Heterologous expression of xylanase gene from bacillus sp. in e. coli bl21 (DE3)

Xylanase gene of 642 bp with molecular weight 23kDa which was isolated from indigenous Bacillus sp. The isolated xylanase gene from indigenous Bacillus sp. was cloned into pET expression vector to obtain a high level expression of this recombinant family 11 xylanase in expression host E.coli BL21 (DE3). This attempt to clone the gene was initiated with the extraction of xylanase gene previously isolated from pGEM®-T easy cloning vector. The cloning vector was digested with restriction endonuclease and the xylanase insert was cloned into pET41(a) and transformed into E.coli BL21(DE3) via heat shock transformation. The expression was attempted to be observed through formation of halos in congo red staining method. Qualitative xylanase screening showed no detectable xylanase activity. This was predicted to be due to reasons like improper framing (frameshift) of cloned xylanase gene to LacZ promoter or incapability of E.coli BL21(DE3) to grow optimally in M9 minimal media with corn cob xylan source. It is highly recommended to get the full sequence of recombinant pET41(a)-Xyn to confirm the position of ligation of xylanase gene. The minimal media should also be altered in salt composition for optimized growth of E.coli BL21 (DE3)

Exacerbated symptoms in Blastocystis sp.-infected patients treated with metronidazole: two case studies

Blastocystis sp. is a gastrointestinal (GI) protozoan parasite reported to cause non-specific GI symptoms including diarrhea, flatulence, abdominal pain, and nausea. Complete eradication of Blastocystis sp. is rather challenging even with the drug of choice, i.e., metronidazole. Here, we report on two Blastocystis sp.-infected individuals, who presented increased parasite load and exacerbated symptoms upon treatment with the usual recommended dosage and regime of metronidazole. The two studies uniquely demonstrate for the first time a cyst count as high as fivefold more than the original cyst count before treatment and show an exacerbation of GI symptoms despite treatment. The study provides additional support in recognizing metronidazole resistance in Blastocystis sp. and its consequences towards the pathogenicity of the parasite

Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity.

Whilst the influence of intestinal microbiota has been shown in many diseases such as irritable bowel syndrome, colorectal cancer, and aging, investigations are still scarce on its role in altering the nature of other infective organisms. Here we studied the association and interaction of Blastocystis sp. and human intestinal microbiota. In this study, we investigated the gut microbiome of Blastocystis sp.-free and Blastocystis sp. ST3-infected individuals who are symptomatic and asymptomatic. We tested if the expression of phenotype and pathogenic characteristics of Blastocystis sp. ST3 was influenced by the alteration of its accompanying microbiota. Blastocystis sp. ST3 infection alters bacterial composition. Its presence in asymptomatic individuals showed a significant effect on microbial richness compared to symptomatic ones. Inferred metagenomic findings suggest that colonization of Blastocystis sp. ST3 could contribute to the alteration of microbial functions. For the first time, we demonstrate the influence of bacteria on Blastocystis sp. pathogenicity. When Blastocystis sp. isolated from a symptomatic individual was co-cultured with bacterial suspension of Blastocystis sp. from an asymptomatic individual, the parasite demonstrated increased growth and reduced potential pathogenic expressions. This study also reveals that Blastocystis sp. infection could influence microbial functions without much effect on the microbiota diversity itself. Our results also demonstrate evidence on the influential role of gut microbiota in altering the characteristics of the parasite, which becomes the basis for the contradictory findings on the parasite's pathogenic role seen across different studies. Our study provides evidence that asymptomatic Blastocystis sp. in a human gut can be triggered to show pathogenic characteristics when influenced by the intestinal microbiota

