T4 Isolates Research Articles

In Vitro Cytopathogenic Activities of Acanthamoeba T3 and T4 Genotypes on HeLa Cell Monolayer.

Amoebic keratitis and encephalitis are mainly caused by free-living amoebae of the genus Acanthamoeba, which consists of both pathogenic and nonpathogenic species. The global distribution, amphizoic properties and the severity of the disease caused by Acanthamoeba species have inspired the scientific community to put more effort into the isolation of Acanthamoeba, besides exploring the direct and indirect parameters that could signify a pathogenic potential. Therefore, this study was performed to characterize the pathogenic potential of Acanthamoeba isolated from contact lens paraphernalia and water sources in Malaysia. Various methodologies were utilized to analyze the thermotolerance and osmotolerance, the secretion level of proteases and the cytopathic effect of trophozoites on the cell monolayer. In addition, the in vitro cytopathogenicity of these isolates was assessed using the LDH-release assay. A total of 14 Acanthamoeba isolates were classified as thermo- and osmotolerant and had presence of serine proteases with a molecular weight of 45-230 kDa. Four T4 genotypes isolated from contact lens paraphernalia recorded the presence of serine-type proteases of 107 kDa and 133 kDa. In contrast, all T3 genotypes isolated from environmental samples showed the presence of a 56 kDa proteolytic enzyme. Remarkably, eight T4 and a single T3 genotype isolates demonstrated a high adhesion percentage of greater than 90%. Moreover, the use of the HeLa cell monolayer showed that four T4 isolates and one T3 isolate achieved a cytopathic effect in the range of 44.9-59.4%, indicating an intermediate-to-high cytotoxicity level. Apart from that, the LDH-release assay revealed that three T4 isolates (CL5, CL54 and CL149) and one T3 isolate (SKA5-SK35) measured an exceptional toxicity level of higher than 40% compared to other isolates. In short, the presence of Acanthamoeba T3 and T4 genotypes with significant pathogenic potential in this study reiterates the essential need to reassess the functionality of other genotypes that were previously classified as nonpathogenic isolates in past research.

Derivatisation of metronidazole enhances cytotoxic effect against Acanthamoeba genotype T4 isolates and leads to cytomorphological changes in trophozoites

Amoebae of the genus Acanthamoeba are worldwide distributed causative agents of serious human infections such as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). To date, treatment of these infections is non-uniform and frequently unsuccessful. Recently, the phosphonium salts were studied for their high levels of antimicrobial activity. This work was aimed to investigate the cytotoxic effect of metronidazole and two phosphonium salts (PS1, PS2) on two clinical Acanthamoeba isolates. The isolates showed distinctly higher susceptibility to both phosphonium salts than to metronidazole. The highest susceptibility was noted to PS1 after 48 h of incubation. Metronidazole derivate PS2 showed higher susceptibility than metronidazole. The values of EC50 of PS2 were approximately twenty times lower than EC50 of metronidazole for Acanthamoeba lugdunensis strain and sixteen times lower for Acanthamoeba quina strain after 48 h. Although the therapeutic effect of metronidazole in Acanthamoeba infections is usually insufficient, its derivatisation can result in a significantly higher amoebicidal effect. Cytomorphological changes of trophozoites after exposure to tested compounds included rounding up of the cells, damage of membrane integrity, presence of pathological protrusions, elongation of the cells or pseudocyst-like stages. Obtained results indicate possible therapeutic potential of studied phosphonium salts.

Species, Sequence Types and Alleles: Dissecting Genetic Variation in Acanthamoeba.

