Parasitol Res DOI 10.1007/s00436-015-4788-1
SHORT COMMUNICATION
Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia Houaida Trabelsi 1 & F. Dendana 1 & S. Neji 1 & H. Sellami 1 & F. Cheikhrouhou 1 & F. Makni 1 & A. Ayadi 1
Received: 6 March 2015 / Accepted: 8 October 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eightyfour water samples were collected over a period of 4 months (September–December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased
* Houaida Trabelsi
[email protected] 1
Fungal and parasitic molecular biology laboratory- Sfax, School of Medicine, Sfax University, 3029 Sfax, Tunisie
awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.
Keywords Free-living amoeba . Hospital water . Acanthamoeba . PCR . Sfax . Tunisia
Introduction Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa which can be found in the soil, in the air, and in the natural and artificial aquatic environments (Visvesvara et al. 2007). Several genera of FLA, such as Acanthamoeba, Naegleria, Balamuthia, and Sappinia are responsible for opportunistic and non-opportunistic infections in humans and other animals (Visvesvara and Maguire 2006). In addition, these FLA serve as hosts for a large number of intracellular pathogenic microorganisms (Greub and Raoult 2004). Acanthamoeba species are the most prevalent amoebae in environmental sources, including different water types (Shuster and Visvesvara 2004). Because of the opportunistic nature of Acanthamoeba and its possible role as a reservoir for human pathogens, monitoring of this protozoan in environments such as hospitals and health care units where people are more debilitated and susceptible to infection, is of importance. However, few studies have focused on water systems, humid areas, or other hospital environments (Michel and Just 1984; Michel et al. 1995; Rohr et al. 1998; Thomas et al. 2006). The aim of this study was to detect FLA by morphological methods and to identify Acanthamoeba spp. by PCR in tap water of the Sfax University Hospital, Tunisia.
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Materials and methods It was a prospective study dealing with 84 water samples collected during a period of 4 months (September–December 2011) from different wards of the Sfax University Hospital: surgical services, medical and surgical intensive care unit, operating theater, and water storage tanks. Sample collection and culture of FLA 500 ml of each water sample was filtered through a cellulose acetate filter, 0.45 μm diameter, under a weak vacuum. The filters were suspended in 8 ml of 1X PAS solution. One milliliter of various amoeba suspensions obtained from each filtered solution was inoculated on 1.5 % non-nutrient agar plates seeded with an E. coli suspension. The samples were incubated at 25 °C and monitored daily for growth of FLA. A piece of agar covered with amoeba was transferred to a slightly modified PYG axenic liquid culture medium with added gentamycin, and incubated at 25 °C (Schuster 2002). The reference strain (Linc-AP1) of Acanthamoeba polyphaga used was isolated by T.J. Rowbotham, Public Health Laboratory, Leeds, United Kingdom. Microscopic examination of amoebae Amoeba trophozoites and cysts were examined directly and after stains (trichrome, Giemsa, nuclear red or methylene blue) by light microscopy at ×100, ×400, and ×1000 magnification. The criteria for identification were based on morphology of cysts and trophozoites according to the Page F.C (1988). The genus Acanthamoeba was identified from the FLA, based on a double-walled and wrinkled or wavy ectocyst and characteristic acanthopodia of trophozoites. DNA extraction and PCR amplification The amoeba cells collected from liquid culture medium PYG were centrifuged (1000g) for 10 min at room temperature and washed three times with phosphate-buffered saline (PBS) pH 7.2. Cell pellets were resuspended in Nuclei Lysis Solution of Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI, USA) or in tissue Lysis Buffer (ATL buffer) of QIAamp DNA Mini® kit and incubated at 56 °C overnight with 10 mg/ml proteinase K. The amoeba DNA was purified and then was stored at −20 °C until used. To confirm the morphological identification of Acanthamoeba, PCR analysis was performed using genus specific primers as previously described (Khan et al. 2001). Primer sequences were Acan F 5′-TTTGAATTCGC TCCAATAGCGTATATTAA-3′ and Acan R 5′-TTTGAATT CAGAAAGAGCTATCAATCTGT-3′ (Kong and Chung
1996). These primers amplify the region of 18S rDNA from Acanthamoeba. PCR reaction was performed in a 50 μl mixture containing 50 ng of template DNA; 10 μl×5 PCR buffer; 50 mM of MgCl2; 0.2 mM each of dATP, dGTP, dCTP, and dTTP; 20 pmol of each primer and 2.5 units GoTaq DNA polymerase (Promega, Madison, USA). The Acan F/Acan R PCR was done in 35 cycles with denaturation (94 °C; 30 s), annealing (58 °C; 1 min), and primer extension (72 °C; 1 min, 30 s). Final elongation (needed for large PCR products) was continued for 5 min at 72 °C. Finally, PCR products were analyzed by electrophoresis in a 1.5 % agarose gel. Sequencing and genotyping of the isolates PCR products of five isolates were purified using a Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA) and sequenced with a Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions. Sequencing analysis was performed using BLASTn in the Genbank database. Genotype identification was based on sequence analysis of DF3 region by comparison to the available Acanthamoeba DNA sequences in GenBank database using ClustalW2 (Larkin et al. 2007). The phylogenetic tree was then constructed by the neighbor-joining (NJ) method.
