Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

Background: Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts.Methods: Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined.Results: All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P \u3c 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54±3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96±0.7 bacteria:amoeba ratio) and E. coli (5.85±1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13±0.89 A. hydrophila:amoeba ratio, 10.13±1.17 E. aerogenes:amoeba ratio, and 11.95±0.7 E. coli:amoeba ratio).Conclusions: A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii. Because cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of A. hydrophila and E. aerogenes to susceptible hosts

Interactions of neuropathogenic Escherichia coli K1 (RS218) and its derivatives lacking genomic islands with phagocytic Acanthamoeba castellanii and nonphagocytic brain endothelial cells

Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, alpha -hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity

Higher DNA methylation of ABO gene promoter is associated with acute myocardial infarction in a hospital-based population in Karachi

Objective: To find out if there is any relationship of methylation status of ABO gene promoter with the risk of acute myocardial infarction (AMI) in a hospital-based Pakistani population in Karachi, Pakistan.Methods: A case control study comprising of 39 adult AMI patients (both males and females; age range 30-70 years) and 39 normal healthy controls (both males and females and similar age range) nested in a large study (to see the relationship of ABO genotypes with AMI) was designed to investigate the methylation status of ABO gene promoter and its association with AMI. The study was carried out at the Aga Khan University, Karachi during July 2018 to June 2019. DNA isolated from samples of AMI patients and normal healthy controls were converted into bisulphite DNA using a kit method. Methylation specific polymerase chain reaction was carried out to determine the methylation status of ABO gene promoter in both cases and controls. Logistic regression was used to find out any association between increased methylation status of ABO gene promoter and risk of AMI.Results: A significantly higher percentage of DNA methylation of the ABO gene promoter was observed in AMI patients as compared to normal healthy controls (82.1% vs. 35.9%; p value \u3c0.001). This higher methylation status of ABO gene promoter was associated with AMI and the odds of AMI in this population were more than 6-fold in subjects with methylated gene promoter compared to those with unmethylated gene promoter after adjusting with age and waist circumference [AOR (95% CI) = 6.27 (1.76-22.3); p value = 0.005].Conclusion: The ABO gene promoter\u27s hypermethylation appears to be increasing the risk of AMI in a hospital-based Pakistani population in Karachi, Pakistan

Anti-acanthamoebic properties of natural and marketed honey in Pakistan

To determine anti-Acanthamoebic activity of natural and marketed honey samples. Natural honey samples were collected directly from the bee hive and marketed honey samples were purchased from the local market in Karachi, Pakistan. Both honey samples were tested for their flavonoid content (quercetin equivalent per g of the extract) and phenolic content (gallic acid equivalent per g). Furthermore, their antioxidant activity was determined by measuring 2,2-diphenyl-1-picrylhydrazyl. Using amoebistatic and amoebicidal assays, the effects of honey samples were tested against growth and viability of Acanthamoeba parasites. Natural honey exhibited potent amoebistatic and amoebicidal effects, in a concentration-dependent manner. Honey-treated A. castellanii showed loss of acanthopodia, following which amoebae detached, rounded up, reduced in size, decreased in cytoplasmic mass and they were observed floating in the culture medium. Importantly, honey-treated amoebae did not revive when inoculated in fresh growth medium, however glycerol-treated amoebae exhibited viable trophozoite and active growth. In contrast, marketed honey samples varied in their efficacy against A. castellanii. The proportion of flavonoid, as determined by quercetin measurements and the proportion of phenolic, as determined by gallic acid measurements was higher in natural honey compared with marketed honey. Similarly, the antioxidant activity, as determined by 2,2-diphenyl-1-picrylhydrazyl scavenging activity was higher in natural honey versus marketed honey. This study shows that natural honey has anti-Acanthamoebic properties and possesses higher flavonoid, phenolic and antioxidant properties compared with the marketed honey. These findings are of concern to the public, health officials, and to the manufacturers regarding production of honey for medical applications

Lead levels in primary teeth of children in Karachi

The majority of children in Karachi have blood lead levels above 10 micrograms/dl, the upper safety limit set by the United States Centers for Disease Control and Prevention. A study was undertaken to evaluate chronic lead exposure in children by measuring lead levels in shed primary teeth collected from primary school children in Karachi. A total of 309 teeth were collected from nine different schools, and analysed for lead content by atomic absorption spectrophotometry with electrothermal atomization. The mean (SD) age of the subjects was 7.6 (1.2) years and the mean lead level was 5.78 micrograms/g of whole tooth (dry weight), ranging from 0.42 to 39.75 micrograms/g. Incisor teeth had a significantly higher mean (SD) lead level, 6.42 (4.19) micrograms/g, than canines and molars which contained 4.91 (5.12) micrograms and 4.50 (2.67) micrograms lead whole tooth (dry weight), respectively. Significant differences were observed between different schools. No difference was observed between boys and girls and the accumulation of lead in teeth was not correlated with chronological age

Review: Apolipoprotein E (Apo E) gene polymorphism and coronary heart disease in Asian populations

