MALAYSIAN JOURNAL OF MICROBIOLOGY
Aims: Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C.
Methodology and results: The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (GmR) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages.
Conclusion, significance and impact of study: The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment.
Aims: The detection of the metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa isolates is crucial for infection control and public health. The present study aimed to investigate the MBL production in carbapenem-resistant P. aeruginosa isolated from various clinical samples in Kastamonu Training and Research Hospital, Turkey.
Methodology and results: Seventy-three carbapenem-resistant P. aeruginosa isolates were recovered from different patients between April 2018 and November 2020. Identification of the isolates was performed by conventional methods (culture examination, determination of Gram reaction, and oxidase test) and an automated system (Vitek 2). Antibiotic susceptibility patterns were determined using the Vitek 2 and the results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The MBL production was phenotypically investigated using the imipenem-EDTA combined disk test. The presence of beta-lactamase IMP (blaIMP), beta-lactamase VIM (blaVIM) and beta-lactamase GIM (blaGIM) genes were determined using PCR to confirm the MBL production. Seventy-one isolates (97%, n=71/73) were resistant to imipenem, sixty-four isolates (88%, n=64/73) to meropenem and sixty-two isolates (85%, n=62/73) to both imipenem and meropenem. Sixty-five isolates (89%, n=65/73) were defined as multidrug-resistant. The MBL production was detected in 57 isolates (78%, n=57/73) phenotypically. However, the blaIMP, blaVIM and blaGIM genes were not detected in all the isolates.
Conclusion, significance and impact of study: It was determined that there were no imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM) and German imipenemase (GIM) type MBLs in carbapenem-resistant P. aeruginosa isolated from Kastamonu Training and Research Hospital. MBL production in carbapenem-resistant P. aeruginosa strains can be investigated phenotypically. However, confirmation of results with molecular tests is especially significant for epidemiological studies.
Aims: Plant growth promoting rhizobacteria (PGPR) is a group of bacteria that colonise plant roots and enhance plant growth by a diverse range of mechanisms. This study aims to determine the capabilities of PGPR isolated from cocoa tree roots and their efficiency in enhancing plant growth under greenhouse conditions.
Methodology and results: Eight samples of healthy cocoa tree roots were collected from different locations in Malaysia. Isolated bacteria were screened based on nitrogen fixation, phosphate and potassium solubilization, and catalase activity. The efficiency of purified PGPR was evaluated from pot experiments of cocoa seedlings under greenhouse conditions. Out of 122 isolates, 18 isolates showed several traits of nitrogen fixation, phosphorus and potassium solubilization and were further screened for other plant growth promoting (PGP) traits like catalase and production of indole acetic acid (IAA). Out of all the PGP trait tests, seven isolates showed the most prominent results for in vitro tests and were further tested in vivo for growth promotion of cocoa seedlings under greenhouse conditions. In the presence of bacterial isolates with 2.5 g of inorganic fertilizer, Leclercia adecarboxylata resulted in increases in plant height, leaf number, root length, stem fresh weight and total fresh and dry weight of cocoa seedlings by 15.68%, 17.14%, 9.48%, 5.67%, 11.84% and 25.12%, respectively.
Conclusion, significance and impact of study: Based on the result, L. adecarboxylata incorporated with selected carrier material improve cocoa seedling growth and biomass. This formulation also reduces the production cost of inorganic fertilizer and increase the application and development of biofertilizer.
Aims: Attention to ice nucleation proteins has increased for more than two decades. Ice nucleation proteins have been utilized for artificial snow-making known as Snowmax™, cryopreservation of tissues and cells, and cloud condensation nuclei. There is a direct relationship between bacterial growth and ice nucleation activity. Therefore, the optimization of the culture medium seems necessary.
Methodology and results: The effect of different carbon and nitrogen sources on the growth of a new native Pseudomonas sp. IRL.INP1 was evaluated by using fractional factorial design, the path of the steepest ascent experiment and central composite design. Ice nucleation activity, biomass and whole-cell protein were identified afterward. The model predicted by response surface methodology indicated that the maximum bacterial growth was observed when sucrose, ammonium sulfate [(NH4)2SO4] and manganese (II) (Mn2+) were utilized at 12.46 g/L, 321.97 mg/L and 938.09 µM, respectively. Also, 1.10 g/L biomass and 0.85 µg/µL whole-cell proteins were gained, and the isolate showed ice nucleation activity 31 sec sooner after optimization.
