Displaying publications 61 - 80 of 107 in total

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  1. Siddiqui R, Yee Ong TY, Maciver S, Khan NA
    Ther Deliv, 2023 Aug;14(8):485-490.
    PMID: 37691579 DOI: 10.4155/tde-2023-0032
    Aim: CNS infections due to parasites often prove fatal. In part, this is due to inefficacy of drugs to cross the blood-brain barrier. Methods: Here, we tested intranasal and intravenous route and compared adverse effects of Amphotericin B administration, through blood biochemistry, liver, kidney and brain histopathological evidence of toxicities in vivo post-administration. Results: It was observed that intranasal route limits the adverse side effects of Amphotericin B, in contrast to intravenous route. Conclusion: As parasites such as Naegleria fowleri exhibit unequivocal affinity toward the olfactory bulb and frontal lobe in the central nervous system, intranasal administration would directly reach amoebae bypassing the blood-brain barrier selectivity and achieve the minimum inhibitory concentration at the target site.
  2. Siddiqui R, Maciver SK, Anuar TS, Khan NA
    Am J Vet Res, 2023 Aug 01;84(8).
    PMID: 37353216 DOI: 10.2460/ajvr.23.03.0061
    OBJECTIVE: The objective of this study was to determine bacterial flora throughout the gastrointestinal tract of a saltwater crocodile (Crocodylus porosus) using 16S rRNA gene analysis.

    ANIMALS: A convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided a healthy male saltwater crocodile, Crocodylus porosus for this study.

    PROCEDURES: Three samples were taken from the oral cavity, 3 samples from the proximal region of the small intestine (jejunum), and 3 samples from the distal part of the large intestine of the gastrointestinal tract of C. porosus were obtained using sterile cotton swabs. Next, swabs were placed in 15 mL sterile centrifuge tubes, individually, and kept on ice for immediate transportation to the laboratory. This was followed by 16S rRNA gene analysis using specific primers (341F-CCTAYGGGRBGCASCAG, and 806R-GGACTACNNGGGTATCTAAT). Amplicons were sequenced on Illumina paired-end platform, and bacterial gastrointestinal communities, the relative abundance of taxa, and principal component and coordinate analysis were performed.

    RESULTS: The findings revealed that bacterial community structures from differing regions exhibited several differences. The number of observed bacterial operational taxonomic units (OTUs) was 153 in the oral cavity, 239 in the small intestine, and 119 in the large intestine of C. porosus. The small intestine reflects the highest richness. In contrast, the large intestine exhibited the least richness of microbial communities. Relative abundance of taxa showed that Proteobacteria, Bacteroidetes, and Firmicutes were dominant in all 3 sample sites. Pseudomonas differed in the oral cavity and the large intestine, with the latter exhibiting less distribution of Pseudomonas. Stenotrophomonas and Castellaniella were higher in the oral cavity, while the relative abundance of Comamonas and Salmonella was higher in the small intestine. Conversely, the relative abundance of Salmonella and Pannonibacter was augmented in the large intestine.

    CLINICAL RELEVANCE: For the first time, this study demonstrates the bacterial diversity along the segments of the gastrointestinal tract of C. porosus. Bacterial flora varies throughout the gastrointestinal tract. Although further studies using large cohorts are warranted; however, our findings suggest that microbiome composition may have the potential as a biomarker in determining the overall health and well-being of C. porosus.

