Mycobacteroides immunogenum is an emerging opportunistic pathogen implicated in nosocomial infections. Comparative genome analyses may provide better insights into its genomic structure, functions and evolution. The present analysis showed that M. immunogenum has an open pan-genome. Approximately 36.8% of putative virulence genes were identified in the accessory regions of M. immunogenum. Phylogenetic analyses revealed two potential novel subspecies of M. immunogenum, supported by evidence from ANIb (average nucleotide identity using blast) and GGDC (Genome to Genome Distance Calculator) analyses. We identified 74 genomic islands (GIs) in Subspecies 1 and 23 GIs in Subspecies 2. All Subspecies 2-harboured GIs were not found in Subspecies 1, indicating that they might have been acquired by Subspecies 2 after their divergence. Subspecies 2 has more defence genes than Subspecies 1, suggesting that it might be more resistant to the insertion of foreign DNA and probably explaining why Subspecies 2 has fewer GIs. Positive selection analysis suggest that M. immunogenum has a lower selection pressure compared to non-pathogenic mycobacteria. Thirteen genes were positively selected and many were involved in virulence.
Burkholderia pseudomallei, a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei, but not Burkholderia thailandensis. Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.
Tuberculosis is a leading public health priority in eastern Malaysia. Knowledge of the genomic epidemiology of tuberculosis can help tailor public health interventions. Our aims were to determine tuberculosis genomic epidemiology and characterize resistance mutations in the ethnically diverse city of Kota Kinabalu, Sabah, located at the nexus of Malaysia, Indonesia, Philippines and Brunei. We used an archive of prospectively collected Mycobacterium tuberculosis samples paired with epidemiological data. We collected sputum and demographic data from consecutive consenting outpatients with pulmonary tuberculosis at the largest tuberculosis clinic from 2012 to 2014, and selected samples from tuberculosis inpatients from the tertiary referral centre during 2012-2014 and 2016-2017. Two hundred and eight M. tuberculosis sequences were available for analysis, representing 8 % of cases notified during the study periods. Whole-genome phylogenetic analysis demonstrated that most strains were lineage 1 (195/208, 93.8 %), with the remainder being lineages 2 (8/208, 3.8 %) or 4 (5/208, 2.4 %). Lineages or sub-lineages were not associated with patient ethnicity. The lineage 1 strains were diverse, with sub-lineage 1.2.1 being dominant (192, 98 %). Lineage 1.2.1.3 isolates were geographically most widely distributed. The greatest diversity occurred in a border town sub-district. The time to the most recent common ancestor for the three major lineage 1.2.1 clades was estimated to be the year 1966 (95 % HPD 1948-1976). An association was found between failure of culture conversion by week 8 of treatment and infection with lineage 2 (4/6, 67 %) compared with lineage 1 strains (4/83, 5 %) (P<0.001), supporting evidence of greater virulence of lineage 2 strains. Eleven potential transmission clusters (SNP difference ≤12) were identified; at least five included people living in different sub-districts. Some linked cases spanned the whole 4-year study period. One cluster involved a multidrug-resistant tuberculosis strain matching a drug-susceptible strain from 3 years earlier. Drug resistance mutations were uncommon, but revealed one phenotype-genotype mismatch in a genotypically multidrug-resistant isolate, and rare nonsense mutations within the katG gene in two isolates. Consistent with the regionally mobile population, M. tuberculosis strains in Kota Kinabalu were diverse, although several lineage 1 strains dominated and were locally well established. Transmission clusters - uncommonly identified, likely attributable to incomplete sampling - showed clustering occurring across the community, not confined to households or sub-districts. The findings indicate that public health priorities should include active case finding and early institution of tuberculosis management in mobile populations, while there is a need to upscale effective contact investigation beyond households to include other contacts within social networks.
