Leptospirosis is an emerging zoonotic disease caused by bacterial species of the genus Leptospira. However, the regulatory mechanisms and pathways underlying the adaptation of pathogenic and non-pathogenic Leptospira spp. in different environmental conditions remain elusive. Leptospira biflexa is a non-pathogenic species of Leptospira that lives exclusively in a natural environment. It is an ideal model not only for exploring molecular mechanisms underlying the environmental survival of Leptospira species but also for identifying virulence factors unique to Leptospira's pathogenic species. In this study, we aim to establish the transcription start site (TSS) landscape and the small RNA (sRNA) profile of L. biflexa serovar Patoc grown to exponential and stationary phases via differential RNA-seq (dRNA-seq) and small RNA-seq (sRNA-seq) analyses, respectively. Our dRNA-seq analysis uncovered a total of 2726 TSSs, which are also used to identify other elements, e.g., promoter and untranslated regions (UTRs). Besides, our sRNA-seq analysis revealed a total of 603 sRNA candidates, comprising 16 promoter-associated sRNAs, 184 5'UTR-derived sRNAs, 230 true intergenic sRNAs, 136 5'UTR-antisense sRNAs, and 130 open reading frame (ORF)-antisense sRNAs. In summary, these findings reflect the transcriptional complexity of L. biflexa serovar Patoc under different growth conditions and help to facilitate our understanding of regulatory networks in L. biflexa. To the best of our knowledge, this is the first study reporting the TSS landscape of L. biflexa. The TSS and sRNA landscapes of L. biflexa can also be compared with its pathogenic counterparts, e.g., L. borgpetersenii and L. interrogans, to identify features contributing to their environmental survival and virulence.
With the rise of antibiotic resistance globally, coupled with evolving and emerging infectious diseases, there is an urgent need for the development of novel antimicrobials. Deep eutectic solvents (DES) are a new generation of eutectic mixtures that depict promising attributes with several biological implications. DES exhibit unique properties such as low toxicity, biodegradability, and high thermal stability. Herein, the antimicrobial properties of DES and their mechanisms of action against a range of microorganisms, including bacteria, amoebae, fungi, viruses, and anti-cancer properties are reviewed. Overall, DES represent a promising class of novel antimicrobial agents as well as possessing other important biological attributes, however, future studies on DES are needed to investigate their underlying antimicrobial mechanism, as well as their in vivo effects, for use in the clinic and public at large.
In today's fast-shifting climate change scenario, crops are exposed to environmental pressures, abiotic and biotic stress. Hence, these will affect the production of agricultural products and give rise to a worldwide economic crisis. The increase in world population has exacerbated the situation with increasing food demand. The use of chemical agents is no longer recommended due to adverse effects towards the environment and health. Biocontrol agents (BCAs) and biostimulants, are feasible options for dealing with yield losses induced by plant stresses, which are becoming more intense due to climate change. BCAs and biostimulants have been recommended due to their dual action in reducing both stresses simultaneously. Although protection against biotic stresses falls outside the generally accepted definition of biostimulant, some microbial and non-microbial biostimulants possess the biocontrol function, which helps reduce biotic pressure on crops. The application of synergisms using BCAs and biostimulants to control crop stresses is rarely explored. Currently, a combined application using both agents offer a great alternative to increase the yield and growth of crops while managing stresses. This article provides an overview of crop stresses and plant stress responses, a general knowledge on synergism, mathematical modelling used for synergy evaluation and type of in vitro and in vivo synergy testing, as well as the application of synergism using BCAs and biostimulants in reducing crop stresses. This review will facilitate an understanding of the combined effect of both agents on improving crop yield and growth and reducing stress while also providing an eco-friendly alternative to agroecosystems.
Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal(r) as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10(-3) to 1.0×10(-2)/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.
Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria's interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0-18 h, 2-13 days, 2-18 weeks, and 6-12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.
Enzymes are often required to function in a particular reaction condition by the industrial procedure. In order to identify critical residues affecting the optimum pH of Staphylococcal lipases, chimeric lipases from homologous lipases were generated via a DNA shuffling strategy. Chimeric 1 included mutations of G166S, K212E, T243A, H271Y. Chimeric 2 consisted of substitutions of K212E, T243A, H271Y. Chimeric 3 contained substitutions of K212E, R359L. From the screening results, the pH profiles for chimeric 1 and 2 lipases were shifted from pH 7 to 6. While the pH of chimeric 3 was shifted to 8. It seems the mutation of K212E in chimeric 1 and 2 decreased the pH to 6 by changing the electrostatic potential surface. Furthermore, chimeric 3 showed 10 ˚C improvement in the optimum temperature due to the rigidification of the catalytic loop through the hydrophobic interaction network. Moreover, the substrate specificity of chimeric 1 and 2 was increased towards the longer carbon length chains due to the mutation of T243A adjacent to the lid region through increasing the flexibility of the lid. Current study illustrated that directed evolution successfully modified lipase properties including optimum pH, temperature and substrate specificity through mutations, especially near catalytic and lid regions.
