Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely, CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterization of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differs significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterization based on ancestral spacer (AS) and TR sequences indicates that E. faecalis with the same CRISPR1-AS genotype do not always harbour the same CRISPR2-AS genotypes and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to MLST, suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in Southeast Asia.

Acinetobacter baumannii carrying the bla NDM-1 gene, which encodes New Delhi metallo-β-lactamase-1 (NDM-1), exhibits resistance to nearly all β-lactams and is not affected by β-lactamase inhibitors, limiting treatment options. The bla NDM-1 gene is often associated with other antimicrobial resistance (AMR) genes, resulting in multidrug-resistant (MDR) phenotypes. We previously reported a large, circa (ca.) 170 kb plasmid co-harbouring bla NDM-1- and bla OXA-58-encoded carbapenemases in clinical MDR Acinetobacter nosocomialis and A. baumannii isolates from a tertiary hospital in Terengganu, Malaysia, in 2015 and 2016, respectively. In this study, we identified four additional MDR A. baumannii isolates from the same hospital (2018-2020) carrying bla NDM-1 on a similar plasmid. Complete genome sequences were obtained using a hybrid assembly of short-read DNA Nanoball Sequencing(DNBSeq) and long-read (Oxford Nanopore Technologies) data. All isolates belonged to distinct, unrelated clonal lineages, indicating the ongoing horizontal transmission of this plasmid. Comparative analysis revealed substantial structural variability among the plasmids, largely driven by insertion sequence elements and mobile xrs (also known as pdif) modules. In two A. baumannii isolates (i.e. AC1932 and AC2014), bla OXA-58 and several adjacent xrs modules were absent. Despite this, carbapenem minimum inhibitory concentrations remained comparable across all isolates, strongly indicating that bla NDM-1 is the primary carbapenem resistance determinant. Notably, in A. baumannii AC1839, we identified a 15,434 bp presumptive transposon carrying a type III cyclic oligonucleotide-based anti-phage signalling system, inserted within the xrs-rich region of the plasmid. AC1839 also carried a separate 11.1 kp plasmid carrying the tetracycline resistance genes tetA(39)-tetR within an xrs module. Identical xrs modules were identified in other unrelated Acinetobacter plasmids. These results underscore the mobility and potential roles of xrs modules in AMR gene dissemination and, more generally, in shaping Acinetobacter plasmid evolution.

The taxonomic classification of a falcon population found in the Mongolian Altai region in Asia has been heavily debated for two centuries and previous studies have been inconclusive, hindering a more informed conservation approach. Here, we generated a chromosome-level gyrfalcon reference genome using the Vertebrate Genomes Project (VGP) assembly pipeline. Using whole genome sequences of 49 falcons from different species and populations, including “Altai” falcons, we analyzed their population structure, admixture patterns, and demographic history. We find that the Altai falcons are genomic mosaics of saker and gyrfalcon ancestries, and carry distinct W and mitochondrial haplotypes that cluster with the lanner falcon. The Altai maternally-inherited haplotypes diverged 422,000 years before present (290,000–550,000 YBP) from the ancestor of sakers and gyrfalcons, both of which, in turn, split 109,000 YBP (70,000–150,000 YBP). The Altai W chromosome has 31 coding variants in 29 genes that may possibly influence important structural, behavioral, and reproductive traits. These findings provide insights into the question of Altai falcons as a candidate distinct species.

Acinetobacter baumannii is an important nosocomial pathogen causing high infections and morbidity among affected individuals and most studies focus on nosocomial strains. However, A. baumannii can also be isolated from community healthy individuals. This study compared the pathogenicity of hospital and community A. baumannii isolates using Galleria mellonella and human cell cultures. The insect model, G. mellonella and in vitro HeLa cell line were used with ten A. baumannii isolates (six community and four hospital isolates from Segamat, Malaysia). G. mellonella killing assays and HeLa cell adherence, invasion, and cytotoxicity assays were performed to investigate the virulence and invasion potential of the isolates. Out of the ten isolates investigated, three community and two hospital isolates were found to be highly virulent in G. mellonella infection model, killing 100 % larvae within 96 hours. These strains were also found to be invasive and have significant cytotoxicity in HeLa cells. Our study revealed that community and hospital isolated A. baumannii could be equally virulent judged by both model systems. Undoubtedly, besides hospital settings, the presence of highly virulent A. baumannii in community reservoirs poses a significant public health risk and requires additional investigation.

