Unlike environmental P. koreensis isolated from soil, which has been studied extensively for its role in promoting plant growth, pathogenic P. koreensis isolated from fish has been rarely reported. Therefore, we investigated and isolated the possible pathogen that is responsible for the diseased state of Tor tambroides. Herein, we reported the morphological and biochemical characteristics, as well as whole-genome sequences of a newly identified P. koreensis strain. We assembled a high-quality draft genome of P. koreensis CM-01 with a contig N50 value of 233,601 bp and 99.5% BUSCO completeness. The genome assembly of P. koreensis CM-01 is consists of 6,171,880 bp with a G+C content of 60.5%. Annotation of the genome identified 5538 protein-coding genes, 3 rRNA genes, 54 tRNAs, and no plasmids were found. Besides these, 39 interspersed repeat and 141 tandem repeat sequences, 6 prophages, 51 genomic islands, 94 insertion sequences, 4 clustered regularly interspaced short palindromic repeats, 5 antibiotic-resistant genes, and 150 virulence genes were also predicted in the P. koreensis CM-01 genome. Culture-based approach showed that CM-01 strain exhibited resistance against ampicillin, aztreonam, clindamycin, and cefoxitin with a calculated multiple antibiotic resistance (MAR) index value of 0.4. In addition, the assembled CM-01 genome was successfully annotated against the Cluster of Orthologous Groups of proteins database, Gene Ontology database, and Kyoto Encyclopedia of Genes and Genome pathway database. A comparative analysis of CM-01 with three representative strains of P. koreensis revealed that 92% of orthologous clusters were conserved among these four genomes, and only the CM-01 strain possesses unique elements related to pathogenicity and virulence. This study provides fundamental phenotypic and genomic information for the newly identified P. koreensis strain.
Campylobacter jejuni is one of the most common causes of bacterial gastrointestinal food-borne infection worldwide. It has been suggested that biofilm formation may play a role in survival of these bacteria in the environment. In this study, the influence of prior modes of growth (planktonic or sessile), temperatures (37 and 42 °C), and nutrient conditions (nutrient broth and Mueller-Hinton broth) on biofilm formation by eight C. jejuni strains with different antibiotic resistance profiles was examined. The ability of these strains to form biofilm on different abiotic surfaces (stainless steel, glass, and polystyrene) as well as factors potentially associated with biofilm formation (bacterial surface hydrophobicity, auto-aggregation, and initial attachment) was also determined. The results showed that cells grown as sessile culture generally have a greater ability to form biofilm (P
In this study, we employed a polyphasic approach to determine the taxonomic position of a newly isolated actinomycete, designated SE31T, obtained from a sediment sample collected at Cape Rochado, Malaysia. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain SE31T belonged to the family Pseudonocardiaceae and exhibited the highest sequence similarity (98.9%) to Sciscionella marina. Further genomic analysis demonstrated a 93.4% average nucleotide identity and 54.4% digital DNA-DNA hybridization relatedness between strain SE31T and S. marina. The chemotaxonomic characteristics of strain SE31T were typical of the genus Sciscionella, including cell-wall chemotype IV (with meso-diaminopimelic acid as the diagnostic diamino acid, and arabinose and galactose as whole-cell sugars). The identified polar lipids of strain SE31T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, and hydroxyphosphatidymethylethanolamine. The primary menaquinone observed was MK-9(H4), and the major cellular fatty acid was iso-C16:0. The genomic DNA size of strain SE31T was determined to be 7.4 Mbp with a G+C content of 68.7%. Based on these comprehensive findings, strain SE31T represents a novel species within the genus Sciscionella, in which the name Sciscionella sediminilitoris sp. nov. is proposed. The type strain of Sciscionella sediminilitoris is SE31T (= DSM 46824T = TBRC 5134T).
The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.
Rescue of (-)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.
Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C(6). Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.
Consumption of probiotics, which are beneficial live microorganisms, has received a lot of attention because of their potential to improve health and wellness. Robust quality control measures are necessary to ensure the safety of probiotics and maximize their health effects. This review delves into the topic of quality management in probiotics, highlighting the significance of sticking to strict guidelines from manufacture to storage to distribution. Probiotic quality standards, Good Manufacturing Practices (GMP) implementation, quality control and testing techniques, and documentation and traceability systems are all discussed in detail. The importance of taking precautions to avoid microbial contamination, meeting all applicable regulations, and clearly marking and packaging probiotic products is also emphasized. In addition, it reviews the clinical evidence supporting the possible health advantages of probiotics and investigates the processes through which probiotics enhance health. The review continues by stressing the significance of educating and informing consumers about probiotics and their proper use in order to maximize health benefits. Probiotics' potential health benefits can be maximized and consumer faith in these helpful microbes can be bolstered by adopting thorough quality management measures to ensure their safety, efficacy, and consistency.
