0.12 mg/L).

Morphological identification of free-living amoebae

Based on morphological characteristics of trophozoites and cysts, typical Acanthamoeba spp. and Vermamoeba spp. (formerly Hartmanella spp.) were observed suspended in Page amoebal saline under an inverted microscope (Figure 1). Overall, 77 (79.4%) of the 97 samples (40 water and 37 biofilm) contained amoebae after amoebal enrichment. Amoebae were generally isolated in all sampling sites, with the exception of one sample from the intensive care unit. Of the FLA positive samples, 6 (7.80%) and 26 (33.8%) were morphologically identified as Acanthamoeba spp. and Vermamoeba spp. respectively. The other FLA (39.0%) could not be identified morphologically. Three samples isolated from the central sterilization service unit (n = 1) and theatres (n = 2) contained both Acanthamoeba and Vermamoeba species (Table 1). From samples positive for FLA, TEM showed the occurrence of potential intracellular bacteria whose identity were not determined in this study (Figure 2).
Table 1

FLA identified by morphological examination from the hospital water distribution system

Sampling site (N)Acanthamoeba spp. (+) (%)Vermamoeba spp. (+) (%)Other FLAa (+) (%)
Inlet (5) 1 (25.0) 
TSSD/CSSD (12)b 2 (16.7) 2 (16.7) 4 (33.3) 
EB unit (16) 5 (31.3) 12 (75.0) 
Theatre complex (42) 3 (7.1) 11 (26.2) 26 (61.9) 
Intensive care unit (10) 1 (10.0) 2 (20.0) 
Renal unit (12) 7 (58.3) 3 (25.0) 
Total 6 (7.80) 26 (33.8) 47 (39.0) 
Sampling site (N)Acanthamoeba spp. (+) (%)Vermamoeba spp. (+) (%)Other FLAa (+) (%)
Inlet (5) 1 (25.0) 
TSSD/CSSD (12)b 2 (16.7) 2 (16.7) 4 (33.3) 
EB unit (16) 5 (31.3) 12 (75.0) 
Theatre complex (42) 3 (7.1) 11 (26.2) 26 (61.9) 
Intensive care unit (10) 1 (10.0) 2 (20.0) 
Renal unit (12) 7 (58.3) 3 (25.0) 
Total 6 (7.80) 26 (33.8) 47 (39.0) 

aAmoebae not identified as morphologically as Acanthamoeba spp. or Vermamoeba spp.

bSamples with both Acanthamoeba spp. and Vermamoeba spp.

EB-endoscopy/bronchoscopy; TSSD-theatre sterilization service unit; CSSD-central sterilization service unit.

Figure 1

(a) Acanthamoeba cysts and (b) Vermamoeba trophozoites (arrows), scale bar 100 μm.

Figure 1

(a) Acanthamoeba cysts and (b) Vermamoeba trophozoites (arrows), scale bar 100 μm.

Figure 2

Transmission electron image of a Acanthamoeba trophozoite with intracellular bacteria (arrows), scale bar 1 μm.

Figure 2

Transmission electron image of a Acanthamoeba trophozoite with intracellular bacteria (arrows), scale bar 1 μm.

Molecular identification of FLA isolates

The 77 samples containing FLA were amplified using the universal primers, Ami6F1, Ami6F2 and Ami9R, for the 18S rRNA PCR and sequenced. Of these samples, 11 (14.3%) did not show bands on agarose gel and 13 (16.9%) were negative after sequence analysis. These samples were considered negative for FLA. Overall, FLA were detected in 53 (68.8%) of the samples analysed by molecular methods. Vermamoeba vermiformis (46.4%) were the most abundant amoebae identified from all the sampling areas, followed by Acanthamoeba spp. (33.8%) and other FLA (24.7%). A total of 23 Vermamoeba vermiformis isolates (GenBank accession numbers shown in Table 2), five Acanthamoeba spp. isolates (GenBank accession numbers: KT183616, KT183582, KT183604, KT183575, KT183576), one isolate each for Amoebozoa sp. (GenBank accession number KT183581) and Naegleria sp. (GenBank accession number KT183611) were identified based on 18 rRNA gene regions from different sampling areas of the hospital. Furthermore, other protozoa Pseudoparamoeba pagei (GenBank accession number KT183621) and Schizoplasmodiopsis amoeboidea (GenBank accession number KT183622) were also detected in four samples but were excluded from this study (Table 2). Neighbor joining (NJ) analysis was performed using the genus specific primer set JDP-1 and JDP2 to show relationships between the Acanthamoebapositive isolates in this study, and reference strains from NCBI GenBank for the genotypes T1–T20. The five Acanthamoeba isolates detected in this study were 91–95% (mean 93%) similar to the T20 genotype sequence (GenBank accession number DQ451162) as evidenced by the NJ tree (Figure 3).
Table 2