Acinetobacter baumannii, a top-priority WHO pathogen, causes life-threatening infections in immunocompromised patients, leading to prolonged hospitalisation and high mortality. Here, we used the Galleria mellonella model to investigate community strain C98 (Ab-C98) virulence via transcriptomic analysis. Ab-C98 showed greater killing and faster colonisation in larvae than the clinical reference strain (ATCC BAA1605). Genes in three iron clusters, acinetobactin, baumannoferrin and the Feo system, were significantly up-regulated. Targeted knockout of siderophore genes (basC, bfnD, and the gene encoding isochorismatase) significantly increased the survival of infected larvae by at least 35.16%, identifying these genes as potential targets for developing anti-virulence agents against A. baumannii.

Despite being a major human pathogen, limited studies have reported RNA modifications in Acinetobacter baumannii. These post-transcriptional modifications play crucial regulatory roles in bacteria and have also been shown to modulate bacterial virulence. Using nanopore sequencing, we characterized RNA modifications in a virulent A. baumannii strain (Ab-C98) under free-living (mid-exponential phase in vitro culture) and during an early stage of infection (3 h post-infection) in Galleria mellonella larvae. Analysis revealed that m5C methylations are essential for ribosome synthesis, while m6A and Ψ are involved in metabolic pathways and translation processes. Iron-chelating genes exbD (m5C and m6A) and feoB (m6A and Ψ) and RNA polymerase subunit rpoC (m6A and Ψ) were selectively modified during infection. This first transcriptome-wide study highlights the potential regulatory roles of m5C, m6A and Ψ modifications in A. baumannii during infection.

COVID19 genomic surveillance is instrumental to better understand transmission dynamics in a setting, detect emergence of new variants and monitor spread of variants at the national, regional and global levels. Complete viral genome sequences are powerful enough to approximate epidemiology and enable informed public health response policies and determine their success. Between 24th November to 9th December 2020, a workplace COVID-19 outbreak, assigned as the Hilir cluster, occurred among healthcare workers (HCWs) in a northeast Malaysian university teaching hospital that was not designated for COVID-19 treatment. Mass screening of 1,292 individuals based on case interviews, contact tracings and nucleic acid testing detected 17 cases from various hospital wards and units. To investigate how COVID19 transmission occurred we whole genome sequenced 14 samples collected from healthcare workers and 5 samples from the concurrent community outbreaks. The genomes of these samples were compared with closely-related publically available genomes from GISAID to gain insights into COVID19 transmission in the hospital and at the local and global scale. The 14 viral sequences obtained from the Hilir cluster were assigned to Pango lineages B.1.524 (7 samples) and B.1.36.16 (7 samples) whereas the community samples were assigned as either B.1.524 and B.1. Phylogenetics revealed multiple introduction of B.1.524 into the workplace, while close relatedness of all B.1.36.16 samples suggested that the introduction of these lineages into the workplace likely stemmed from a single introduction. These lines of genomic evidences contradicted with the proposed transmission route, underlining the central role of genomics in COVID-19 or any future pandemics surveillance. The study also highlight the difficulty in enforcing and maintaining isolation methods in a hospital setting.

Weedy rice is a major problem in paddy fields around the world. It is well known that weedy rice appears to grow faster and mature earlier than cultivated rice. It is possible that differences in the root microbial genetics are correlated with this characteristic. This study incorporated 16S rRNA amplicon sequencing to study the microbial composition in the rhizosphere and endosphere of rice root. No significant difference was found between the microbiota associated with weedy and cultivated rice lines grown in the same field. It was found that the endosphere had less microbial diversity compared to the rhizosphere. The major groups of bacteria found in the endosphere are from the phylum Proteobacteria, Myxococcota, Chloroflexota, and Actinobacteria. In addition, by analyzing the microbiome of japonica rice grown in the field in a temperate climate, we found that despite differences in genotype and location, some bacterial taxa were found to be common and these members of the putative rice core microbiome can also be detected by in situ hybridization. The delineation of a core microbiome in the endosphere of rice suggests that these bacterial taxa might be important in the life cycle of a wide range of rice types.