Apolipoprotein E (Apo E) is a basic component of very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL). It plays an important role in the clearance of cholesterol from circulation, and thereby slows down the process of atherosclerosis. Its 3 isoforms, E2, E3 and E4 are encoded by 3 alleles, e2, e3 and e4, respectively. E3 is the most common isoform in most populations in the world, while E2 is the least frequent isoform. A meta-analysis of several studies conducted on Asian populations revealed that carriers of e4 allele had 42% higher risk of coronary heart disease (CHD), while e2 allele had no significant association with this disease. Epidemiological studies performed in this region showed that E3 was the most prevalent isoform and most likely to be involved in CHD in Asia. Studies conducted in India indicated that individuals with e3/4 and e4/4 genotypes had considerably higher prevalence of dyslipidemia as compared to other genotypes, and hypertensive patients had high frequency of e4 allele. However, several other studies failed to show a relationship between ApoE gene polymorphism and CHD. Inadequate statistical power, low allele frequency, CHD phenotypes could be some of the possible reasons for conflicting results of some of these studies. This area of research is wide open and epidemiological studies with large sample size would be required to ascertain the relationships between ApoE gene polymorphism and CHD

Presence of rotavirus and free-living amoebae in the water supplies of Karachi, Pakistan

Rotavirus and pathogenic free-living amoebae are causative agents of important health problems, especially for developing countries like Pakistan where the population has limited access to clean water supplies. Here, we evaluated the prevalence of rotavirus and free-living amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) in drinking water supplies of Karachi, Pakistan. Six water filtration plants that supply drinking water to the population of Karachi were investigated. Additionally, drinking water samples from households were analyzed for the presence of rotavirus and free-living amoebae. Rotavirus was present in 35% of the water samples collected from water filtration plants; however, domestic tap water samples had a prevalence of only 5%. Out of 20 water samples from filtration plants, 13 (65%) were positive for Acanthamoeba spp., and one (5%) was positive for B. mandrillaris. Out of 20 drinking water samples collected from different areas of Karachi, 35% were positive for Acanthamoeba spp. Rotavirus was detected in 5% of the drinking water samples tested. Overall, these findings showed for the first time the presence of rotavirus, in addition to pathogenic free-living amoebae in drinking water supplies of Karachi that could be an important public health risk for the affected population

Presence of rotavirus and free-living amoebae in the water supplies of Karachi, Pakistan

Rotavirus and pathogenic free-living amoebae are causative agents of important health problems, especially for developing countries like Pakistan where the population has limited access to clean water supplies. Here, we evaluated the prevalence of rotavirus and free-living amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) in drinking water supplies of Karachi, Pakistan. Six water filtration plants that supply drinking water to the population of Karachi were investigated. Additionally, drinking water samples from households were analyzed for the presence of rotavirus and free-living amoebae. Rotavirus was present in 35% of the water samples collected from water filtration plants; however, domestic tap water samples had a prevalence of only 5%. Out of 20 water samples from filtration plants, 13 (65%) were positive for Acanthamoeba spp., and one (5%) was positive for B. mandrillaris. Out of 20 drinking water samples collected from different areas of Karachi, 35% were positive for Acanthamoeba spp. Rotavirus was detected in 5% of the drinking water samples tested. Overall, these findings showed for the first time the presence of rotavirus, in addition to pathogenic free-living amoebae in drinking water supplies of Karachi that could be an important public health risk for the affected population

Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts

Acanthamoeba cysts are highly resistant to contact lens disinfecting solutions. Acanthamoeba cyst wall is partially made of 1,4 beta-glucan (i.e., cellulose) and other complex polysaccharides making it a hardy shell that protects the resident amoeba. Here, we hypothesize that targeting the cyst wall structure in addition to antiamoebic compound would improve the efficacy of marketed contact lens disinfecting solutions. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents abolished viability of Acanthamoeba castellanii cysts and trophozoites. When tested alone, none of the agents nor contact lens disinfecting solutions completely destroyed A. castellanii cysts and trophozoites. The absence of cyst wall-degrading enzymes in marketed contact lens disinfecting solutions render them ineffective against Acanthamoeba cysts. It is concluded that the addition of cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy in decreasing the incidence of Acanthamoeba effectively

Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts

Acanthamoeba cysts are highly resistant to contact lens disinfecting solutions. Acanthamoeba cyst wall is partially made of 1,4 β-glucan (i.e., cellulose) and other complex polysaccharides making it a hardy shell that protects the resident amoeba. Here, we hypothesize that targeting the cyst wall structure in addition to antiamoebic compound would improve the efficacy of marketed contact lens disinfecting solutions. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents abolished viability of Acanthamoeba castellanii cysts and trophozoites. When tested alone, none of the agents nor contact lens disinfecting solutions completely destroyed A. castellanii cysts and trophozoites. The absence of cyst wall-degrading enzymes in marketed contact lens disinfecting solutions render them ineffective against Acanthamoeba cysts. It is concluded that the addition of cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy in decreasing the incidence of Acanthamoeba effectively