Conclusion, significance and impact of study: Ice nucleation proteins are growth-dependent and the growth condition optimization leads to higher bacterial cells growth. Therefore, best bacterial growth was obtained when proper carbon and nitrogen sources were used, and ice nucleation activity was observed in shorter time. This is the first study concerning ice nucleation activity optimization using different carbon and nitrogen sources.
Aims: Arthropods guts, such as termite harbor diverse microorganisms including those that are capable of fixing atmospheric nitrogen (N2). Nitrogen-fixing bacteria can help termite to overcome their shortage of dietary N by providing fixed N2. Nitrogenase enzyme is responsible for this trait and encoded by nif genes which are highly conserved and are primarily used in the identification of N2-fixing microorganisms. Here, we characterized N2-fixing bacteria isolated from the hindguts of termite Coptotermes gestroi.
Methodology and results: A total of 46 bacterial isolates were obtained after a primary screening based on their ability to grow on Burk’s media. Subsequently, the nifH gene from two of these isolates, namely S7 and S20, were successfully amplified and sequenced. Molecular phylogenetic analysis of 16S rRNA gene sequence revealed that isolate S7 is closely related to Ralstonia pickettii ATCC27511 (99.34% similarity, 1059 bp), whereas isolate S20 is closely related to Microbacterium sp. NCCP-451 (LC488936) (99.06% similarity, 948 bp). Besides that, the recA gene of isolate S7 is closely related to Ralstonia pickettii 12D (CP001644) (100% similarity, 442 bp) and the type strain of Ralstonia pickettii (ATCC 27511) (NZ KN050646) (98.97% similarity, 438 bp). Meanwhile, nifH gene of isolate S7 showed highest similarity to the uncultured bacterium NR1606 (AF035490) (99.93% similarity, 277 bp). Moreover, the nifH gene of isolate S20 is clearly separated from Azoarcus sp. and distantly related to Microbacterium sp. The incongruence between the partial 16S rRNA and nifH gene sequences could indicate the possibility of horizontal transfer of nif genes.
Conclusion, significance and impact of study: The phylogenetic incongruence between housekeeping genes (16S rRNA and RecA) and nifH gene in these bacteria provides new insight on potential horizontal gene transfer (HGT) activity taking place in bacterial communities particularly in the guts of arthropods. The finding of this study on potential HGT can also aid in the prediction of origins and evolution of gene transfer among bacteria.
Aims: Acinetobacter baumannii has been identified as one of the six most pathogenic bacteria that is the cause of most hospital bacterial infections according to Infectious Disease Society of America (IDSA). These nosocomial pathogens are notorious worldwide due to its ability in causing lethal infections among immunocompromised patients and its resistance to many strong antibiotics. This study aims to compare the expressed proteins of two A. baumannii strain, ATCC 19606 and a pathogenic clinically isolated strain known as AB-13.
Methodology and results: AB-13 clinically strain was isolated from the lower respiratory tract of a patient with pneumonia. In this study, the proteomic profile of both ATCC 19606 and AB-13 are produced using 2-dimensional gel electrophoresis. The total protein contents were extracted, quantified and separated using 2-DE with a pH range of 4–7 to acquire the proteomic profile for comparison. The final analytical gel was analysed using Delta2D software and among the 324 protein spots successfully resolved, 10 spots exhibited signs of differential expression with 7 spots found to be downregulated and 3 spots upregulated (p< 0.01). These differences could signify the evolution AB-13 has undergone as it acquires traits ultimately aiding in its survivability, antimicrobial resistance and pathogenicity within varied environments especially during infections.
Conclusion, significance and impact of study: These findings support the presence of variation in AB-13 from a proteomic perspective, highlighting the pathogen’s evolution improving survivability and pathogenicity, warranting in-depth exploration towards understanding A. baumannii virulence and pathogenicity.
Aims: This study aimed to evaluate the effect of electroporation on the growth characteristics and antimicrobial activity of lactic acid bacteria (LAB) including Bifidobacterium longum ATCC 15707, Lactobacillus acidophilus ATCC 314, Lactobacillus casei ATCC 393 and Lactobacillus fermentum ATCC 14931.
Methodology and results: Electroporation with the strength of electric field at 1.0–3.0 kV/cm for 2–4 millisecond were applied on the bacterial cultures. All bacterial cultures showed significant (P<0.05) increased in cell viability (40%–325%) upon electroporation. Such treatment also increased the acidity of the cell where the pH of cells decreased upon treatment. In tandem with the increased viability, electroporated bacterial cultures also showed higher proteolytic activity compared to the control (P<0.05). The electroporation treatment also increased (P<0.05) the bacteriocin activity of treated cells compared to the control. However, the molecular weight of bacteriocins produced were not affected by electroporation. Treated cells also possessed better antimicrobial activity. According to the results collected, all treated LAB strains showed 11.5%–113.8% higher (P<0.05) inhibitory activity compared to untreated control against tested pathogenic bacteria, Escherichia coli and Listeria monocytogenes that commonly associated with food contamination. Microarray data analysis showed that electroporation regulated the entities encoding for surface protein and transporter.