  3. Anwar A, Khan NA, Alharbi AM, Alhazmi A, Siddiqui R
    Int Ophthalmol, 2024 Mar 15;44(1):140.
    PMID: 38491335 DOI: 10.1007/s10792-024-03062-4
    Keratitis is corneal inflammatory disease which may be caused by several reason such as an injury, allergy, as well as a microbial infection. Besides these, overexposure to ultraviolet light and unhygienic practice of contact lenses are also associated with keratitis. Based on the cause of keratitis, different lines of treatments are recommended. Photodynamic therapy is a promising approach that utilizes light activated compounds to instigate either killing or healing mechanism to treat various diseases including both communicable and non-communicable diseases. This review focuses on clinically-important patent applications and the recent literature for the use of photodynamic therapy against keratitis.
  4. Ali SM, Siddiqui R, Ong SK, Shah MR, Anwar A, Heard PJ, et al.
    Appl Microbiol Biotechnol, 2017 Jan;101(1):253-286.
    PMID: 27743045 DOI: 10.1007/s00253-016-7872-2
    Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential sources of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic Escherichia coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analysed. Among hundreds of compounds, only a few homologous compounds were identified that contained the isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole-containing analogs, sulfonamides, furanones, and flavanones and known to possess broad-spectrum antimicrobial properties and anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization, and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs.
  5. Akbar N, Khan NA, Sagathevan K, Iqbal M, Tawab A, Siddiqui R
    Sci Rep, 2019 11 18;9(1):17012.
    PMID: 31740685 DOI: 10.1038/s41598-019-52738-w
    Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.
  6. Siddiqui R, Roberts SK, Ong TYY, Mungroo MR, Anwar A, Khan NA
    Parasit Vectors, 2019 Nov 14;12(1):538.
    PMID: 31727139 DOI: 10.1186/s13071-019-3785-0
    BACKGROUND: Acanthamoeba is well known to produce a blinding keratitis and serious brain infection known as encephalitis. Effective treatment is problematic, and can continue up to a year, and even then, recurrence can ensue. Partly, this is due to the capability of vegetative amoebae to convert into resistant cysts. Cysts can persist in an inactive form for decades while retaining their pathogenicity. It is not clear how Acanthamoeba cysts monitor environmental changes, and determine favourable conditions leading to their emergence as viable trophozoites.

    METHODS: The role of ion transporters in the encystation and excystation of Acanthamoeba remains unclear. Here, we investigated the role of sodium, potassium and calcium ion transporters as well as proton pump inhibitors on A. castellanii encystation and excystation and their effects on trophozoites.

    RESULTS: Remarkably 3',4'-dichlorobenzamil hydrochloride a sodium-calcium exchange inhibitor, completely abolished excystation of Acanthamoeba. Furthermore, lanthanum oxide and stevioside hydrate, both potassium transport inhibitors, resulted in the partial inhibition of Acanthamoeba excystation. Conversely, none of the ion transport inhibitors affected encystation or had any effects on Acanthamoeba trophozoites viability.

    CONCLUSIONS: The present study indicates that ion transporters are involved in sensory perception of A. castellanii suggesting their value as potential therapeutic targets to block cellular differentiation that presents a significant challenge in the successful prognosis of Acanthamoeba infections.