pks+ Escherichia coli (E. coli) triggers genomic instability in normal colon cells which leads to colorectal cancer (CRC) tumorigenesis. Previously, we reported a significant presentation of pks+ E. coli strains in CRC patients' biopsies as compared to healthy cohorts. In this work, using an in vitro infection model, we further explored the ability of these strains in modulating cell cycle arrest and activation of apoptotic mediators in both primary colon epithelial cells (PCE) and CRC cells (HCT-116). Sixteen strains, of which eight tumours and the matching non-malignant tissues, respectively, from eight pks+ E. coli CRC patients were subjected to BrDU staining and cell cycle analysis via flow cytometry, while a subset of these strains underwent analysis of apoptotic mediators including caspase proteins, cellular reactive oxygen species (cROS) and mitochondrial membrane potential (MMP) via spectrophotometry as well as proinflammatory cytokines via flow cytometry. Data revealed that all strains exerted S-phase cell cycle blockade in both cells and G2/M phase in PCE cells only. Moreover, more significant upregulation of Caspase 9, cROS, proinflammatory cytokines and prominent downregulation of MMP were detected in HCT-116 cells indicating the potential role of pks related bacterial toxin as anticancer agent as compared to PCE cells which undergo cellular senescence leading to cell death without apparent upregulation of apoptotic mediators. These findings suggest the existence of discrepancies underlying the mechanism of action of pks+ E. coli on both cancer and normal cell lines. This work propounds the rationale to further understand the mechanism underlying pks+ E. coli-mediated CRC tumorigenesis and cancer killing.
Carotenoids are a diverse group of lipid-soluble pigments that exhibit potent biological activities such as antioxidant, anti-inflammatory, and provitamin A activities. The potent health benefits of carotenoids result in the surge in the market demands for carotenoids, especially natural carotenoids from sustainable sources. Microbial carotenoids have attracted considerable interests for many industrial applications because of the low costs and ease of scaling-up with shorter production time. There is a growing interest in the search of new and sustainable microbial sources and cost-efficient production strategies following the high economical values and vast commercial applications of carotenoids. This article presents a review on the industrial production strategies of microbial carotenoids from microalgae, fungi, and bacteria sources. The industrial significance of the mass production of microbial carotenoids is also discussed. The structure, classification, and biosynthesis pathway of the carotenoids are also presented in this review.
Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.
Palm and palm-kernel oils and their olein and stearin fractions were suitable as the main carbon sources for growth and production of clavulanic acid by Streptomyces clavuligerus. However, oleic and lauric acids were not utilized for growth. A spontaneous mutant, which was selected for higher cephamycin C production, also produced more clavulanic acid with these oils in the medium.
Microbial degradation is considered as an attractive method to eliminate exposure to mycotoxin that cause a serious threat in agriculture global industry and severe human health problems. Compared with other more prominent mycotoxin compounds, fusaric acid (FA) biodegradation has not been widely investigated. In this study, a fusaric acid-degrading bacterium Burkholderia sp. IMCC1007 was identified by 16 S rRNA gene sequencing and its detoxification characteristics were evaluated. This strain able to utilize FA as sole energy and carbon source with growth rate (µ) of 0.18 h- 1. Approximately 93% from the initial substrate FA concentration was almost degraded to the residual about 4.87 mg L- 1 after 12 h of incubation. The optimal degradation conditions for pH and temperature were recorded at 6.0 with 30 °C respectively. An efficient FA degradation of strain IMCC1007 suggested its potential significance to detoxification development. Accroding to LC-MS/Q-TOF analysis, FA was bio-transformed to 4-hydroxybenzoic acid (C7H6O3) and other possible metabolites. Plant treated with detoxified FA products exhibited reduction of wilting index, mitigating against FA phytoxicity effect on plant growth and photosynthesis activity. Phytotoxicity bioassay suggested that degradation product of IMCC1007 was not a potent harmful compound towards plants as compared to the parent compound, FA. As a conslusion, our study provides a new insight into the practical application of biodetoxifcation agent in controlling mycotoxin contamination.
