Two different tolerance experiments were applied to 22 different strains as those that tolerate different temperatures (thermotolerants) and different concentrations of mannitol (osmotolerants). Subgroups of the T4 genotype, most isolated from environmental sources, all strains in the T5, T11 and T12 genotypes grew at 37°C and 0.5 M mannitol. A few strains grew at 39°C and 1 M mannitol, only two strains were observed to grow at 41°C. In previous studies, for tolerance tests applied to Acanthamoeba strains, those that tolerate 37–41° C temperature and 0.5 M-1 M mannitol were accepted as potential pathogenic species (Kiss, Barna, Vargha, & Török, 2014; Tawfeek, Bishara, Sarhan, Taher, & Khayyal, 2016; Vijayakumar, 2018). In a different study, it was reported that all of the strains isolated from the soil in South Florida grew at 37°C (Booton, et al., 2004). Booton et al. (2004) showed that 22.2% of the strains isolated from tap water in Cairo, Egypt were osmotolerant and 50% thermotolerant, but these rates reached 15.2% and 58% in the Delta region (Al-Herrawy et al., 2013). In different studies, it has been shown that 66% of the strains isolated from the soil in Ankara, Turkey are both osmotolerant and thermotolerant (Kilic, Tanyuksel, Sissons, Jayasekera, & Khan, 2004). This inconsistency in the results of all tolerance studies is thought to be related to the fact that different Acanthamoeba species isolated from various sources from each study may have different physiological characteristics. In addition, although there are many studies on tolerance tests, there is no study comparing the osmotolerance and thermotolerance characteristics of Acanthamoeba strains with T4 (Neff, A, B, D, E), T5, T11 and T12 genotypes, both between genotype groups and within species with the same genotype.
For an amoeba to be considered potentially pathogenic, it must exhibit thermotolerant and osmotolerant properties, among several factors. Because these features show the behavior of the amoeba under stressful conditions (Todd, et al., 2015). While many studies have shown that Acanthamoeba can grow at 37°C and above to exhibit pathogenic potential in thermotolerance experiments, Schuster (2002) reported that some clinical specimens grow better at 30°C (Todd, et al., 2015). It is known that the strains that can cause Acanthamoeba keratitis can grow at 37°C, because the temperature of the human eye is 34°C on average (Walochnik et al., 2000). Since all strains in the thermotolerance experiments in this study grew at 37°C, they may have the potential to cause AK. Two of the five Acanthamoeba isolates isolated from keratitis cases were higher temperature thermotolerant, while the other three isolates were less thermotolerant and osmotolerant. Ledee et al. suggested that this may be due to the exposure of keratitis cornea samples to drugs that may alter physiological properties. Since the human body temperature is 37°C in granulomatous amoebic encephalitis infection, it has been widely accepted that thermotolerance is a prerequisite for pathogenicity (Walochnik, et al., 2000). In the study by Pumidonming et al., it was emphasized that strains that did not grow at this temperature most likely did not cause disease, and even did not cause infection in strains that reproduced (Pumidonming, Koehsler, & Walochnik, 2010).
The T4 genotype is the most common genotype in both keratitis and central nervous system infections. Potential pathogenicity may be in question in this genotype since both in this study and in the cases where Acanthamoeba infections, which are the most common in the world and more than 90% of isolated cases, gave positive results in tolerance tests (Rocha-Cabrera, 2015; Putaporntip, et al., 2021; Kao, et al., 2012). In the literature, it has been supported by these studies that T4 strains, which have a very high growth rate at 37–42°C and 1 M mannitol, reproduce both in clinical and environmental sources (Rocha-Cabrera, et al., 2015; Retana-Moreira, et al., 2015; Landell, Salton, Caumo, Broetto, & Rott, 2013; Behniafar, Niyyati, & Lasjerdi, 2015; Pumidonming, Koehsler, & Walochnik, 2010). Although not directly related to pathogenicity, the thermotolerant property of the genus Acanthamoeba has the ability to reproduce at 37°C or higher for some clinical specimens (Putaporntip, et al., 2021). However, as a result of this research, T4D (SVS16) and T11 (Z009) genotypes obtained from environmental sources showed reproduction at high temperature. While all subgroups of T4 genotype were grown in 0.5 M mannitol, only 56.25% (9/16) of them were grown in 1 M mannitol. Its tolerant state at 0.5 M mannitol is the result of its growth in both environmental and clinical strains. However, the most important finding in this study was that there was no growth in 1 M mannitol in T4B and T4E genotypes. The strains in these two sub-genotypes could not tolerate 1 M mannitol as they were isolated from keratitis cases. At the same time, since there are no tolerance experiments for subgroups of T4 genotype in the literature, this study will shed light on the studies to be done on this subject.