Results Morphological study FLA were detected in 45 samples (53.5 %) as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), and water storage tanks (6.6 %). Seven different morphotypes were detected as follows: acanthopodial (89 %), monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Two or more morphotypes were associated in the same culture sample in 73.3 % of cases. The Acanthamoeba genus was detected in 40 samples (47.6 %): 79.4 % in surgical services; 17.6 % in operating theater and surgical intensive care unit. No Acanthmoeba strain was detected in the medical intensive care unit. Molecular study PCR amplification by the specific primers (Acan-F/Acan-R) confirmed the genus Acanthamoeba in 54 samples (64.2 %). Two products of 700 bp and/or 900 bp were detected (Fig. 1):
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M
T-
T+
Schg4 BM2 Sur3 SN4 Smax1 Sop1 Sur6
900 bp 700 bp
Fig. 1 PCR amplification using Acanthamoeba genus-specific primers pair Acan- F/Acan-R. M: size markers (100 bp DNA ladder); T– negative control (without DNA); T+ Acanthamoeba polyphaga (linc AP-1); Schg4 General surgery department (sample 4); BM2 Maxillofacial operating
theater (sample 2); Sur 3 Urology department (sample 3); SN4 Neurology department (sample 4); Smax1 Maxillofacial surgery department (sample 1); Sop1 Orthopedic surgery department (sample 1); Sur6 Urology department (sample 6)
one band of 700 bp or 900 bp was detected in 49 samples and the two bands were detected at the same time in five samples. The five distinct PCR products that we subsequently sequenced showed that these sequences were 99 % identical to Acanthamoeba sp. deposited in GenBank (Table 1). The results obtained from genotyping of these five samples showed that these strains belong to T4, T10, and T11 genotypes. Table 1 shows the genotypes and accession numbers.
especially when supplied from storage tanks, is a potential source of amoeba contamination. In our investigation, FLA were detected in 53.5 % of water samples, with Acanthamoeba accounting for 47.6 %. These results were similar to the study of Rohr et al. (1998) who found FLA in 52 % of hospital hot water samples and in 47 % of moist sanitary areas. Bagheri et al. (2010) found Acanthamoeba in 48 % of samples collected from the tap water of several Iranian hospitals. In another Iranian study of hospital wards with immunocompromised patients, FLA were detected in 59.2 % of samples among which 89.1 % were Acanthamoeba T4 genotype (Lasjerdi et al. 2011). In a study in Portugal, Carlesso et al. (2010) isolated FLA in 35 % of hospital environment samples. Of these, Acanthamoeba was present in 23 % of samples among which 40 % were potential pathogens. The considerable spreading of FLA in these different studies can be due to high resistance of amoeba, particularly the Acanthamoeba genus, against water chlorination as well as tolerance to relativity high temperatures (Ma et al. 1990). In our sampling, amoebae were not found in the tap water of the medical intensive care unit, very likely due to the presence of 0.1 μm porosity filters in the faucets. Consequently, these filters were recommended in all hospital water systems for limiting the passage of different FLA. We were interested to the molecular study of Acanthamoeba spp., which is pathogenic. The product lengths were 700 bp