  7. Rajendran K, Anwar A, Khan NA, Aslam Z, Raza Shah M, Siddiqui R
    ACS Chem Neurosci, 2020 08 19;11(16):2431-2437.
    PMID: 31347828 DOI: 10.1021/acschemneuro.9b00289
    Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis (PAM) which almost always results in death. N. fowleri is also known as "brain-eating amoeba" due to its literal infestation of the brain leading to an inflammatory response in the brain tissues. Currently, there is no single drug that is available to treat PAM, and most treatments are combinations of antifungal, anticancer, and anti-inflammatory drugs. Recently nanotechnology has gained attention in chemotherapeutic research converging on drug delivery, while oleic acid (OA) has shown positive effects on the human immune system and inflammatory processes. In continuation of our recent research in which we reported the effects of oleic acid conjugated with silver nanoparticles (OA-AgNPs) against free-living amoeba Acanthamoeba castellanii, in this report, we show their antiamoebic effects against N. fowleri. OA alone and its nanoconjugates were tested against the amoeba by using amoebicidal and host cell cytopathogenicity assays. Trypan blue exclusion assay was used to determine cell viability. The results revealed that OA-AgNPs exhibited significantly enhanced antiamoebic effects (P < 0.05) against N. fowleri as compared to OA alone. Evidently, lactate dehydrogenase release shows reduced N. fowleri-mediated host cell cytotoxicity. Based on our study, we anticipate that further studies on OA-AgNPs could potentially provide an alternative treatment of PAM.
  8. Anwar A, Shahbaz MS, Saad SM, Kanwal, Khan KM, Siddiqui R, et al.
    Eur J Med Chem, 2019 Nov 15;182:111575.
    PMID: 31415900 DOI: 10.1016/j.ejmech.2019.111575
    We report one-pot synthesis of a series of new 3-aryl-8-methylquinazolin-4(3H)-ones (QNZ) and their antimicrobial activity against Acanthamoeba castellanii belonging to T4 genotype. A library of fifteen synthetic derivatives of QNZs was synthesized, and their structural elucidation was performed by using nuclear magnetic resonance (NMR) spectroscopy and electron impact mass spectrometry (EI-MS). Elemental analyses and high-resolution mass spectrometry data of all derivatives were found to be in agreeable range. Amoebicidal assays performed at concentrations ranging from 50 to 100 μg/mL revealed that all derivatives of QNZ significantly decreased the viability of A. castellanii and QNZ 2, 5, 8, and 13 were found to have efficient antiamoebic effects. Field emission scanning electron microscopy (FESEM) imaging of amoeba treated with compounds 5 and 15 showed that these compounds cause structural alterations on the walls of A. castellanii. Furthermore, several QNZs inhibited the encystation and excystationas as well as abolished A. castellanii-mediated host cells cytopathogenicity in human cells. Whereas, these QNZs showed negligible cytotoxicity when tested against human cells in vitro. Hence, this study identified potential lead molecules having promising properties for drug development against A. castellanii. A brief structure-activity relationship is also developed to optimize the hit of most potent compounds from the library. To the best of our knowledge, it is first of its kind medicinal chemistry approach on a single class of compounds i.e., quinazolinone against keratitis and brain infection causing free-living amoeba, A. castellanii.
  9. Anwar A, Ting ELS, Anwar A, Ain NU, Faizi S, Shah MR, et al.
    AMB Express, 2020 Feb 03;10(1):24.
    PMID: 32016777 DOI: 10.1186/s13568-020-0960-9
    Acanthamoeba spp. are the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis (GAE). The current options to treat Acanthamoeba infections have limited success. Silver nanoparticles show antimicrobial effects and enhance the efficacy of their payload at the specific biological targets. Natural folk plants have been widely used for treating diseases as the phytochemicals from several plants have been shown to exhibit amoebicidal effects. Herein, we used natural products of plant or commercial sources including quercetin (QT), kolavenic acid (PGEA) isolated from plant extracts of Polyalthia longifolia var pendula and crude plant methanolic extract of Caesalpinia pulcherrima (CPFLM) as antiacanthamoebic agents. Furthermore, these plant-based materials were conjugated with silver nanoparticles (AgNPs) to determine the effects of the natural compounds and their nanoconjugates against