Biodiversity of FLA isolated from different sources of a South African hospital

OriginGenBank accession no.Published closest described species
Inlet KT183594 Vermamoeba vermiformis 
TSSD/CSSD KT183577 Vermamoeba vermiformis 
 KT183616  Acanthamoeba sp. 
Endoscopy/Bronchoscopy unit KT183578 Vermamoeba vermiformis 
 KT183608 Vermamoeba vermiformis 
 KT183579 Vermamoeba vermiformis 
 KT183580 Vermamoeba vermiformis 
 KT183588 Vermamoeba vermiformis 
 KT183581  Amoebozoa sp. 
 KT183582  Acanthamoeba sp. 
Theatre complex KT183583 Vermamoeba vermiformis 
 KT183585 Vermamoeba vermiformis 
 KT183587 Vermamoeba vermiformis 
 KT183606 Vermamoeba vermiformis 
 KT183607 Vermamoeba vermiformis 
 KT183592 Vermamoeba vermiformis 
 KT183603 Vermamoeba vermiformis 
 KT183604  Acanthamoeba sp. 
 KT183575  Acanthamoeba sp. 
 KT183621  Pseudoparamoeba pagei 
 KT183611  Naegleria sp. 
Intensive care unit KT183618 Vermamoeba vermiformis 
 KT183574 Vermamoeba vermiformis 
 KT183593 Vermamoeba vermiformis 
 KT183591 Vermamoeba vermiformis 
Renal unit KT183576  Acanthamoeba sp. 
 KT183612 Vermamoeba vermiformis 
 KT183600 Vermamoeba vermiformis 
 KT183625 Vermamoeba vermiformis 
 KT183620 Vermamoeba vermiformis 
 KT183619 Vermamoeba vermiformis 
 KT183622  Schizoplasmodiopsis amoeboidea 
 KT183584  Pseudoparamoeba pagei 
OriginGenBank accession no.Published closest described species
Inlet KT183594 Vermamoeba vermiformis 
TSSD/CSSD KT183577 Vermamoeba vermiformis 
 KT183616  Acanthamoeba sp. 
Endoscopy/Bronchoscopy unit KT183578 Vermamoeba vermiformis 
 KT183608 Vermamoeba vermiformis 
 KT183579 Vermamoeba vermiformis 
 KT183580 Vermamoeba vermiformis 
 KT183588 Vermamoeba vermiformis 
 KT183581  Amoebozoa sp. 
 KT183582  Acanthamoeba sp. 
Theatre complex KT183583 Vermamoeba vermiformis 
 KT183585 Vermamoeba vermiformis 
 KT183587 Vermamoeba vermiformis 
 KT183606 Vermamoeba vermiformis 
 KT183607 Vermamoeba vermiformis 
 KT183592 Vermamoeba vermiformis 
 KT183603 Vermamoeba vermiformis 
 KT183604  Acanthamoeba sp. 
 KT183575  Acanthamoeba sp. 
 KT183621  Pseudoparamoeba pagei 
 KT183611  Naegleria sp. 
Intensive care unit KT183618 Vermamoeba vermiformis 
 KT183574 Vermamoeba vermiformis 
 KT183593 Vermamoeba vermiformis 
 KT183591 Vermamoeba vermiformis 
Renal unit KT183576  Acanthamoeba sp. 
 KT183612 Vermamoeba vermiformis 
 KT183600 Vermamoeba vermiformis 
 KT183625 Vermamoeba vermiformis 
 KT183620 Vermamoeba vermiformis 
 KT183619 Vermamoeba vermiformis 
 KT183622  Schizoplasmodiopsis amoeboidea 
 KT183584  Pseudoparamoeba pagei 
Figure 3