Conclusion, significance and impact of study: The results from this study suggested that electroporation could enhance the growth characteristics and antimicrobial activity of LAB by modifying the surface regions of the cells. This result may serve as the reference for food manufacturers to opt for effective biopreservation method and produce food with extended shelf life.
Aims: Piper sarmentosum or locally known as Kaduk, is a tropical herb plant that was investigated for its phenolic content by previous researchers. The present study aimed at the analysis of crude methanolic extract of P. sarmentosum leaves for phenolic compounds identification and its anti-amoebic properties against pathogenic Acanthamoeba castellanii.
Methodology and results: Folin-Ciocalteu assay was used to determine P. sarmentosum leaves methanolic extract (PSLME)’s total phenolic content (TPC). The extract was further characterized by using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses to determine the chemical constituents in methanolic PSLME extract. The cytotoxicity of the extract was evaluated through the determination of inhibition concentration for half of cell population (IC50) of pathogenic A. castellanii followed by cell morphological analysis using inverted light and scanning electron microscopies. Acridine-orange/Propidium iodide (AOPI) staining was also conducted to determine the integrity of cell membrane for quantitative analysis. The results demonstrated that the TPC from PSLME was 142.72 mg [GAE]/g with a total of 33 phenolic compounds identified. The IC50 value obtained for A. castellanii was low (74.64 μg/mL) which indicates promising anti-acanthamoebic activity. Microscopy analyses showed that the plant extract caused cells encystment, in which exhibited by distinctive morphological changes on the cells shape and organelle, as well as shortening of acanthopodia. The dual staining and its quantitative analysis prove compromised membrane integrity in the treated amoeba.
Conclusion, significance and impact of study: This finding provides the evidence that PSLME contains active phenolic compounds contributing to the anti-acanthamoebic activity on pathogenic Acanthamoeba species.
Aims: The occurrence of bacterial disease in shrimp ponds is a major problem faced in shrimp farming. Thus, the aims of this study were to isolate and evaluate antibiotic resistant profile of Vibrio harveyi strain isolated from shrimp pond water, as well as to study the potential anti-Vibrio activity of Combretum quadrangulare Kurz. (CQ) and Mimosa pudica (MP) leaves extracts.
Methodology and results: Vibrio harveyi WSC103 was isolated from water in white shrimp (Litopenaeus vannamei) culture pond and identified using 16S rRNA gene sequencing analysis. This strain showed characteristics of multidrug-resistant (7 antibiotics). It had become more sensitive to antibiotics (9 out of 10 antibiotics) after plasmid curing. It is showed CQ and MP leaves extracts contain potent bioactive compounds (tannins, flavonoids, steroids, cardiac glycosides and alkaloids) against V. harveyi WSC103. The aqueous, 95% ethanolic and 75% acetone extracts of CQ (MIC value of 3.13–12.50 mg/mL) and MP (MIC value of 3.13–25.00 mg/mL) leaves revealed strong vibriostatic activity, but aqueous and 95% ethanolic extracts in both plants showed vibriocidal activity. The 95% ethanolic extract of both CQ and MP leaves displayed the excellent vibriocidal property with MBC value of 100 mg/mL with zone of inhibition at 11.44 ± 1.01 and 11.78 ± 1.01 mm by agar disc diffusion.
Conclusion, significance and impact of study: The isolated Vibrio harveyi WSC103 was successfully characterized as a novel multidrug-resistant strain. The ethanolic C. quadrangulare Kurz. and M. pudica extracts exhibited prominent vibriostatic and vibriocidal capacities. These finding is proven that C. quadrangulare Kurz. and M. pudica extracts would be an alternative anti-Vibrio agent for aquaculture infectious treatment.
Aims: Chromium salt possesses unique characteristics that render it useful in numerous applications in several industrial processes, especially tanning of animal hides which act as a major source of hexavalent chromium toxicity in environment. This study aimed to evaluate the efficiency of loofah immobilized Cladosporium cladosporioides CEL14 in remediate tannery wastewater which contains hexavalent chromium.