Background Rice (Oryza sativa) seed lipids contain about 25% saturated fatty acids, dominated by palmitic acid. Reduction of saturated fat consumption is recommended for health concerns and so reducing palmitic acid content is of interest for the rice bran oil industry. In higher plants, palmitoyl-acyl carrier protein thioesterase (FatB) controls the release of palmitic acid from acyl carrier protein and its accumulation in oil. Results In this study, four rice OsFatB genes were identified, and their expression patterns were analyzed in seed by the reverse-transcription quantitative PCR. Two types of rice lines were obtained with down-regulated OsFatB expression: an OsFatB1 knockout mutant obtained via Tos17 insertion and transgenic rice lines in which expression of the four OsFatB genes using RNAi technology. Both types exhibited partial reductions of palmitic acid content (11% and 21–55%, respectively) and a slight reduction in myristic acid content. Increased oleic and linoleic acid content was observed in the transgenic rice lines but not in the Tos17 insertion line. Conclusion These results demonstrated that the palmitic acid content is controlled by multiple OsFatBs, and the contribution of OsFatB1 is limited.

Acinetobacter baumannii is a common cause of multidrug-resistant (MDR) nosocomial infections around the world. However, little is known about the persistence and dynamics of A. baumannii in a healthy community. This study investigated the role of the community as a prospective reservoir for A. baumannii and explored possible links between hospital and community isolates. A total of 12 independent A. baumannii strains were isolated from human faecal samples from the community in Segamat, Malaysia, in 2018 and 2019. Another 15 were obtained in 2020 from patients at the co-located tertiary public hospital. The antimicrobial resistance profile and biofilm formation ability were analysed, and the relatedness of community and hospital isolates was determined using whole-genome sequencing (WGS). Antibiotic profile analysis revealed that 12 out of 15 hospital isolates were MDR, but none of the community isolates were MDR. However, phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) and a pangenome analysis of core genes showed clustering between four community and two hospital strains. Such clustering of strains from two different settings based on their genomes suggests that these strains could persist in both. WGS revealed 41 potential resistance genes on average in the hospital strains, but fewer (n=32) were detected in the community strains. In contrast, 68 virulence genes were commonly seen in strains from both sources. This study highlights the possible transmission threat to public health posed by virulent A. baumannii present in the gut of asymptomatic individuals in the community.

Durian ( Durio zibethinus Murr.) fruits are famous for their unique aroma. This study analysed the Durian fruit transcriptome to discover the expression patterns of genes and to understand their regulation. Three developmental stages of Durian fruit, namely, early [90 days post-anthesis (DPA)], mature (120 DPA), and ripen (127 DPA), were studied. The Illumina HiSeq platform was used for sequencing. The sequence data were analysed using four different mapping aligners and statistical methods: CLC Genomic Workbench, HISAT2+DESeq2, Tophat+Cufflinks, and HISAT2+edgeR. The analyses showed that over 110 million clean reads were mapped to the Durian genome, yielding 19,976, 11,394, 17,833, and 24,351 differentially expressed genes during 90-127 days post-anthesis. Many identified differentially expressed genes were linked to the fruit ripening processes. The data analysis suggests that most genes with increased expression at the ripening stage were primarily involved in the metabolism of cofactors and vitamins, nucleotide metabolism, and carbohydrate metabolism. Significantly expressed genes from the young to mature stage were mainly associated with carbohydrate metabolism, amino acid metabolism, and cofactor and vitamin metabolism. The transcriptome data will serve as a foundation for understanding Durian fruit development-specific genes and could be helpful in fruit’s trait improvement.

Galleria mellonella larvae have been increasingly used in research, including microbial infection studies. They act as suitable preliminary infection models to study host-pathogen interactions due to their advantages, such as the ability to survive at 37°C mimicking human body temperature, their immune system shares similarities with mammalian immune systems, and their short life cycle allowing large-scale studies. Here, we present a protocol for simple rearing and maintenance of G. mellonella without requiring special instruments and specialized training. This allows the continuous supply of healthy G. mellonella for research purposes. Besides, this protocol also provides detailed procedures on the (i) G. mellonella infection assays (killing assay and bacterial burden assay) for virulence studies and (ii) bacterial cell harvesting from infected larvae and RNA extraction for bacterial gene expression studies during infection. Our protocol could not only be used in the studies of A. baumannii virulence but can also be modified according to different bacterial strains.