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between isolates of Acanthamoeba could be quantitatively assessed by comparing sequences of the nuclear 18S rRNA gene, ultimately producing 22 sequence types, designated T1 through T22. The concept of sequence types helps our understanding of Acanthamoeba evolution. Nevertheless, substantial variation in the 18S rRNA gene differentiates many isolates within each sequence type. Because the majority of isolates with sequences in the international DNA databases have been studied for only a small segment of the gene, designated ASA.S1, genetic variation within this hypervariable region of the 18S rRNA gene has been scrutinized. In 2002, we first categorized variation in this region in a sample of T3 and T4 isolates from Hong Kong, observing ten “alleles” within type T4 and five “alleles” within T3. Subsequently, confusion occurred when different labs applied redundant numerical labels to identify different alleles. A more unified approach was required. We have tabulated alleles occurring in the sequences submitted to the international DNA databases, and determined their frequencies. Over 150 alleles have occurred more than once within 3500+ isolates of sequence type T4. Results from smaller samples of other sequence types (T3, T5, T11 and T15, and supergroup T2/6) have also been obtained. Our results provide new insights into the evolutionary history of Acanthamoeba, further illuminating the degree of genetic separation between significant taxonomic units within the genus, perhaps eventually elucidating what constitutes a species of Acanthamoeba.

Isolation of fungi producing hormone Indole Acetic Acid (IAA) on sugarcane bagasse and filter cake

Sugarcane bagasse and filter cake are the residual results of the process of extracting sugarcane stems into sugar which is widely used as an organic material that is beneficial to plants. The purpose of this study was to determine the ability of fungi that live in sugarcane bagasse and filter cake to produce Indole Acetic Acid (IAA) hormone. A bagasse sample of 1 gram was crushed and suspended in 99 ml of pure aquades water and a 10 gram sample of filter cake was suspended in 90 ml of pure aquades. 1 ml was taken and put into 9 ml of sterile distilled water and then mixed using vortex and diluted 10−5 to 10−6, 0.1 ml pipette then poured into a cup containing Potato Dextrose Agar (PDA) media. Purification was carried out by removing a fungi colony on a sterile PDA media and then morphologically characterized. Qualitatively testing IAA production capability by culturing 1 full loop on the PDA media, then put it into PDB media and added L-Triptophan and Salkowski reagents. Supernatant was stored for 24 hours at room temperature and dark. The change in color of the superoxide to pink indicates IAA and the quantity test was performed using a spectrophotometer λ 530 nm to measure absorbance. The results show that bagasse and filter cake isolates had morphological differences. All isolates tested had the ability to produce IAA. The average production of the hormone IAA of isolate from sugarcane bagasse was 1.063 – 3.469 ppm while filter cake was 0.891-1.547 ppm. The highest IAA production produced by T4 isolates of 3.469 ppm and fungi isolates which produced the lowest IAA hormone was B21 isolates of 0.047 ppm. T4 isolates from sugarcane bagasse have good potential to be developed for use in plants.

Potentially pathogenic Acanthamoeba genotype T4 isolated from dental units and emergency combination showers.

BACKGROUND Acanthamoeba is the genus of free-living amoebae that is most frequently isolated in nature. To date, 20 Acanthamoeba genotypes have been described. Genotype T4 is responsible for approximately 90% of encephalitis and keratitis cases. Due to the ubiquitous presence of amoebae, isolation from environmental sources is not uncommon; to determine the clinical importance of an isolation, it is necessary to have evidence of the pathogenic potential of amoebae.OBJECTIVEThe aim of this study was to physiologically characterise 8 Acanthamoeba T4 isolates obtained from dental units and emergency combination showers and to determine their pathogenic potential by employing different laboratory techniques.METHODSEight axenic cultures of Acanthamoeba genotype T4 were used in pathogenic potential assays. Osmotolerance, thermotolerance, determination and characterisation of extracellular proteases and evaluation of cytopathic effects in MDCK cells were performed.FINDINGSAll of the isolates were osmotolerant, thermotolerant and had serine proteases from 44-122 kDa. Two isolates had cytopathic effects on the MDCK cell monolayer.MAIN CONCLUSIONThe presence of Acanthamoeba T4 with pathogenic potential in areas such as those tested in this study reaffirms the need for adequate cleaning and maintenance protocols to reduce the possibility of infection with free-living amoebae.