T5 genotype is the second most common genotype after T4 genotype. This genotype has been isolated from both environmental and clinical cases (Booton, Visvesvara, Byers, Kelly, & Fuerst, 2005; Siddiqui ve Khan, 2012). Of the four isolates of the T5 genotype in this study, only one was isolated from the clinical case. It has been shown in the literature that T5 isolates can grow at high temperatures (Pumidonming, Koehsler, & Walochnik, 2010; Possamai, Loss, Costa, Falqueto, & Furst, 2018; Booton, et al., 2004). In the studies, none of the strains that were thermotolerant at 40°C could tolerate 1 M mannitol (Pumidonming, Koehsler, & Walochnik, 2010). In this study, these strains of both clinical and environmental origin were able to grow up to a maximum of 39°C and 0.5 M mannitol in tolerance experiments.
The T11 genotype is among the genotypes found both in environmental sources and considered as causative agents of keratitis (Booton, et al., 2004; Pumidonming, et al., 2010). It has been reported that strains with this genotype can grow at 37–40°C and tolerate 0.5 M mannitol (Milanez, et al., 2020; Booton, et al., 2004; Possamai, et al., 2018; Hajialilo, et al., 2016). In addition, Hajialilo et al. showed that strains with the T11 genotype did not grow at 37°C and 0.5 M mannitol, while Possamai et al. showed that they tolerated 1 M mannitol (Hajialilo, et al., 2016; Possamai, et al., 2018). In our study, only one isolate of T11 genotype was used and this strain was isolated from an environmental source. This isolate was able to tolerate all temperatures, but only 0.5 M mannitol in osmotolerance experiments.
Since the T12 genotype is the genotype that causes encephalitis in humans, it is more common in clinical cases, while its environmental niche is unknown (Booton, Visvesvara, Byers, Kelly, & Fuerst, 2005; Satitpitakul, Putaporntip, & Jongwutiwes, 2021; Blaschitz, et al., 2006; Gavarāne, et al., 2018; Kao, et al., 2012). At the same time, this genotype, which has the most different genotype, is known to be quite lethal (Yu, et al., 1999; Gavarāne, et al., 2018). In the literature, a thermotolerance test was applied to this genotype on a strain isolated from a keratitis case and it was determined that it could reproduce at 42°C. In our study, a single strain showed growth ability at 37°C and 39°C and 0.5 M mannitol in tolerance tests.
It is thought that in vitro growth of Acanthamoeba samples may be associated with virulence, partially under high temperature and osmotic stress. Because the virulence of an isolate is relatively related to its ability to adapt to and survive the tissues of the mammalian host (Khan, 2006; Khan ve Tareen, 2003). It is thought that in vivo experiments are needed to determine the pathogenic potential of samples isolated from environmental sources (Landell, Salton, Caumo, Broetto, & Rott, 2013). At the same time, higher ambient temperature is thought to increase the growth of thermotolerant Acanthamoeba (Landell, Salton, Caumo, Broetto, & Rott, 2013). It has been suggested that these tolerant strains may have evolved through natural selection to adapt to heat stress in their niche (Landell, Salton, Caumo, Broetto, & Rott, 2013)
The temperatures used in the thermotolerance tests in this study were 37°C, 39°C, and 41°C, respectively, and the concentrations used in the osmotolerance tests were 0.5 M and 1 M mannitol. According to the results of tolerance studies in the literature, for Acanthamoeba to be considered as a potential pathogenic species, it must be able to tolerate both these high temperatures and high mannitol concentrations (Tawfeek, Bishara, Sarhan, Taher, & Khayyal, 2016; Vijayakumar, 2018). However, in our study three of the four strains (3/4) in T4B and T4E genotypes isolated from keratitis cases had no growth at both 39°C − 41°C and 1 M mannitol, we think classification as insignificant according to high temperature and high mannitol concentrations for researchers to associate these studies with pathogenicity.