a clinical isolate of A. castellanii from a keratitis patient (ATCC 50492) belonging to the T4 genotype. The compounds were conjugated with AgNPs and characterized by using ultraviolet visible spectrophotometry and atomic force microscopy. Quercetin coated silver nanoparticles (QT-AgNPs) showed characteristic surface plasmon resonance band at 443 nm and the average size distribution was found to be around 45 nm. The natural compounds alone and their nanoconjugates were tested for the viability of amoebae, encystation and excystation activity against A. castellanii. The natural compounds showed significant growth inhibition of A. castellanii while QT-AgNPs specifically exhibited enhanced antiamoebic effects as well as interrupted the encystation and excystation activity of the amoebae. Interestingly, these compounds and nanoconjugates did not exhibit in vitro cytotoxic effects against human cells. Plant-based compounds and extracts could be an interesting strategy in development of alternative therapeutics against Acanthamoeba infections.
  10. Abdelnasir S, Anwar A, Kawish M, Anwar A, Shah MR, Siddiqui R, et al.
    AMB Express, 2020 Jul 17;10(1):127.
    PMID: 32681358 DOI: 10.1186/s13568-020-01061-z
    Acanthamoeba castellanii can cause granulomatous amoebic encephalitis and Acanthamoeba keratitis. Currently, no single drug has been developed to effectively treat infections caused by Acanthamoeba. Recent studies have shown that drugs conjugated with nanoparticles exhibit potent in vitro antiamoebic activity against pathogenic free-living amoebae. In this study, we have developed a nano drug delivery system based on iron oxide nanoparticles conjugated with metronidazole which were further loaded with amphotericin B to produce enhanced antiamoebic effects against Acanthamoeba castellanii. The results showed that metronidazole-nanoparticles-amphotericin B (Met-MNPs-Amp) significantly inhibited the viability of these amoebae as compared to the respective controls including drugs and nanoparticles alone. Met-MNPs-Amp exhibited IC50 at 50 μg/mL against both A. castellanii trophozoites and cysts. Furthermore, these nanoparticles did not affect the viability of rat and human cells and showed safe hemolytic activity. Hence, the results obtained in this study have potential utility in drug development against infections caused by Acanthamoeba castellanii. A combination of drugs can lead to successful prognosis against these largely neglected infections. Future studies will determine the value of conjugating molecules with diagnostic and therapeutic potential to provide theranostic approaches against these serious infections.
  11. Mohd Hussain RH, Afiqah WN, Abdul Ghani MK, Khan NA, Siddiqui R, Anuar TS
    Saudi J Biol Sci, 2021 Apr;28(4):2352-2359.
    PMID: 33911949 DOI: 10.1016/j.sjbs.2021.01.030
    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.
  12. Kanwal, Mungroo MR, Anwar A, Ali F, Khan S, Abdullah MA, et al.
    Exp Parasitol, 2020 Nov;218:107979.
    PMID: 32866583 DOI: 10.1016/j.exppara.2020.107979
    Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae that can cause life-threatening infections involving the central nervous system. The high mortality rates of these infections demonstrate an urgent need for novel treatment options against the amoebae. Considering that indole and thiazole compounds possess wide range of antiparasitic properties, novel bisindole and thiazole derivatives were synthesized and evaluated against the amoebae. The antiamoebic properties of four synthetic compounds i.e., two new bisindoles (2-Bromo-4-(di (1H-indol-3-yl)methyl)phenol (denoted as A1) and 2-Bromo-4-(di (1H-indol-3-yl)methyl)-6-methoxyphenol (A2)) and two known thiazole (4-(3-Nitrophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (A3) and 4-(Biphenyl-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (A4)) were evaluated against B. mandrillaris and N. fowleri. The ability of silver nanoparticle (AgNPs) conjugation to enrich antiamoebic activities of the compounds was also investigated. The synthetic heterocyclic compounds demonstrated up to 53% and 69% antiamoebic activities against B. mandrillaris and N. fowleri respectively, while resulting in up to 57% and 68% amoebistatic activities, respectively. Antiamoebic activities of the compounds were enhanced by up to 71% and 51% against B. mandrillaris and N. fowleri respectively, after conjugation with AgNPs. These compounds exhibited potential antiamoebic effects against B. mandrillaris and N. fowleri and conjugation of synthetic heterocyclic compounds with AgNPs enhanced their activity against the amoebae.