The virulence of Vibrio parahaemolyticus is variable depending on its virulence determinants. A V. parahaemolyticus strain, in which the virulence is governed by the pirA and pirB genes, can cause acute hepatopancreatic necrosis disease (AHPND) in shrimps. Some V. parahaemolyticus that are non-AHPND strains also cause shrimp diseases and result in huge economic losses, while their pathogenicity and pathogenesis remain unclear. In this study, a non-AHPND V. parahaemolyticus, TJA114, was isolated from diseased Penaeus vannamei associated with a high mortality. To understand its virulence and adaptation to the external environment, whole-genome sequencing of this isolate was conducted, and its phenotypic profiles including pathogenicity, growth characteristics and nutritional requirements were investigated. Shrimps following artificial infection with this isolate presented similar clinical symptoms to the naturally diseased ones and generated obvious pathological lesions. The growth characteristics indicated that the isolate TJA114 could grow well under different salinity (10-55 p.p.t.), temperature (23-37 °C) and pH (6-10) conditions. Phenotype MicroArray results showed that this isolate could utilize a variety of carbon sources, amino acids and a range of substrates to help itself adapt to the high hyperosmotic and alkaline environments. Antimicrobial-susceptibility test showed that it was a multidrug-resistant bacterium. The whole-genomic analysis showed that this V. parahaemolyticus possessed many important functional genes associated with multidrug resistance, stress response, adhesions, haemolysis, putative secreted proteases, dedicated protein secretion systems and a variety of nutritional metabolic mechanisms. These annotated functional genes were confirmed by the phenotypic profiles. The results in this study indicated that this V. parahaemolyticus isolate possesses a high pathogenicity and strong environmental adaptability.
Burkholderia pseudomallei is a Gram-negative soil-dwelling bacillus that causes melioidosis, a frequently fatal infectious disease, in tropical and subtropical regions. Previous studies have identified the overall genetic and evolutionary characteristics of B. pseudomallei on a global scale, including its origin and transmission routes. However, beyond its known hyperendemicity foci in northern Australia and Southeast Asia, the distribution and genetic characteristics of B. pseudomallei in most tropical regions remain poorly understood, including in southern China. Here, we sequenced the genomes of 122 B. pseudomallei strains collected from Hainan, an island in southern China, in 2002-2018, to investigate the population structure, relationships with global strains, local epidemiology, and virulence and antimicrobial-resistance factors. A phylogenetic analysis and hierarchical clustering divided the Hainan strains into nine phylogenic groups (PGs), 80 % of which were concentrated within five major groups (group 1: corresponding to minor sequence types [STs], 12.3 %; group 3: ST46 and ST50, 31.1 %; group 9: ST58, 13.1 %; group 11: ST55, 8.2 %; group 15: mainly ST658, 15.6%). A phylogenetic analysis that included global strains suggested that B. pseudomallei in Hainan originated from Southeast Asian countries, transmitted in multiple historical importation events. We also identified several mutual transmission events between Hainan and Southeast Asian countries in recent years, including three importation events from Thailand and Singapore to Hainan and three exportation events from Hainan to Singapore, Malaysia, and Taiwan island. A statistical analysis of the temporal distribution showed that the Hainan strains of groups 3, 9, and 15 have dominated the disease epidemic locally in the last 5 years. The spatial distribution of the Hainan strains demonstrated that some PGs are distributed in different cities on Hainan island, and by combining phylogenic and geographic distribution information, we detected 21 between-city transmission events, indicating its frequent local transmission. The detection of virulence factor genes showed that 56 % of the Hainan strains in group 1 encode a B. pseudomallei-specific adherence factor, boaB, confirming the specific pathogenic characteristics of the Hainan strains in group 1. An analysis of the antimicrobial-resistance potential of B. pseudomallei showed that various kinds of alterations were identified in clinically relevant antibiotic resistance factors, such as AmrR, PenA and PBP3, etc. Our results clarify the population structure, local epidemiology, and pathogenic characteristics of B. pseudomallei in Hainan, providing further insight into its regional and global transmission networks and improving our knowledge of its global phylogeography.