Methodology and results: A total of 18 fungal species were isolated from three different sites of tannery wastewater in Egypt, of which C. cladosporioides CEL14 was the most capable species of chromate remediation with 81% after 7 days of incubation as free cells. The experiments were conducted in minimum salt medium supplemented with 200 ppm chromate in the form of potassium dichromate. Different process parameters studies demonstrated that chromate was completely removed at 30 °C, pH 6, 0.1% malt extract and 0.2% glucose after 7 days of incubation with 20% inoculum size. After that, C. cladosporioides was immobilized on a natural support material (loofah). The removal ability of chromate was enhanced through permanent viable immobilization on loofah pieces, which showing complete removal of chromate within 3 days. The toxicity assessment of treated tannery effluents revealed that 74% of Brassica napus seeds were germinated upon exposure to the treated effluent.
Conclusion, significance and impact of study: This study revealed that C. cladosporioides CEL14 isolate has high potential as bioremediating agent against toxic hexavalent chromium. The removal ability of toxic chromate was enhanced through permanent viable immobilization on loofah pieces. This technology is simple, cost effective, efficient and environmentally friendly. The loofah immobilized with C. cladosporioides CEL14 has potential to be applied in wastewater treatment of small-scale tanneries after onsite trials.
Aims: Andrographis paniculata (AP), a medicinal herb was selected to investigate the antifungal activity on selected dermatophyte fungi. The phytochemical screening was also carried out to evaluate its chemical constituents.
Methodology and results: The potato dextrose agar (PDA) incorporated with aqueous, ethanol and methanol AP extracts at concentrations 0.99% (v/v), 1.96% (v/v) and 7.41% (v/v) were used for selected fungi culturing; Trichophyton mentagrophytes, T. rubrum, T. interdigitale, Microsporum fulvum, M. nanum, M. gypseum, M. canis, Fusarium solani and Aspergillus fumigatus. Phytochemical screening showed the presence of flavonoids, saponins and tannins in the ethanol extract and flavonoids alone in both aqueous and methanol extracts. Studies on antifungal effects indicated that the ethanol extract significantly increased the mycelial inhibition percentage of all tested fungi, especially at a concentration of 7.41% (v/v). All ethanol AP extract concentrations inhibited M. gypseum and M. canis (p<0.05) with at least 36.00% mycelial inhibition. In aqueous AP extract, it significantly increased the mycelial inhibition of T. mentagrophytes, T. interdigitale and M. gypseum (p<0.05), while the methanol AP extract significantly inhibited all fungi at a concentration of 7.41% (v/v) except for T. rubrum, M. gypseum and F. solani (p<0.05). No spore sedimentation was recorded for the fungal spores of T. rubrum, M. nanum, T. mentagrophytes, M. gypseum and T. interdigitale at 7.41% (v/v) ethanol AP.
Conclusion, significance and impact of study: It is concluded that the ethanol AP extract contained phytochemical constituents and showed the highest antifungal activity. In addition, this extract has a great potential to treat dermatophytes effectively.
Aims: This study aims to isolate, characterize and screen the plant growth-promoting bacteria from Zingiberaceae plants. Plant promoting activities such as indole-3-acetic acid (IAA), phosphate solubilization, zinc solubilization and nitrogen-fixing capabilities are determined and the IAA production of selected isolates are optimized.
Methodology and results: Endophytic bacteria were isolated from the plant samples by surface sterilization on nutrient agar (NA) plates and incubated at 30 °C for 2–3 days. The bacteria were identified based on their phenotypic characteristics and 16S rRNA gene sequence analyses. All isolates were identified as genera Bacillus, Lysinibacillus, Kerstersia, Klebsiella and Brucella. The isolates exhibited phosphate solubilization (1.5 ± 0.75–37.5 ± 8.75 Solubilization Index, SI), zinc solubilization (2.5 ± 0–60 ± 1.5 SI) and IAA production (0.1 ± 0.2–115.7 ± 1.6 µg/mL), while 3 isolates possessed nitrogen-fixing capabilities. Five isolates (PHAS-2, PWS-2, PWR-2, PHBS-2 and SCG-2) were selected for IAA optimization. Isolate PWR-2 produced the maximum IAA at 447.7 ± 0 µg/mL when tryptophan concentration was maintained at 1.0%.
Conclusion, significance and impact of study: Genera of bacteria included Bacillus, Lysinibacillus, Kerstersia, Klebsiella and Brucella were successfully isolated from Zingiberaceae plants. All the isolates showed the capability to produce IAA, while some isolates exhibited phosphate solubilization and zinc solubilization, and a few possessed nitrogen-fixing capabilities. The potential IAA production isolates could be applied for the enhancement of agricultural production that will be becoming a more widely accepted practice.