  13. Akbar N, Siddiqui R, Iqbal M, Sagathevan K, Kim KS, Habib F, et al.
    ACS Omega, 2021 May 11;6(18):12261-12273.
    PMID: 34056379 DOI: 10.1021/acsomega.1c01137
    Among several animals, Rattus rattus (rat) lives in polluted environments and feeds on organic waste/small invertebrates, suggesting the presence of inherent mechanisms to thwart infections. In this study, we isolated gut bacteria of rats for their antibacterial activities. Using antibacterial assays, the findings showed that the conditioned media from selected bacteria exhibited bactericidal activities against Gram-negative (Escherichia coli K1, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and Salmonella enterica) and Gram-positive (Bacillus cereus, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes) pathogenic bacteria. The conditioned media retained their antibacterial properties upon heat treatment at boiling temperature for 10 min. Using MTT assays, the conditioned media showed minimal cytotoxic effects against human keratinocyte cells. Active conditioned media were subjected to tandem mass spectrometry, and the results showed that conditioned media from Bacillus subtilis produced a large repertoire of surfactin and iturin A (lipopeptides) molecules. To our knowledge, this is the first report of isolation of lipopeptides from bacteria isolated from the rat gut. In short, these findings are important and provide a platform to develop effective antibacterial drugs.
  14. Khan NA, Soopramanien M, Maciver SK, Anuar TS, Sagathevan K, Siddiqui R
    Molecules, 2021 Aug 18;26(16).
    PMID: 34443585 DOI: 10.3390/molecules26164999
    Crocodiles are remarkable animals that have the ability to endure extremely harsh conditions and can survive up to a 100 years while being exposed to noxious agents that are detrimental to Homo sapiens. Besides their immunity, we postulate that the microbial gut flora of crocodiles may produce substances with protective effects. In this study, we isolated and characterized selected bacteria colonizing the gastrointestinal tract of Crocodylusporosus and demonstrated their inhibitory effects against three different cancerous cell lineages. Using liquid chromatography-mass spectrometry, several molecules were identified. For the first time, we report partial analyses of crocodile's gut bacterial molecules.
  15. Abdelnasir S, Mungroo MR, Shahabuddin S, Siddiqui R, Khan NA, Anwar A
    ACS Chem Neurosci, 2021 Oct 06;12(19):3579-3587.
    PMID: 34545742 DOI: 10.1021/acschemneuro.1c00179
    Free-living amoebae include Acanthamoeba castellanii and Naegleria fowleri that are opportunistic protozoa responsible for life-threatening central nervous system infections with mortality rates over 90%. The rising number of cases and high mortality rates are indicative of the critical unmet need for the development of efficient drugs in order to avert future deaths. In this study, we assess the anti-amoebic capacity of a conducting polymer nanocomposite comprising polyaniline (PANI) and hexagonal boron nitride (hBN) against A. castellanii and N. fowleri. We observed significant amoebicidal and cysticidal effects using 100 μg/mL PANI/hBN (P < 0.05). Further, the nanocomposite demonstrated negligible cytotoxicity toward HaCaT and primary human corneal epithelial cells (pHCECs). In evaluating the mode of inhibition of A. castellanii due to treatment with PANI/hBN, increased intracellular reactive oxygen species (ROS) was measured and scanning microscopy visualized the formation of pores in the amoebae. Overall, this study is suggestive of the potential of the PANI/hBN nanocomposite as a promising therapy for amoeba infections.
  16. Siddiqui R, Abjani F, Yeo CI, Tiekink ER, Khan NA
    J Negat Results Biomed, 2017 Apr 03;16(1):6.
    PMID: 28366172 DOI: 10.1186/s12952-017-0070-7
    BACKGROUND: Gold compounds have shown promise in the treatment of non-communicable diseases such as rheumatoid arthritis and cancer, and are considered of value as anti-microbial agents against Gram-negative and Gram-positive bacteria, and have anti-parasitic properties against Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, Leishmania infantinum, Giardia lamblia, and Entamoeba histolytica. They are known to affect enzymatic activities that are required for the cellular respiration processes.