Acute hepatopancreatic necrosis disease (AHPND) is a severe disease affecting recently stocked cultured shrimps. The disease is mainly caused by V. parahaemolyticus that harbors the pVA1 plasmid; this plasmid contains the pirA and pirB genes, which encode a delta-endotoxin. AHPND originated in China in 2009 and has since spread to several other Asian countries and recently to Latin America (2013). Many Asian strains have been sequenced, and their sequences are publicly accessible in scientific databases, but only four strains from Latin America have been reported. In this study, we analyzed nine pVA1-harboring V. parahaemolyticus sequences from strains isolated in Mexico along with the 38 previously available pVA1-harboring V. parahaemolyticus sequences and the reference strain RIMD 2210633. The studied sequences were clustered into three phylogenetic clades (Latin American, Malaysian, and Cosmopolitan) through pangenomic and phylogenomic analysis. The nucleotide sequence alignment of the pVA1 plasmids harbored by the Asian and Latin American strains confirmed that the main structural difference in the plasmid between the Asian and Latin American strains is the absence of the Tn3 transposon in the Asian strains; in addition, some deletions in the pirAB region were found in two of the Latin American strains. Our study represents the most robust and inclusive phylogenomic analysis of pVA1-harboring V. parahaemolyticus conducted to date and provides insight into the epidemiology of AHPND. In addition, this study highlights that disease diagnosis through the detection of the pirA and pirB genes is an inadequate approach due to the instability of these genes.
We constructed recombinant phage particles displaying the Bacillus thuringiensis Cry1Ba4 active toxin using the pfUSE5 and pComb3X phagemid vectors. The recombinant phage particles were screened and evaluated for displayed biologically active Cry1Ba4 toxin against the target insect larvae. Concurrent expression of Cry1Ba4 protoxin was carried out using the pETBlue -2 plasmid expression vector in Escherichia coli Tuner (DE3)pLacI and the protoxin was successfully expressed at a size of 129 kDa. In the bioassay, 3.30 mg crude extract of Cry1Ba4 protoxin, 9.35 x 10(9) TU and 7.70 x 10(9) TU of induced recombinant phage particles carrying Cry1Ba4 active toxin displayed on pComb3X and pFUSE5, respectively, demonstrated mortality of greater than 85% against Plutella xylostella (third-instar) within 48 hours. Thus, we have successfully displayed the Cry1Ba4 activated toxin on the surface of a phage and demonstrated toxicity towards larvae.
Enteromorpha prolifera blooms considerably affected coastal environments in recent years. However, the effects of E. prolifera on microbial ecology and function remained unknown. In this study, metagenomic sequencing was used to investigate the effect of E. prolifera bloom on the microbial communities and functional genes in an aquaculture environment. Results showed that E. prolifera bloom could significantly alter the microbial composition and abundance, and heterotrophic bacteria comprised the major groups in the E. prolifera bloom pond, which was dominated by Actinomycetales and Flavobacteriales. The study indicated that viruses played an important role in shaping the microbial community and diversity during E. prolifera bloom. These viruses affected various dominant microbial taxa (such as Rhodobacteraceae, Synechococcus, and Prochlorococcus), which produced an obvious impact on potential nutrient transformation. Functional annotation analysis indicated that E. prolifera bloom would considerably shift the metabolism function by altering the structure and abundance of the microbial community. E. prolifera bloom pond had the low ability of potential metabolic capabilities of nitrogen, sulfur, and phosphate, whereas promoted gene abundance of genetic information processing. These changes in the microbial community and function could produce serious effect on aquaculture ecosystem.
Phycosphere hosts the boundary of unique holobionts harboring dynamic algae-bacteria interactions. During our investigating the microbial consortia composition of phycosphere microbiota (PM) derived from diverse harmful algal blooms (HAB) dinoflagellates, a novel rod-shaped, motile and faint yellow-pigmented bacterium, designated as strain LZ-6 T, was isolated from HAB Alexandrium catenella LZT09 which produces high levels paralytic shellfish poisoning toxins. Phylogenetic analysis based on 16S rRNA gene and two housekeeping genes, rpoA and pheS sequences showed that the novel isolate shared the highest gene similarity with Marinobacter shengliensis CGMCC 1.12758 T (99.6%) with the similarity values of 99.6%, 99.9% and 98.5%, respectively. Further phylogenomic calculations of average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strains LZ-6 T and the type strain of M. shengliensis were 95.9%, 96.4% and 68.5%, respectively. However, combined phenotypic and chemotaxonomic characterizations revealed that the new isolate was obviously different from the type strain of M. shengliensis. The obtained taxonomic evidences supported that strain LZ-6 T represents a novel subspecies of M. shengliensis, for which the name is proposed, Marinobacter shengliensis subsp. alexandrii subsp. nov. with the type strain LZ-6 T (= CCTCC AB 2018388TT = KCTC 72197 T). This proposal automatically creates Marinobacter shengliensis subsp. shengliensis for which the type strain is SL013A34A2T (= LMG 27740 T = CGMCC 1.12758 T).
