Iranian J Parasitol: Vol. 6, No.4, 2011, pp.84-89 Iranian J Parasitol Tehran University of Medical Open access Journal at Sciences Publication http:// ijpa.tums.ac.ir Iranian Society of Parasitology http:// tums.ac.ir http:// isp.tums.ac.ir Short Communication First Report of Vannellidae Amoebae (Vannella Spp.) Isolated From Biofilm Source Z Lasjerdi1, *M Niyyati1, A Haghighi1, F Zaeri2, E Nazemalhosseini Mojarad3 on Wednesday, April 18, 2012 1Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 School of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3Research Center for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sci- ences, Tehran, Iran (Received 25 May 2011; accepted 14 Nov 2011) ABSTRACT http://journals.tums.ac.ir/ Background: Members of the Vannellidae family are free-living amoebae (FLA) distributed mainly in water and soil sources. The present study reports the first isolation of this genus in the biofilm source from hospital environment in Tehran, Iran. Methods: Biofilm samples were collected from hospital environment. Cultivation was performed in non-nutrient agar covered with a heat-killed Escherichia coli. Cloning of the suspected amoe- Downloaded from bae was done. PCR amplification and Homology analysis using the Basic Local Alignment Search Tool (BLASTn) was performed to search for the most similar reference sequences. Results: Microscopic examination showed numerous fan-shaped amoebae and peculiar cysts different to the usual Archiveshape of typical FLA. Sequence a nalofysis of th e SIDPCR- product revealed that the suspected amoebae are highly homologous with Vannella spp. gene (99% identity and 100% query coverage) available in the gene bank database. Conclusion: Although Vannella spp. is not proved to be pathogenic itself, but they are capable of harboring pathogenic intracellular organisms such as Microsporidian parasites. Thus, identifica- tion of such amoebae can be of clinical importance, as they could lead to transmission of other pathogens to human. Keywords: Vannella, Biofilm, Iran *Corresponding author: Tel: +989122407432; Fax: +982122294951, E-mail: [email protected] 84 www.SID.ir Lasjerdi et al. : First Report of Vannellidae Amoebae … Introduction ree-living amoebae (FLA) include It should be mentioned that amoebae belong various taxa with high distribution in to this genera could act as a trojan horse for F the environmental sources such as microbial world and they could support their fresh water, hot springs, sea water, mineral endosymbiont proliferation (15). This prop- water, soil, dust, clay and moist habitats erty makes non-pathogenic FLA as relevant such as biofilms (1-3). The ubiquitous nature organisms for human being. Indeed, the car- of these organisms leads to the common rier property of Vannella for intracellular exposure of human to FLA (4). However, microorganism such as pathogenic Micro- various genera are known as potentially sporidian parasites is of utmost clinical pathogenic organism including Acanthamo- importance (15, 16). eba spp., Balamuthia mandrillaris, The present study is the first report regarding Naegleria fowleri and Sappinia pedata (1, 5). the isolation of Vannellidae amoebae To date, 17 different genotypes of (Vannellids) in the biofilm sources in Iran Acanthamoeba has been identified according using both morphological tests and molecu- to the diagnostic fragment 3 (DF3) of the lar approaches. on Wednesday, April 18, 2012 18S rRNA gene (6) and most of these genotypes have been proved to be patho- Material and Methods genic in human being (2). Recently, other genera of FLA have been identified as Sample Processing pathogens of cornea and Central Nervous Biofilm samples were collected using sterile System (CNS) (7, 8). Indeed, recent reports swap from hospital environment in our pre- of mixed corneal infection due to Acantha- vious study (17). Briefly, biofilms were dis- moeba with Vahlkampfia and Hartmannella solved in distilled sterile water, incubated for http://journals.tums.ac.ir/ and also brain involvement due to Paravahl- an hour and filtrated using membrane filters kampfia francinae lead to the more attention (Pore size 1.2 µ). Filters were then placed to other FLA, worldwide (7-9). Other fami- upside down on to non-nutrient agar covered lies within FLA include Vannellidae amoe- with a heat- killed Escherichia coli. Incuba- bae (Vannellids) which were previously tion was performed at 30 °C. Plates were introduced as the family belonged to the then monitored for the presence of FLA us- Downloaded from Thecamoebidae. However, to date Vannelli- ing both light and inverted microscope. dae is a totally different family from Theca- moebidae (10). Vannellidae contains several Microscopic examination genera such as VannArchiveella, platyamoeba and One ofplate sh owSIDed numerous fan-shaped Pessonella (10). The genus Vannella was amoebae and peculiar cysts different to the first described by Bovee, 1965 (10, 11) and usual shape of typical FLA and thus fresh occur mainly in water sources (marine and smear of the suspected culture-plate was ex- fresh water). This genus contains approxi- amined under inverted microscope. Sus- mately 40 described species identified in pected amoebae were then submitted to marine and fresh water sources (10, 12, 13). cloning to obtain a single line cell and to The trophozoite form is rather fan-shaped eliminate bacterial and fungi contamination and some of them are capable to form cysts of plates for subsequent evaluation accord- (12, 14). Few researches reported the pres- ing to our previous studies (17, 18). ence of these amoebae in soil samples (12). 