    METHODS: Anti-amoebic effects of phosphanegold(I) thiolates were tested against clinical isolate of A. castellanii belonging to the T4 genotype by employing viability assays, growth inhibition assays, encystation assays, excystation assays, and zymographic assays.

    RESULTS: The treatment of A. castellanii with the phosphanegold(I) thiolates tested (i) had no effect on the viability of A. castellanii as determined by Trypan blue exclusion test, (ii) did not affect amoebae growth using PYG growth medium, (iii) did not inhibit cellular differentiation, and (iv) had no effect on the extracellular proteolytic activities of A. castellanii.

    CONCLUSION: Being free-living amoeba, A. castellanii is a versatile respirator and possesses respiratory mechanisms that adapt to various aerobic and anaerobic environments to avoid toxic threats and adverse conditions. For the first time, our findings showed that A. castellanii exhibits resistance to the toxic effects of gold compounds and could prove to be an attractive model to study mechanisms of metal resistance in eukaryotic cells.

  17. Gabriel S, Rasheed AK, Siddiqui R, Appaturi JN, Fen LB, Khan NA
    Parasitol Res, 2018 Jun;117(6):1801-1811.
    PMID: 29675682 DOI: 10.1007/s00436-018-5864-0
    Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.
  18. Anwar A, Chi Fung L, Anwar A, Jagadish P, Numan A, Khalid M, et al.
    Pathogens, 2019 Nov 22;8(4).
    PMID: 31766722 DOI: 10.3390/pathogens8040260
    T4 genotype Acanthamoeba are opportunistic pathogens that cause two types of infections, including vision-threatening Acanthamoeba keratitis (AK) and a fatal brain infection known as granulomatous amoebic encephalitis (GAE). Due to the existence of ineffective treatments against Acanthamoeba, it has become a potential threat to all contact lens users and immunocompromised patients. Metal nanoparticles have been proven to have various antimicrobial properties against bacteria, fungi, and parasites. Previously, different types of cobalt nanoparticles showed some promise as anti-acanthamoebic agents. In this study, the objectives were to synthesize and characterize the size, morphology, and crystalline structure of cobalt phosphate nanoparticles, as well as to determine the effects of different sizes of cobalt metal-based nanoparticles against A. castellanii. Cobalt phosphate octahydrate (CHP), Co3(PO4)2•8H2O, was synthesized by ultrasonication using a horn sonicator, then three different sizes of cobalt phosphates Co3(PO4)2 were produced through calcination of Co3(PO4)2•8H2O at 200 °C, 400 °C and 600 °C (CP2, CP4, CP6). These three types of cobalt phosphate nanoparticles were characterized using a field emission scanning electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analysis. Next, the synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against A. castellanii, as well as cell cytotoxicity. The overall results showed that 1.30 ± 0.70 µm of CHP microflakes demonstrated the best anti-acanthemoebic effects at 100 µg/mL, followed by 612.50 ± 165.94 nm large CP6 nanograins. However, amongst the three tested cobalt phosphates, Co3(PO4)2, the smaller nanoparticles had stronger antiamoebic effects against A. castellanii. During cell cytotoxicity analysis, CHP exhibited only 15% cytotoxicity against HeLa cells, whereas CP6 caused 46% (the highest) cell cytotoxicity at the highest concentration, respectively. Moreover, the composition and morphology of nanoparticles is suggested to be important in determining their anti-acathamoebic effects. However, the molecular mechanisms of cobalt phosphate nanoparticles are still unidentified. Nevertheless, the results suggested that cobalt phosphate nanoparticles hold potential for development of nanodrugs against Acanthamoeba.
  19. Mohd Hussain RH, Ishak AR, Abdul Ghani MK, Ahmed Khan N, Siddiqui R, Shahrul Anuar T
    J Water Health, 2019 Oct;17(5):813-825.
    PMID: 31638031 DOI: 10.2166/wh.2019.214
    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.
  20. Anwar A, Abdalla SAO, Aslam Z, Shah MR, Siddiqui R, Khan NA
    Parasitol Res, 2019 Jul;118(7):2295-2304.
    PMID: 31093751 DOI: 10.1007/s00436-019-06329-3
    Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid-conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid-AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5 μM concentration of oleic acid alone and oleic acid-conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid-conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5 μM. Furthermore, oleic acid and oleic acid-conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid-conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.
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