Phylogenetic relationships of the PCR products of Acanthamoeba species obtained from environmental samples and reference strains for Acanthamoeba subtype T1–T20 from the NCBI GenBank inferred by neighbor-joining analysis using MEGA 6.06. Sample labeling consists of the Taxa, the accession number (in brackets) and the corresponding subtype. The length of the branch indicates the evolutionary resemblance between each sample.

Figure 3

Phylogenetic relationships of the PCR products of Acanthamoeba species obtained from environmental samples and reference strains for Acanthamoeba subtype T1–T20 from the NCBI GenBank inferred by neighbor-joining analysis using MEGA 6.06. Sample labeling consists of the Taxa, the accession number (in brackets) and the corresponding subtype. The length of the branch indicates the evolutionary resemblance between each sample.

DISCUSSION

The presence of FLA in environmental waters such as rivers, lakes, drinking water purification plants, domestic tap water and marine waters has been well documented worldwide. Therefore, it is important to study their ecology to prevent exposure and outbreaks of FLA associated diseases and the potential spread of amoeba resistant bacteria. This study focused on the occurrence of FLA in a hospital water distribution system, a potential source of nosocomial infections that has been overlooked by many studies, particularly in Africa. To our knowledge this is the first study to investigate the occurrence of FLA in water and biofilm from a South African hospital water system.

FLA were isolated from 77 (79.4%) of the samples analyzed. The prevalence of FLA in this study is higher than in studies done by Rohr et al. (1998) in Germany and Lasheras et al. (2006) in France where amoebae were detected in 29 of 56 (52%) and in 73 of 106 (68.9%) hospital water samples respectively. Another study by Thomas et al. (2006) also detected amoebae from 15 of 200 samples (7.5%) in a Swiss hospital. These studies analyzed water from hospital hot water systems, compared to the current work which analyzed samples from a cold water system where the mean water temperature was 23.1 °C (range, 14.4–27.3 °C). Thomas et al. (2006) reported a higher prevalence of FLA in cold water compared to hot water, with a prevalence of FLA significantly lower for temperatures above 60 °C. This might explain the higher prevalence of FLA in our study compared to those other studies. Furthermore, the variability in the prevalence values of FLA can also be explained by the difference in water source, geographical areas and the different methods used in the identification of FLA. The occurrence of biofilms in water systems have also been shown to provide better conditions for FLA multiplication compared to bacteria suspended in water systems (Pickup et al. 2007). A recent study by Ovrutsky et al. (2013) detected amoebae in 14.8% of hospital samples that were analysed. Of the positive samples, 12 (80.0%) were biofilm samples while three (20.0%) were water samples. This was in agreement with our study as there was a high detection rate in biofilm samples, that is, 37 (80.4%) compared to water samples in which amoebae were detected in 40 (78.4%) samples.