Discussion To the best of our knowledge, this is the first study reporting on the morphological, molecular detection and identification of FLA and Acanthamoeba genus in the hospital tap water of Tunisia. Previous studies were performed in a dialysis unit and in dental unit water (Dendena et al. 2008; Trabelsi et al. 2010). Worldwide, a few studies conducted to detect FLA in hospital water systems and therapeutic pools have been reported (Visvesvara et al. 1990; Rohr et al. 1998; Thomas et al. 2006; Bagheri et al. 2010; Lasjerdi et al. 2011; Carlesso et al. 2010). But, there are no documented reports concerning hospital water-related diseases and their direct relation with the presence of FLA. Health care facilities and hospitals acquire their water from municipal systems and/or from their own storage tanks. Water piping in hospitals is often old and damaged. So, tap water, Table 1 Sample ID
Molecular data from Acanthamoeba isolated in hospital water Collection site
Acanthamoeba isolated
Gen bank accession N°
Genotype
Smax 1
Maxillofacial department
Acanthamoeba sp. MN-2012 strain
JN585816.1
T10
Schg1 Scv1 Sur12 Sur6
General surgery department Cardiovascular surgery department Urology department Urology department
Acanthamoeba sp. FA03 Acanthamoeba sp. FA03 Acanthamoeba sp. FA03 Acanthamoeba sp. FA03
DQ264391.1 DQ264391.1 DQ264391.1 DQ264391.1
T11 T4 T4 T4
Smax1 Maxillofacial surgery department (sample 1), Schg1 General surgery department (sample 1), Scv1 Cardiovascular surgery department (sample 1), Sur12 Urology department (sample 6), Sur6 Urology department (sample 6)
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and 900 bp. The band size of the A. polyphaga reference strain was 700 bp. This same band was detected in our samples. The presence of the two bands in five samples could correspond to an association of two different 18S Acanthamoeba spp. Costa et al. (2010) found 930 bp and 1.100 bp products in samples taken from hospital dust. According to these authors, 1100 bp products corresponded to group I and 930 bp corresponded to group II. Because the PCR method cannot be used for species identification, the variation in fragment lengths probably reflects the phylogenetic distance between Acanthamoeba groups, which has been corroborated by analysis of sequence variation in 18S rDNA (Stothard et al. 1998). Genotyping Acanthamoeba species is a useful tool for studying taxonomic and epidemiological relationships. In our study, three isolates belonged to the T4 genotype. Worldwide, it was the predominant genotype isolated in environmental, hospital samples and in AK (Acanthamoeba keratitis) (Maghsood et al. 2005; Kao et al. 2013; Reyes-Batlle et al. 2014; Dendana et al. 2013; Niyyati et al. 2009a; Risler et al. 2013; Walochnik et al. 2015; Derda et al. 2015). Similarly, it has been the major genotype associated with non-keratitis infections such as granulomatous amoebic encephalitis (GAE) and other infection (Booton et al. 2005). One of our isolates belongs to the T10 genotype. This genotype, that is correlated with the named species Acanthamoeba culbertsoni, was isolated in a few studies from environment samples, keratitis, and brain infection (Booton et al. 2005; Nuprasert et al. 2010; Walochnik et al. 2015). The T11 genotype was isolated from environmental sources in some studies (Niyyati et al. 2009a; Niyyati et al. 2009b; Kao et al. 2013; Todd et al. 2015; Leońska-Duniec et al. 2015; Cruz and Rivera 2014). It was known to be associated, particularly, with AK (Maghsood et al. 2005; Booton et al. 2005; Lorenzo-Morales et al. 2011; Walochnik et al. 2015). In conclusion, the widespread occurrence of FLA in hospital water in the current study indicates that disinfection procedures and hygiene measures are insufficient to remove or destroy these protozoa. Therefore, monitoring the presence of FLA in hospital units, as well as evaluating the pathogenicity of the isolates, can be an approach to alert the health professionals to improve the disinfection methods and minimize the risks from pathogenic FLA.
Compliance with ethical standards Conflicts of interest The authors declare that they have no competing interests.
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