Divalent cations (Mg2+, Ca2+) protect bacterial outer membrane damage by polymyxin B

Polymyxin B interacts with divalent cations by displacing cations from their binding sites in the lipopolysaccharide (LPS) molecules. It leads to the disorganization of the outer membrane component of the Gram negative bacteria, which releases LPS component from bacterial surface causing severe membrane leakage and finally cell death. In this paper, the reversible activity of PMB was investigated in the presence of access divalent cations such as Mg2+ or Ca2+. Membrane damage and cell disruption were monitored by detecting leakage of the outer membrane and cytoplasmic enzyme marker (β-lactamase and β-galactosidase), release of LPS component (KDO) from bacterial cells and bacterial survival. With the presence of divalent cations, leakage of enzyme markers and LPS released was significantly reduced when bacteria were exposed to PMB. The survival curve also significantly increased. The inhibitory of damage caused by PMB also depended on the type of divalent cation present. Ca2+ has been shown to be more effective in protecting the bacteria cells than Mg2+. This is because Ca2+ appears more frequently as a constituent of the structural components of the bacteria. In conclusion, the presence of higher concentration of divalent cation particularly with Ca2+ inhibits PMB activity and maintained bacterial survival

Acanthamoeba species isolated from marine water in Malaysia exhibit distinct genotypes and variable physiological properties

The present study identifies the Acanthamoeba genotypes and their pathogenic potential in five marine waters in Malaysia. Fifty water samples were collected between January and May 2019. Physical parameters of water quality were measured in situ, whereas chemical and microbiological analyses were conducted in the laboratory. All samples had undergone filtration using nitrocellulose membrane and were tested for Acanthamoeba using cultivation and polymerase chain reaction by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using physiological tolerance tests. Thirty-six (72.0%) samples were positive for Acanthamoeba. Total coliforms (p = 0.013) and pH level (p = 0.023) displayed significant correlation with Acanthamoeba presence. Phylogenetic analysis showed that 27 samples belonged to genotype T4, four (T11), two (T18) and one from each genotype T5, T15 and T20. Thermo- and osmo-tolerance tests signified that three (8.3%) Acanthamoeba strains displayed highly pathogenic attributes. This study is the first investigation in Malaysia describing Acanthamoeba detection in marine water with molecular techniques and genotyping. The study outcomes revealed that the marine water in Malaysia could be an integral source of Acanthamoeba strains potentially pathogenic in humans. Thus, the potential risk of this water should be monitored routinely in each region

Acanthamoeba species isolated from marine water in Malaysia exhibit distinct genotypes and variable physiological properties

The present study identifies the Acanthamoeba genotypes and their pathogenic potential in five marine waters in Malaysia. Fifty water samples were collected between January and May 2019. Physical parameters of water quality were measured in situ, whereas chemical and microbiological analyses were conducted in the laboratory. All samples had undergone filtration using nitrocellulose membrane and were tested for Acanthamoeba using cultivation and polymerase chain reaction by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using physiological tolerance tests. Thirty-six (72.0%) samples were positive for Acanthamoeba. Total coliforms (p = 0.013) and pH level (p = 0.023) displayed significant correlation with Acanthamoeba presence. Phylogenetic analysis showed that 27 samples belonged to genotype T4, four (T11), two (T18) and one from each genotype T5, T15 and T20. Thermo- and osmo-tolerance tests signified that three (8.3%) Acanthamoeba strains displayed highly pathogenic attributes. This study is the first investigation in Malaysia describing Acanthamoeba detection in marine water with molecular techniques and genotyping. The study outcomes revealed that the marine water in Malaysia could be an integral source of Acanthamoeba strains potentially pathogenic in humans. Thus, the potential risk of this water should be monitored routinely in each region

In vitro effects of multi-purpose contact lens disinfecting solutions towards survivability of Acanthamoeba genotype T4 in Malaysia