Four types of mycelial extracts were derived from the airlift liquid fermentation (ALF) of Pleurotus flabellatus, namely exopolysaccharide (EX), endopolysaccharide (EN), hot water (WE), and hot alkali (AE) extracts. Such extracts were screened for their active components and biological potential. EN proved to be most effective in inhibition of lipid peroxidation (EC50 = 1.71 ± 0.02 mg/mL) and in Cupric ion reducing antioxidant capacity (CUPRAC) assay (EC50 = 2.91 ± 0.01 mg TE/g). AE exhibited most pronounced ability to chelate ferrous ions (EC50 = 4.96 ± 0.08 mg/mL) and to scavenge ABTS radicals (EC50 = 3.36 ± 0.03 mg TE/g). β-glucans and total phenols contributed most to the chelating ability and quenching of ABTS radicals. Inhibition of lipid peroxidation correlated best with total glucans, total proteins, and β-glucans. Total proteins contributed most to CUPRAC antioxidant capacity. Antifungal effect was determined against Candida albicans ATCC 10231 (MIC: 0.019-0.625 mg/mL; MFC: 0.039-2.5 mg/mL), and towards C. albicans clinical isolate (MIC and MFC: 10.0-20.0 mg/mL). Comparison of cytotoxicity against colorectal carcinoma HCT 116 cells (IC50: 1.8 ± 0.3-24.6 ± 4.2 mg/mL) and normal lung MRC-5 fibroblasts (IC50: 17.0 ± 4.2-42.1 ± 6.1 mg/mL) showed that EN, and especially AE possess selective anticancer activity (SI values 3.41 and 9.44, respectively). Slight genotoxicity was observed only for AE and EX, indicating the low risk concerning this feature. Notable antioxidative and anticandidal activities, selective cytotoxicity against colorectal carcinoma cells, and absence/low genotoxicity pointed out that ALF-cultivated P. flabellatus mycelium could be considered as a valuable source of bioactive substances.
Over two hundred bacteria were isolated from the halosphere, rhizosphere and endophyte of Malaysian maize plantation and screened for phytases activity. Thirty isolates with high detectable phytase activity were chosen for media optimization study and species identification. Ten types of bacterial phytase producers have been discovered in this study, which provides opportunity for characterization of new phytase(s) and various commercial and environmental applications. The majority of the bacterial isolates with high detectable phytase activity were of endophyte origin and 1.6% of the total isolates showed phytase activity of more than 1 U/ml. Most of the strains produced extra-cellular phytase and Staphylococcus lentus ASUIA 279 showed the highest phytase activity of 1.913 U/ml. All 30 species used in media optimization study exhibit favorable enzyme production when 1% rice bran was included in the growth media.
To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.
Mosquitoes are infectious vectors for a wide range of pathogens and parasites thereby transmitting several diseases including malaria, dengue, Zika, Japanese encephalitis and chikungunya which pose a major public health concern. Mostly synthetic insecticides are usually applied as a primary control strategy to manage vector-borne diseases. However excessive and non-judicious usage of such chemically derived insecticides has led to serious environmental and health issues owing to their biomagnification ability and increased toxicity towards non-target organisms. In this context, many such bioactive compounds originating from entomopathogenic microbes serve as an alternative strategy and environmentally benign tool for vector control. In the present paper, the entomopathogenic fungus, Lecanicillium lecanii (LL) was processed to make the granules. Developed 4% LL granules have been characterized using the technique of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The developed formulation was also subjected to an accelerated temperature study at 40 °C and was found to be stable for 3 months. Further, GCMS of the L. lecanii was also performed to screen the potential biomolecules present. The developed formulation was found to be lethal against Anopheles culicifacies with an LC50 value of 11.836 µg/mL. The findings from SEM and histopathology also substantiated the mortality effects. Further, the SEM EDX (energy dispersive X-ray) studies revealed that the treated larvae have lower nitrogen content which is correlated to a lower level of chitin whereas the control ones has higher chitin content and healthy membranes. The developed LL granule formulation exhibited high toxicity against Anopheles mosquitoes. The granule formulations can be used as an effective biocontrol strategy against malaria-causing mosquitoes.
Pleurotus citrinopileatus (yellow oyster mushroom) has an attractive shape and yellow colour but the fragile texture complicates packaging, and its strong aroma is unappealing to consumers. This study aimed to improve the characteristics and yield of P. citrinopileatus by interspecies mating between monokaryotic cultures of P. citrinopileatus and P. pulmonarius. Ten monokaryon cultures of the parental lines were crossed in all combinations to obtain hybrids. Eleven compatible mating pairs were obtained and cultivated to observe their sporophore morphology and yield. The selected hybrid, i.e. P1xC9, was beige in colour while hybrid P3xC8 was yellow in colour. Their sporophores had less offensive aroma, improved texture and higher yield. The DNA sequences of these hybrids were found to be in the same clade as the P. citrinopileatus parent with a bootstrap value of 99%. High bootstrap values indicate high genetic homology between hybrids and the P. citrinopileatus parent. The biological efficiencies of these hybrids P1xC9 (70.97%) and P3xC8 (52.14%) were also higher than the P. citrinopileatus parent (35.63%). Interspecies hybrids obtained by this mating technique can lead to better strains of mushrooms for genetic improvement of the Pleurotus species.