Burkholderia pseudomallei, a Gram-negative pathogen, is the causative agent of melioidosis in humans. This bacterium can be isolated from the soil, stagnant and salt-water bodies, and human and animal clinical specimens. While extensive studies have contributed to our understanding of B. pseudomallei pathogenesis, little is known about how a harmless soil bacterium adapts when it shifts to a human host and exhibits its virulence. The bacterium's large genome encodes an array of factors that support the pathogen's ability to survive under stressful conditions, including the host's internal milieu. In this study, we performed comparative transcriptome analysis of B. pseudomallei cultured in human plasma versus soil extract media to provide insights into B. pseudomallei gene expression that governs bacterial adaptation and infectivity in the host. A total of 455 genes were differentially regulated; genes upregulated in B. pseudomallei grown in human plasma are involved in energy metabolism and cellular processes, whilst the downregulated genes mostly include fatty acid and phospholipid metabolism, amino acid biosynthesis and regulatory function proteins. Further analysis identified a significant upregulation of biofilm-related genes in plasma, which was validated using the biofilm-forming assay and scanning electron microscopy. In addition, genes encoding known virulence factors such as capsular polysaccharide and flagella were also overexpressed, suggesting an overall enhancement of B. pseudomallei virulence potential when present in human plasma. This ex vivo gene expression profile provides comprehensive information on B. pseudomallei's adaptation when shifted from the environment to the host. The induction of biofilm formation under host conditions may explain the difficulty in treating septic melioidosis.
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.
Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia-positive and Borrelia-negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis, Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella-like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.
While fleas are often perceived simply as a biting nuisance and a cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat (Ctenocephalides felis) and dog (Ctenocephalides canis) fleas, as well as Ctenocephalides orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp., including Rickettsia felis (agent of flea-borne spotted fever) and Rickettsia asembonensis , a suspected pathogen. These Rickettsia spp. are members of a phylogenetic clade known as the ‘transitional group’, which includes both human pathogens and arthropod-specific endosymbionts. The relatively depauperate flea microbiome can also contain other endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis-derived pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain (wCori), and the C. orientis mitochondrion; all were obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which (wCfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades (‘supergroups’), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the USA. Analysis of loci under positive selection in the transitional group revealed genes involved in host–pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited large-scale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides-associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.
Ethnicity is consistently reported as a strong determinant of human gut microbiota. However, the bulk of these studies are from Western countries, where microbiota variations are mainly driven by relatively recent migration events. Malaysia is a multicultural society, but differences in gut microbiota persist across ethnicities. We hypothesized that migrant ethnic groups continue to share fundamental gut traits with the population in the country of origin due to shared cultural practices despite subsequent geographical separation. To test this hypothesis, the 16S rRNA gene amplicons from 16 studies comprising three major ethnic groups in Malaysia were analysed, covering 636 Chinese, 248 Indian and 123 Malay individuals from four countries (China, India, Indonesia and Malaysia). A confounder-adjusted permutational multivariate analysis of variance (PERMANOVA) detected a significant association between ethnicity and the gut microbiota (PERMANOVA R2=0.005, pseudo-F=2.643, P=0.001). A sparse partial least squares - discriminant analysis model trained using the gut microbiota of individuals from China, India and Indonesia (representation of Chinese, Indian and Malay ethnic group, respectively) showed a better-than-random performance in classifying Malaysian of Chinese descent, although the performance for Indian and Malay were modest (true prediction rate, Chinese=0.60, Indian=0.49, Malay=0.44). Separately, differential abundance analysis singled out Ligilactobacillus as being elevated in Indians. We postulate that despite the strong influence of geographical factors on the gut microbiota, cultural similarity due to a shared ethnic origin drives the presence of a shared gut microbiota composition. The interplay of these factors will likely depend on the circumstances of particular groups of migrants.
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.