Morphological, genotypic, and physiological characterization of Acanthamoeba isolates from keratitis patients and the domestic environment in Vitoria, Espírito Santo, Brazil

Amoebae of the genus Acanthamoeba are free-living protozoa that can cause granulomatous encephalitis and keratitis in humans. In this study, four clinical and three household dust isolates obtained in Vitória, Espírito Santo, Brazil were characterized by their morphological, genotypic, and physiological properties. All isolates belonged to group II according to Pussard and Pons’ cyst morphology. Analysis of their 18S rDNA sequence identified one isolate from household dust as genotype T11 and the others six samples as genotype T4. Five T4 isolates presented a highly variable region (DF3) in 18S rDNA identical to those previously described. Physiological assays carried out with trophozoites in co-culture with bacteria or in axenic conditions showed all samples tolerated temperatures up to 37°C, regardless of culture method. One keratitis isolate grew at 42°C in co-culture with bacteria. Most isolates in co-culture survived at 1.0M, except a T11 isolate, which tolerated up to 0.5M. The isolates did not grow at 42°C and did not tolerate 0.5M and 1.0M under axenic condition. This is the first report of 18S rRNA gene genotyping applied to Acanthamoeba isolated from keratitis patients in Brazil. The results also indicated that osmo-tolerance is dependent on the culture system.

Identification of Free-Living Amoebae and Amoeba-Associated Bacteria from Reservoirs and Water Treatment Plants by Molecular Techniques

The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Antagonist potential of Trichoderma indigenous isolates for biological control of Phytophthora palmivora the causative agent of black pod disease on cocoa ( Theobroma cacao L.) in Côte d'Ivoire

The biodiversity of Trichoderma isolates from cocoa rhizosphere in cocoa production areas of Cote d’Ivoire, and their antagonist potential with Phytophthora palmivora using in vitro assays and bioassays, were investigated and screened for field trials. A total of 135 isolates were analysed at the species level by using sequence analysis of ITS1 and 2 of the rRNA region and a fragment of translation elongation factor 1a (tef1) gene. Sixty-four isolates were identified as T. virens, 60 as T. harzianum, 7 as T. spirale, two as T. asperellum and two unidentified. Forty-three Trichoderma isolates were in vitro confronted with P. palmivora on agar plates, and their antagonist activity was further evaluated by the damages on leaf discs and detached pods. Twenty-five isolates reduced the mycelial growth of P. Palmivora more than 50%. The isolate T17 assigned to T .virens was the best to reduce mycelium growth upto 97.9%. All of Trichoderma isolates with the exception of isolate T39 reduced foliar sensitivity to P. palmivora. Tweenty-six Trichoderma isolates reduced the pod sensitivity to P. palmivora more than 50%. Based on the combined analysis, T. virens T7, T. harzianum T40, T. asperellum T54 and T. spirale T4 isolates were selected for field trials.

Variable Responses of Acanthamoeba Strains to Three Multipurpose Lens Cleaning Solutions

A novel method has been developed to compare Acanthamoeba strain differences by testing their susceptibility to multipurpose contact lens cleaning solutions (MPS). This method uses surface-attached amoebae that mimic cells attached to a contact lens. In brief, acanthamoebae were grown on non-nutrient agar plates with Escherichia coli prey. Blocks of agar with cysts or trophozoites (approximately 50 cells) were cut out and transferred to MPS [ReNu MultiPlus; (Bausch & Lomb, Rochester, NY), Complete (Allergan, Irvine, CA), and Opti-Free Express (Alcon, Ft. Worth, TX)] for up to 24 h treatment. After neutralizing in Dey Engley broth (Difco), blocks were washed in amoeba saline and reinoculated onto fresh prey bacteria. Positive growth was indicative of survival. Testing showed that the efficacy of the three MPS was different. Opti-Free was the most effective against cysts, followed by ReNu while Complete was relatively ineffective. Not surprisingly, trophozoites were more susceptible than cysts. Findings for individual MPS complement previous work with other methods. This study has also identified that environmental strains vary in their sensitivity to disinfecting solutions. Overall, Acanthamoeba genotypes T3, T5, and T11 from the environment were more resistant than the T4 isolates from the cornea and beach. This resilience supports previous work on temperature tolerance, in which T3 and T5, and T11 acanthamoebae grew in temperatures up to 41 degrees C. Investigators should therefore consider the strain genotype and its source before embarking on MPS sensitivity testing.

Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt

The free-living amoebae of the genus Acanthamoeba include non-pathogenic and pathogenic species and has been recently classified into 15 different genotypes, T1–T15. In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in freshwater sources associated with human activities in the Nile Delta region, Egypt. Identification of Acanthamoeba was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized using temperature and osmotolerance assays and PCR reactions with two primer pairs specific to Acanthamoeba pathogenesis. Isolates genotypes were also determined after ribosomal DNA sequencing. These data revealed that isolates belong to T1, T2, T3, T4 and T7 genotypes. As expected, T4 isolates exhibited the most pathogenic traits and were osmotolerant, temperature tolerant and expressed extracellular serine proteases. This is the first report presenting environmental distribution of Acanthamoeba genotypes in Egypt.

Phylogenetic Analysis of Canine Parvovirus VP2 Gene in Taiwan

Canine parvovirus (CPV) is a non-enveloped virus with a single-stranded DNA genome and causes infectious enteritis in dog. In this study, 36 isolates of CPV infection were obtained in Taichung, Taiwan from 2003 to 2004. Using primers that can distinguish subtypes of CPV, we amplified part of viral VP2 gene by polymerase chain reaction (PCR) and the PCR product was sequenced; results demonstrated that two isolates could be classified as type 2a of CPV and the others were type 2b. The complete coding region of VP2 gene of type 2b was also sequenced, and phylogenetic analysis of these DNA sequences revealed that our Taichung isolate was close to the V-120, FPV-314, 97-008, Taiwan 9, LCPV-T1, and T4 isolates; however, because of the degeneracy of codons, the amino acid sequences of Taichung isolate was similar to that of the 97-008 isolate from Japan. It is known that two important amino acid residues (Asn-426 in type 2a and Asp-426 in type 2b; Ile-555 in type 2a and Val-555 in type 2b) are the determinants for the discrimination of type 2a or type 2b. After scrutinizing the complete VP2 gene of our Taichung isolate, we found the VP2 protein of the Taichung isolate did possess this molecular feature of type 2b virus. Previous studies reported that type 2a virus was the major type in Taiwan; our finding showed that CPV type 2b was the predominant type in the middle part of Taiwan. Moreover, a unique Ala-489 in VP2 of our Taichung isolate was found, contrasting to a Val-489 in the VP2 of other strains.

Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates

The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5%) belonged to T4, two (15.3%) belonged to T3 and three (23%) belonged to the T2 genotype. In contrast, the majority of 12 environmental isolates tested in the present study belonged to T2 (7/12, 58.3%), followed by 4/12 T4 isolates (33.3%). In addition, the genotypes of six new Acanthamoeba isolates from UK keratitis cases were determined. Of these, five (83.3%) belonged to T4 and one was T3 (16.6%), supporting the expected high frequency of T4 in Acanthamoeba keratitis. In total, the genotypes of 24 Acanthamoeba keratitis isolates from the UK and Iran were determined. Of these, 17 belonged to T4 (70.8%), three belonged to T2 (12.5%), three belonged to T3 (12.5%) and one belonged to T11 (4.1%), confirming that T4 is the predominant genotype (S2=4.167; P=0.0412) in Acanthamoeba keratitis.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis.

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.

Emm Typing of group A streptococcus clinical isolates: identification of dominant types for throat and skin isolates.

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990-1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty-four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm13w, and TNT/emm58 were predominant among 80 skin isolates. emm-type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.