Acinetobacter species are widely known opportunistic pathogens causing severe community and healthcare-associated infections. One such emerging pathogen, Acinetobacter colistiniresistens, is known to exhibit intrinsic resistance to colistin. We investigated the molecular characteristics of A. colistiniresistens strain C-214, isolated from the fecal sample of a healthy community member, as part of a cohort study being conducted in Segamat, Malaysia. Comparison of the whole genome sequence of C-214 with other A. colistiniresistens sequences retrieved from the NCBI database showed 95% sequence identity or more with many of the genome sequences representing that species. Use of the Galleria mellonella killing assay showed that C-214 was pathogenic in this model infection system. The strain C-214 had a colistin and polymyxin B MIC of 32 and 16 mg/L, respectively. Besides, it was resistant to cefotaxime, amikacin, and tetracycline and showed moderate biofilm-producing ability. Different genes associated with virulence or resistance to major classes of antibiotics were detected. We observed mutations in lpxA/C/D in C-214 and other A. colistiniresistens strains as probable causes of colistin resistance, but the biological effects of these mutations require further investigation. This study provides genomic insights into A. colistiniresistens, a potentially pathogenic bacterium isolated from a community member and notes the public health threat it may pose.

The prevalence of antibiotic-resistant bacteria in Southeast Asia is a significant concern, yet there is limited research on the gut resistome and its correlation with lifestyle and environmental factors in the region. This study aimed to profile the gut resistome of 200 individuals in Malaysia using shotgun metagenomic sequencing and investigate its association with questionnaire data comprising demographic and lifestyle variables. A total of 1038 antibiotic resistance genes from 26 classes were detected with a mean carriage rate of 1.74 ± 1.18 gene copies per cell per person. Correlation analysis identified 14 environmental factors, including hygiene habits, health parameters, and intestinal colonization, that were significantly associated with the resistome (adjusted multivariate PERMANOVA, p < 0.05). Notably, individuals with positive yeast cultures exhibited a reduced copy number of 15 antibiotic resistance genes. Network analysis highlighted Escherichia coli as a major resistome network hub, with a positive correlation to 36 antibiotic-resistance genes. Our findings suggest that E. coli may play a pivotal role in shaping the resistome dynamics in Segamat, Malaysia, and its abundance is strongly associated with the community’s health and lifestyle habits. Furthermore, the presence of yeast appears to be associated with the suppression of antibiotic-resistance genes.

Urban-dwelling birds can be useful biomonitors to assess the impact of the urbanisation on both public and wildlife health. Widely distributed urban bird species, the House crow, was studied for heavy metal accumulation levels from nine cities of South Asia, Southeast Asia and Africa that border the Indian Ocean. Feathers were spectroscopically investigated for the deposition of ten heavy metals, i.e. As, Zn, Pb, Cd, Ni, iron Fe, Mn, Cr, Cu and Li. Fe and Zn were found to be the most prevalent metals in all sites. Measured concentrations of Pb (4.38–14.77 mg kg −1 ) overall, and Fe (935.66 mg kg −1 ) and Cu (67.17 mg kg −1 ) at some studied sites were above the toxicity levels reported lethal in avian toxicological studies. Multivariate analysis and linear models supported geographical location as a significant predictor for the level of most of the metals. Zn and Cu, generally and Pb, Cd, Mn, Cr at some sites exhibited potential bioaccumulation from surrounding environments. Inter-species comparisons strengthen the inference that the House crow is a reliable bioindicator species for the qualitative assessment of local urban environmental pollution and could be a useful tool for inter-regional monitoring programs.

Fogging with insecticides is one of the main control measures for adult mosquito populations employed in countries that are affected by dengue. In many such countries, urban communities are increasingly characterised by high-density residence in high-rise condominia. Although fogging is typically applied at the ground level, its efficacy in three-dimensional urban environments is poorly understood. Here, we investigated the effect of fogging on vector mosquito distribution and abundance in high-rise condominia by conducting a before-after fogging survey. We showed that although mosquitoes were significantly concentrated at the lower levels in high-rise condominia, they were found throughout the three-dimensional environments. Fogging did not significantly alter this distribution or abundance pattern across any floor level. Thus, any fogging effect was short-lived as mosquito populations recovered within a few days before the subsequent scheduled treatment. In addition, increasing fogging frequency within practicable limits did not prolong the intended control effect. As urban mosquitoes are increasingly insusceptible to fogging due to insecticide resistance and vertical avoidance, this study demonstrates the need to implement other mosquito control strategies for high-rise condominia to manage mosquito populations.