85 www.SID.ir Iranian J Parasitol: Vol. 6, No.4, 2011, pp.84-89 DNA extraction, PCR amplification and and to obtain free-bacteria and fungi plate. sequencing analysis Flabellate shaped trophozoites measured Cloned plate containing suspected amoebae about 20 µ and cysts were detected in clump were then scraped using pH 7 PBS. Pellet of manner (Fig. 1). Microscopic examination amoebae were then obtained using 3000 rpm revealed our suspected amoebae as Vannelli- centrifuge and DNA extraction were done dae. Sequence analysis of the PCR product using Chelex kit (Instagen matrix, Biorad). (Fig. 2) confirmed that the suspected amoe- PCR were performed by NA primers which bae are highly homologous with Vannella amplify a partial 18s rRNA gene of some spp. gene (99% identity and 100% query FLA such as Vannella and Hartmannella. coverage) available in the gene bank data- The primers sequences were: NA1: 5′-GCT base (Accession number: AY929909.1; CCA ATA GCG TAT ATT strain 4362V/II 18S small subunit ribosomal AA-3′ and NA2: 5′-AGA AAG AGC TAT RNA gene) and this confirms our micro- CAATCT GT-3′ (17). PCR reactions were scopic observation. The new gene have been performed in 30 µl Ampliqone (Taq DNA deposited in the gene bank database under Polymerase Master Mix RED, Denmark) as accession number: JN009668. a ready-made mixture. Briefly, 25 µl of Taq This is the first report of Vannella spp. iso- on Wednesday, April 18, 2012 Master mix were used with 10 ng template lated from biofilm sources in Iran. Our pre- DNA, 0.1 µM of each primer and distilled vious study showed that biofilm could har- water. PCR cycling conditions included ini- bor various free-living amoebae such as tial denaturing step of 94oC for 1 min, fol- Acanthamoeba T4 and T5 genotype and lowed by 35 repetition cycles at 94oC for 35 Vahlkampfia (17). The present study proved s, annealing at 58oC for 45 s, and at 72oC for that Vannella also could proliferate in such 1 min. PCR products were electrophoresed habitats. Indeed, microorganism within bio- using 2% agarose gel stained with a solution films could be a great source of food for http://journals.tums.ac.ir/ of ethidium bromide and visualized under FLA and therefore biofilm sources intro- UV light. duced as serious hazard in hospital environ- PCR product was submitted to sequencing ment (15). Improved disinfection measures using the ABI 3130X automatic sequencer in are recommended in health-care settings to the Research Center for Gastroenterology avoid aggregation of biofilms. Previous re- and Liver Diseases, Shahid Beheshti Univer- searches isolated Vannella from marine and Downloaded from sity of Medical Sciences, Tehran. Homology fresh water environments (10, 12). Few stu- analysis using the Basic Local Alignment dies also report the isolation of Vannella Search Tool (BLASTn) was performed to from soil samples (12). Although, there is no search for the most Archivesimilar reference se- rep ortof of hum an SIDinfection due to Vannella, quences. but it is worthy to mention that this genus could be a carrier of other pathogens such as Results and Discussion Microsporidian- like protozoa (15, 16). In support of this, Hoffman et al. (1998) re- Plate culture revealed fan-shape trophozoites searches revealed that Vannella could be in- and unusual cysts after one week of incuba- fected by Microsporidian organisms. The tion. Thecamoeba were detected according sources of Vannella containing Microspori- to ovoid and flat shape of their trophozoites dian organisms in Hoffman et al. study were and therefore cloning have been done to res- a domestic tap- water supply (16). This is cue our targeted amoebae from Thecamoeba important since Vannella could protect their endosymbiont from chlorine and harsh envi- 86 www.SID.ir Lasjerdi et al. : First Report of Vannellidae Amoebae … ronment and this can lead to multiplying of In conclusion, although Vannella is not pathogenic organism inside the host amoe- proved to be pathogenic yet, but they are bae. Indeed, infected amoebae can act as a capable of harboring pathogenic organisms vehicle for transmission of microorganisms such as Microsporidian parasites and thus to cornea. Subsequently, Scheid (2007) re- identification of this genus could lead to fur- ported a case of keratitis in a contact lens ther studies regarding bacteria and amoebae wearer without a history of corneal trauma relations.