In a previous study, notable differences of several physicochemical properties, as well as the community structure of ammonia oxidizing bacteria as judged by 16S rRNA gene analysis, were observed among several disused tin-mining ponds located in the town of Kampar, Malaysia. These variations were associated with the presence of aquatic vegetation as well as past secondary activities that occurred at the ponds. Here, methane oxidizing bacteria (MOB), which are direct participants in the nutrient cycles of aquatic environments and biological indicators of environmental variations, have been characterised via analysis of pmoA functional genes in the same environments. The MOB communities associated with disused tin-mining ponds that were exposed to varying secondary activities were examined in comparison to those in ponds that were left to nature. Comparing the sequence and phylogenetic analysis of the pmoA clone libraries at the different ponds (idle, lotus-cultivated and post-aquaculture), we found pmoA genes indicating the presence of type I and type II MOB at all study sites, but type Ib sequences affiliated with the Methylococcus/Methylocaldum lineage were most ubiquitous (46.7 % of clones). Based on rarefaction analysis and diversity indices, the disused mining pond with lotus culture was observed to harbor the highest richness of MOB. However, varying secondary activity or sample type did not show a strong variation in community patterns as compared to the ammonia oxidizers in our previous study.
Disused tin-mining ponds make up a significant amount of water bodies in Malaysia particularly at the Kinta Valley in the state of Perak where tin-mining activities were the most extensive, and these abundantly available water sources are widely used in the field of aquaculture and agriculture. However, the natural ecology and physicochemical conditions of these ponds, many of which have been altered due to secondary post-mining activities, remains to be explored. As ammonia-oxidizing bacteria (AOB) are directly related to the nutrient cycles of aquatic environments and are useful bioindicators of environmental variations, the focus of this study was to identify AOBs associated with disused tin-mining ponds that have a history of different secondary activities in comparison to ponds which were left untouched and remained as part of the landscape. The 16S rDNA gene was used to detect AOBs in the sediment and water sampled from the three types of disused mining ponds, namely ponds without secondary activity, ponds that were used for lotus cultivation and post-aquaculture ponds. When the varying pond types were compared with the sequence and phylogenetic analysis of the AOB clone libraries, both Nitrosomonas and Nitrosospira-like AOB were detected though Nitrosospira spp. was seen to be the most ubiquitous AOB as it was present in all ponds types. However, AOBs were not detected in the sediments of idle ponds. Based on rarefaction analysis and diversity indices, the disused mining pond with lotus culture indicated the highest richness of AOBs. Canonical correspondence analysis indicated that among the physicochemical properties of the pond sites, TAN and nitrite were shown to be the main factors that influenced the community structure of AOBs in these disused tin-mining ponds.
Bud rot disease is a damaging disease of oil palm in Colombia. The pathogen responsible for this disease is a species of oomyctes, Phytophthora palmivora which is also the causal pathogen of several tropical crop diseases such as fruit rot and stem canker of cocoa, rubber, durian and jackfruit. No outbreaks of bud rot have been reported in oil palm in Malaysia or other Southeast Asian countries, despite this particular species being present in the region. Analysis of the genomic sequences of several genetic markers; the internal transcribe spacer regions (ITS) of the ribosomal RNA gene cluster, beta-tubulin gene, translation elongation factor 1 alpha gene (EF-1α), cytochrome c oxidase subunit I & II (COXI and COXII) gene cluster along with amplified fragment length polymorphism (AFLP) analyses have been carried out to investigate the genetic diversity and variation of P. palmivora isolates from around the world and from different hosts in comparison to Colombian oil palm isolates, as one of the steps in understanding why this species of oomycetes causes devastating damage to oil palm in Latin America but not in other regions. Phylogenetic analyses of these regions showed that the Colombian oil palm isolates were not separated from Malaysian isolates. AFLP analysis and a new marker PPHPAV, targeting an unclassified hypothetical protein, was found to be able to differentiate Malaysian and Colombian isolates and showed a clear clade separations. Despite this, pathogenicity studies did not show any significant differences in the level of aggressiveness of different isolates against oil palm in glasshouse tests.