The taxonomic classification of a falcon population found in the Altai region in Asia has been heavily debated for two centuries and previous studies have been inconclusive, hindering a more informed conservation approach. Here, we generated a chromosome-level gyrfalcon reference genome using the Vertebrate Genomes Project (VGP) assembly pipeline. Using whole genome sequences of 44 falcons from different species and populations, including “Altai” falcons, we analyzed their population structure, admixture patterns and demographic history. We find that the Altai falcons are genomically mosaic of saker and gyrfalcon ancestries, and carry distinct W- and mitochondrial-haplotypes that cluster with the lanner falcon. The Altai haplotype diverged 422,000 years ago from the ancestor of sakers and gyrfalcons, both of which, in turn, split 109,000 years ago. The Altai W chromosome includes coding variants that may influence important structural, behavioral and reproductive traits. These findings support the designation of Altai falcons as a distinct falcon species ( Falco altaicus ).

Melissa officinalis L. (lemon balm) is a renowned medical herb enriched with phenolic compounds, particularly rosmarinic acid (RA) which is known for its antimicrobial and antioxidant activities. This study aimed at improving the medicinal characteristics of M. officinalis by enhancing the production of the total phenolic content (TPC) and RA as well as evaluating the activities of RA-regulatory enzymes, following a series of light treatments, using i) three different spectrally tuned light treatments, ii) white light-emitting diode (LED), and iii) sunlight. Phytochemical analyses revealed that the blue-enriched light treatment (40% red, 35% blue, 15% green and 10% far-red) significantly elevated the TPC, RA, and antioxidant capacity of M. officinalis. Additionally, the application of spectrally tunable LEDs also induced the malondialdehyde level and sugar concentration with minimal effect on the chlorophyll content and antimicrobial activity. Increased activities of RA-regulatory enzymes, namely phenylalanine ammonia-lyase, tyrosine aminotransferase, and hydroxyphenylpyruvate reductase, were associated with RA production. These findings suggest that the elicitation based on spectrally tunable LEDs has promising potential for promoting phenolic compounds production in M. officinalis.

Objectives This study profiled the prevalence of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) in the community and compared their resistome and genomic profiles with isolates from clinical patients through whole-genome sequencing. Methods Fecal samples from 233 community dwellers from Segamat, a town in southern Malaysia, were obtained between May through August 2018. Putative ESBL strains were screened and tested using antibiotic susceptibility tests. Additionally, eight clinical ESBL-EC were obtained from a hospital in the same district between June through October 2020. Whole-genome sequencing was then conducted on selected ESBL-EC from both settings (n = 40) for pan-genome comparison, cluster analysis, and resistome profiling. Results A mean ESBL-EC carriage rate of 17.82% (95% CI: 10.48%– 24.11%) was observed in the community and was consistent across demographic factors. Whole-genome sequences of the ESBL-EC (n = 40) enabled the detection of multiple plasmid replicon groups (n = 28), resistance genes (n = 34) and virulence factors (n = 335), with no significant difference in the number of genes carried between the community and clinical isolates (plasmid replicon groups, p = 0.13; resistance genes, p = 0.47; virulence factors, p = 0.94). Virulence gene marker analysis detected the presence of extraintestinal pathogenic E. coli (ExPEC), uropathogenic E. coli (UPEC), and enteroaggregative E. coli (EAEC) in both the community and clinical isolates. Multiple blaCTX-M variants were observed, dominated by blaCTX-M-27 (n = 12), blaCTX-M-65 (n = 10), and blaCTX-M-15 (n = 9). The clinical and community isolates did not cluster together based on the pan-genome comparison, suggesting isolates from the two settings were clonally unrelated. However, cluster analysis based on carried plasmids, resistance genes and phenotypic susceptibility profiles identified four distinct clusters, with similar patterns between the community and clinical isolates. Conclusion ESBL-EC from the clinical and community settings shared similar resistome profiles, suggesting the frequent exchange of genetic materials through horizontal gene transfer.

The human gut contains a complex microbiota dominated by bacteriophages but also containing other viruses and bacteria and fungi. There are a growing number of techniques for the extraction, sequencing, and analysis of the virome but currently no standardized protocols. This study established an effective workflow for virome analysis to investigate the virome of stool samples from two understudied ethnic groups from Malaysia: the Jakun and Jehai Orang Asli. By using the virome extraction and analysis workflow with the Oxford Nanopore Technology, long-read sequencing successfully captured close to full-length viral genomes. The virome composition of the two indigenous Malaysian communities were remarkably different from those found in other parts of the world. Additionally, plant viruses found in the viromes of these individuals were attributed to traditional food-seeking methods. This study establishes a human gut virome workflow and extends insights into the healthy human gut virome, laying the groundwork for comparative studies.