The incidence of Acanthamoeba keratitis has been increasing since the previous decades, especially among contact lens users. This infection is majorly caused by the use of ineffective contact lens disinfecting solution. Thus, this study was conducted to evaluate the in vitro effects of multi-purpose disinfecting solutions (MPDS) against Acanthamoeba trophozoites and cysts. Acanthamoeba genotype T4 isolated from contact lens paraphernalia and an environmental strains were propagated for trophozoite or cyst-containing culture and adjusted in final concentration of 1 × 105 cells/ml. Amoebicidal and cysticidal assays were conducted by incubating trophozoites and cysts with OPTI-FREE® Express®, ReNu® Fresh™, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive according to the manufacturer’s minimum recommended disinfectant time (MMRDT) for up to 12 h at 30 ⁰C. Trypan blue hemocytometer-based microscopic counts determined amoebicidal and cysticidal effects. The viability of Acanthamoeba trophozoites and cysts was confirmed by re-inoculated them in the 1.5% non-nutrient agar plates. It was found that none of the MPDS showed amoebicidal and cysticidal effects during the MMRDT. However, OPTI-FREE® Express® demonstrated a significant differences in average cell reduction for both stages within MMRDT. When subjected to 12 h exposure, both OPTI-FREE® Express® and ReNu® Fresh™ led to significant reduction in the number of trophozoite and cyst cells. Notably, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive did appreciably improve the solution effectiveness towards trophozoite cells when incubated for 12 h. All MPDS were largely ineffective, with 100% survival of all isolates at MMRDT, while OPTI-FREE® Express® showed limited amoebicidal activity against the contact lens paraphernalia isolate, however, it was more against the environmental strains after 12 h incubation time. The commercially available MPDS employed in this research offered minimal effectiveness against the protozoa despite the contact time. Improvement or development of new solution should consider the adjustment of the appropriate disinfectant concentration, adequate exposure time or the incorporation of novel chemical elements, which are effective against Acanthamoeba for accelerated disinfecting and more reduction of potential exposure of contact lens users to Acanthamoeba keratitis

Occurrence and molecular characterisation of Acanthamoeba isolated from recreational hot springs in Malaysia: evidence of pathogenic potential.

This study aimed to identify the Acanthamoeba genotypes and their pathogenic potential in five recreational hot springs in Peninsular Malaysia. Fifty water samples were collected between April and September 2018. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. All samples were filtered through the nitrocellulose membrane and tested for Acanthamoeba using both cultivation and polymerase chain reaction (PCR) by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using thermo- and osmotolerance tests. Thirty-eight (76.0%) samples were positive for Acanthamoeba. Water temperature (P = 0.035), chemical oxygen demand (P = 0.026), sulphate (P = 0.002) and Escherichia coli (P < 0.001) were found to be significantly correlated with the presence of Acanthamoeba. Phylogenetic analysis revealed that 24 samples belonged to genotype T4, nine (T15), two (T3) and one from each genotype T5, T11 and T17. Thermo- and osmotolerance tests showed that 6 (15.79%) of the Acanthamoeba strains were highly pathogenic. The existence of Acanthamoeba in recreational hot springs should be considered as a health threat among the public especially for high-risk people. Periodic surveillance of hot spring waters and posting warning signs by health authorities is recommended to prevent disease related to pathogenic Acanthamoeba

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

Morphological and molecular characterization of Acanthamoeba isolated from contact lens paraphernalia in Malaysia: Highlighting the pathogenic potential of T4 genotype

Objective: To determine the morphological and molecular characterization of Acanthamoeba isolates from contact lens paraphernalia in Malaysia and to investigate their pathogenic potential based on the physiological tolerance.Methods: One hundred and eighty contact lens wearers donated their contact lens, lens storage cases and lens solutions between 2018 and 2019. The samples were inoculated onto 1.5% non-nutrient agar plates for 14 d. Polymerase chain reaction (PCR) was performed and the amplified PCR products were sequenced and compared with the published sequences in GenBank. The pathogenic potential of positive isolates was further tested using temperature-tolerance and osmo-tolerance assays. Acanthamoeba species were categorized into three distinct morphological groups established by Pussard and Pons.Results: Acanthamoeba was successfully isolated from 14 (7.8%) culture-positive samples in which 11 belong to morphological group II and 3 belong to morphological group III, respectively. The sequencing of 18S ribosomal RNA gene led to the identification of the T4 genotype in all the isolated strains. In vitro assays revealed that 9 (64.3%) Acanthamoeba isolates were able to grow at 42 °C and 1 M mannitol and were thus considered to be highly pathogenic.Conclusions: To the best of our knowledge, this is the first report identifying the Acanthamoeba genotype and their pathogenic potential among contact lens wearers in Malaysia. The potentially pathogenic T4 genotype isolated in this study is the most predominant genotype responsible for human ocular infection worldwide. Hence, increasing attention should be aimed at the prevention of contamination by Acanthamoeba and the disinfection of contact lens paraphernalia