Using both culture-based and molecular-based methods, genera belonging to Acanthamoeba and Vermamoeba (formerly Hartmanella) and Naegleria were detected. The universal primers, Ami6F1, Ami6F2 and Ami9R, used in this study did not specify if the only Naegleria sp. (GenBank accession number KT183611) isolate belonged to the pathogenic N. fowleri. The other medically important FLA, B. mandrillaris, and Sappinia sp. were also not detected probably because of their low prevalence in the environment, especially in water (Visvesvara 2013). In addition, the incubation temperature (32 °C) used in this study may have not been adequate to allow the growth of other FLA, particularly Naegleria spp., which grow better at higher temperatures. The most prevalent genus in the current study was Vermamoeba detected with both culture based (33.8%) and molecular methods (46.4%). Our findings are in agreement with a study from Turkey where the genus Vermamoeba was detected in 24 out of 33 detected FLA from 150 drinking water samples. Similar results were described by Thomas et al. (2006) in a study carried out in Switzerland to determine the biodiversity of FLA in a hospital water network where V. vermiformis had a prevalence of 86.7%. However, the genus Vermamoeba is not necessarily the most prevalent FLA in the environment. Several studies have reported Acanthamoeba to be the most common FLA in air, soil and water (Thomas et al. 2006; Coşkun et al. 2013). The higher prevalence of Vermamoeba compared to Acanthamoeba in our study can be explained by the fact that we used universal FLA primers, which might be more effective in amplification of Vermamoeba than Acanthamoeba. The genus Vermamoeba is not usually associated with human disease and has been isolated in human tissues without the proof that the isolates are the cause of infection (Cabello-Vílchez et al. 2014). However, cytopathogenicity has been observed on keratocytes and has been reported as a causative agent of a form of keratitis, which can lead to corneal ulcers (Kennedy et al. 1995; Abedkhojasteh et al. 2013). Based on phylogenetic analysis, the five samples positive for Acanthamoeba spp. closely resembled the T20 genotype. This is, to our knowledge, the first report of this genotype in Africa. This genotype was recently described by Fuerst et al. (2015) and contains several Acanthamoeba strains previously miss-assigned to genotypes T16 or T4. The T20 genotype contains highly pathogenic strains responsible for Acanthamoeba keratitis, granulomatous amoebic encephalitis and respiratory infections in humans. Infections that affect a number of organs and tissues have also been reported in other vertebrates (Visvesvara et al. 2007, 2013). Therefore, the presence of the genera Vermamoeba and Acanthamoeba is a possible risk factor to a large population of immune-compromised individuals and medical personnel who might be exposed to these opportunistic FLA. Patients and health care personnel exposure can occur during drinking (ingestion), showering (inhalation or aspiration aerosols) or bathing and washing of hands (contact) (Anaissie et al. 2002). The persistent nature of amoebal cysts means that these populations are frequently exposed to FLA. An example of their persistent nature was shown by Acanthamoeba cysts reactivation after being stored in a completely dry environment for over 20 years (Visvesvara 2013). Furthermore, the residual chlorine range of 0.01–0.35 mg/L in water samples analyzed in this study is not sufficient in eliminating FLA as they have been shown to survive concentrations as high as 100 mg/L for 10 min of exposure (Storey et al. 2004).

In addition to causing opportunistic infections, both Vermamoeba and Acanthamoeba are known to act as reservoirs and transmitters of nosocomial pathogens including Legionella, Pseudomonas aeruginosa and mycobacteria (Greub & Raoul 2004; Lamoth & Greub 2010) Although this study did not identify specific intracellular bacteria that can be harboured by FLA, TEM showed their presence inside an amoeba host (Figure 2). These FLA are known to protect amoeba resistant pathogens from disinfectants and increase their virulence and resistance to antibiotics compared to microorganisms not propagated inside amoebae. Therefore the occurrence of FLA in a hospital network presents a potential public health risk which should be monitored to limit the risk of nosocomial infection and disease outbreaks.

CONCLUSIONS

FLA belonging to the genera Acanthamoeba and Vermamoeba were isolated from 77 (79.4%) of the samples analysed based on morphological characteristics. These FLA had the potential to contain intracellular bacteria. From positive samples, the FLA Acanthamoeba, Vermamoeba and Naegleria were detected using 18S rRNA PCR and sequence analysis. Vermamoeba were the most abundant FLA followed by Acanthamoeba and then the only Naegleria species. Further sequencing of Acanthamoeba with genus-specific closely resembled the T20 pathogenic genotype. Therefore patients and healthcare workers might be exposed to pathogenic amoebae in the hospital water system.

ACKNOWLEDGEMENTS

We thank the National Institute for Occupational Health and the University of Johannesburg for providing facilities for this project and the National Research Foundation for providing a bursary. We also acknowledge the Water Research Commission for funding the project and Monica Birkhead for her assistance in the transmission electron microscopy work

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Supplementary data