Resistance towards metronidazole in Blastocystis sp.: A pathogenic consequence

Blastocsytis sp. is a protozoan parasite that has been linked to common gastrointestinal illnesses. Metronidazole, the first line therapy, was reported to show frequent inefficacy. Previously, Blastocystis sp. isolated from different population showed varying metronidazole resistance. However, the effect of metronidazole treatment on pathogenic potentials of Blastocystis sp. isolated from different populations, which is known to have different gut environment, is unclear. This study investigates the in vitro effect of metronidazole on the pathogenic potentials of Blastocystis sp. isolated from urban and orang asli individuals. Blastocystis sp. ST 3 isolated from symptomatic and asymptomatic individuals were treated with a range of metronidazole concentration. The parasites’ growth characteristics, apoptotic rate, specific protease activity and the ability to proliferate cancer cells were analyzed upon treatment with 0.001 mg/l metronidazole. The study demonstrates that Blastocystis sp. isolates showed increase in the parasite numbers especially the amoebic forms (only in urban isolates) after treating with metronidazole at the concentration of 0.001 mg/ml. High number of cells in post-treated isolates coincided with increase of apoptosis. There was a significant increase in cysteine protease of Blastocystis sp. isolates upon treatment despite the initial predominance of serine protease in asymptomatic isolates. Metronidazole resistant Blastocystis sp. also showed significant increase in cancer cell proliferation. Resistance to metronidazole did not show significant different influence on the pathogenicity between Blastocystis sp. isolated from urban and orang asli individual. However, an increase in parasite numbers, higher amoebic forms, cysteine protease and ability to proliferate cancer cells implicates a pathogenic role. The study provides evidence for the first time, the effect of metronidazole towards enhancing pathogenic potentials in Blastocystis sp. when isolated from different gut environment. This necessitates the need for reassessment of metronidazole treatment modalities. © 2019 Rajamanikam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Distinct Phenotypic Variation of Blastocystis sp. ST3 from Urban and Orang Asli Population—An Influential Consideration during Sample Collection in Surveys

Blastocystis sp. is a globally distributed protozoan parasite with uncertain pathogenicity. Phenotypic variation in Blastocystis sp. suggests its adaptation; however, the phenotypic features of Blastocystis sp. ST3 from a distinct source of isolation is unknown. Blastocystis sp. isolated from individuals in urban and orang asli (indigenous population in Selangor, Malaysia) settlements were studied for phenotypic characteristics such as growth profile, morphology, ultrastructure, and resistance to harsh conditions. Subsequently, pathogenic potentials, such as in protease activity and the ability to stimulate the proliferation of cancer cells, were assessed. Higher parasite counts with granular and apoptotic forms were found in Blastocystis sp. from orang asli individuals. Cells with fuzzy coats and amoebic structures which seemingly implicate increased interaction with bacteria were seen predominantly in urban symptomatic persons. Also, Blastocystis sp. from orang asli isolates resisted harsh environments, suggesting longer co-adaptation to the hosts. Urban and orang asli symptomatic isolates possessed a predominance of only cysteine protease, whereas all the asymptomatic isolates showed significantly higher cysteine, serine, or aspartic protease activity. However, only solubilized antigen from urban symptomatic isolates showed significant stimulation of cancer cell proliferation. For the first time, our findings demonstrate significant phenotypic variation in a single subtype, ST3 of Blastocystis sp., isolated from urban and orang asli populations that are known to have distinct gut microbial compositions. The outcome emphasizes the importance of identifying people’s locations and lifestyles during sample collection before forming conclusions on the prevailing data and implicating subtypes to pathogenicity. The environment plays a significant role in Blastocystis sp. infection

Systematic Review and Meta-Analysis: Epidemiology of Human <i>Blastocystis</i> spp. Infection in Malaysia

Blastocystis spp. is a unicellular enteric protozoan parasite in humans with a controversial role in disease etiology. It is common in developing countries among immunocompromised patients and people who have close contact with animals. In this study, we have systematically reviewed previous studies on the distribution and genotypes of human Blastocystis infection in Peninsular Malaysia. Studies examining the prevalence of Blastocystis in diverse demographics, including rural, urban, comorbid conditions, and high-risk populations, were taken into consideration. The infection has been reported in nine states; the total percentage of infection was 17.8% (1671/9397), with the most cases in Pahang (27.3%) and the least in Johor (3.4%). Molecular studies revealed the presence of six subtypes: ST1, ST2, ST3, ST4, ST5, and ST6. ST3 was reported as the predominant subtype in all the states, with a prevalence of 54.7% (338/618). The findings provide greater clarity on the epidemiology of Blastocystis in Malaysia, which will help in policy making towards planning and strategizing control measures against the parasite