Increased prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and their distribution in both hospital and community settings. Discovery and development of new anti-MRSA agents as alternatives to the very few antibiotics left in the armamentarium are, thus, urgently required. Recently, an efflux mechanism in MRSA has been identified as one of the main contributors of resistance towards various structurally unrelated antibiotics. The potential of reserpine (a phytoalkaloid) as efflux pump inhibitor (EPI) against various microbes remains limited as the concentration needed for inhibition is toxic to humans. This study therefore aimed to evaluate 13 alkaloid compounds as potential inhibitory agents and/or potential EPIs against a panel of three MRSA isolates which not only differ in their susceptibility to vancomycin (amongst the last drugs available to treat serious MRSA infection), but also exhibited active efflux activity. Results indicated berberine's moderate inhibitiory activity against two MRSA isolates scoring a minimum inhibitory concentration (MIC) value of 125 microg/ml. Notable efflux inhibitory activity (ranging from two- to eightfold Ethidium Bromide MIC reduction) meanwhile was detected from quinine, piperine and harmaline using reserpine as the positive control. Findings from this study support the opinion that a vast number of potential phytocompounds with pharmacological potential await discovery. Therapeutic application of these compounds, however, warrants further investigation to ascertain their pharmacodynamics and safety aspects.
The emerging aquaculture industry is in need of non-antibiotic-based disease control approaches to minimize the risk of antibiotic-resistant bacteria. Bacterial infections mainly caused by Vibrio spp. have caused mass mortalities of fish especially during the larval stages. The objectives of this study were to verify the potential of symbiotic probiont strains, isolated from microalgae (Amphora, Chlorella, and Spirulina) for suppressing the growth of Vibrio spp. and at the same time ascertain their abilities to enhance microalgal biomass by mutualistic interactions through microalgae-bacteria symbiosis. In addition, in vivo studies on Artemia bioencapsulated with probiont strains (single strain and mix strains) and microalgae were evaluated. The selected potential probionts were identified as Lysinibacillus fusiformis strain A-1 (LFA-1), Bacillus sp. strain A-2 (BA-2), Lysinibacillus fusiformis strain Cl-3 (LFCl-3), and Bacillus pocheonensis strain S-2 (BPS-2) using 16s rRNA. The cell densities of Amphora culture supplemented with BA-2 and Chlorella culture supplemented with LFCl-3 were higher than those of the controls. Artemia bioencapsulated with mix strains (LFA-1 + BA-2 + LFCl-3 + BPS-2) and Amphora demonstrated the highest survival rate compared to the controls, after being challenged with V. harveyi (60 ± 4%) and V. parahaemolyticus (78 ± 2%). Our study postulated that BA-2 and LFCl-3 were found to be good promoting bacteria for microalgal growth and microalgae serve as a vector to transport probiotic into Artemia. Moreover, mixture of potential probionts is beneficial for Artemia supplementation in conferring protection to Artemia nauplii against pathogenic Vibrios.
Bactrocera carambolae is a highly polyphagous fruit pest of agricultural importance. This study reports the bacterial communities associated with the developmental stages of B. carambolae. The microbiota of the developmental stages were investigated by targeted 16S rRNA gene (V3-V4 region) sequencing using the Illumina MiSeq. At 97% similarity, there were 19 bacterial phyla and unassigned bacteria, comprising 39 classes, 86 orders, 159 families and 311 genera. The bacterial composition varied among the specimens of developmental stage and across developmental stages as well as exuviae. Four phyla of bacteria (with relative abundance of ≥1% in at least one specimen)-Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria-were recovered from the larva, pupa, adult stages and exuviae. Proteobacteria was the predominant phylum in all the developmental stages as well as the exuviae. Enterobacteriaceae (Proteobacteria) was the predominant family in the adult flies while the family [Weeksellaceae] (Bacteroidetes) was predominant in the larval and pupal stages. Among the genera occurring in more than one developmental stage of B. carambolae, Erwinia was more abundant in the larval stage, Halomonas more abundant in adult female, Stenotrophomonas more abundant in adult male, and Chryseobacterium more abundant in the larval and pupal stages. The results indicate transmission of bacteria OTUs from immatures to the newly emerged adults, and from exuviae to the environment.
Cold stress leads to the disruption of the cellular homeostasis in plants and generation of reactive oxygen species (ROS) and productivity losses. In the present study, 94 psychrotrophic phosphorus-solubilizing bacteria with multiple plant growth-promoting (PGP) capabilities were isolated from rhizosphere of wheat. The most efficient strain EU-KL94 showing highest amount of solubilized phosphorus and maximum number of PGP attributes was identified using 16S rRNA sequencing as Ochrobactrum thiophenivorans. Ochrobactrum thiophenivorans EU-KL94 along with recommended doses of the chemical fertilizers as controls were used for alleviation of cold stress in oats. The strain improved the root and shoot length, dry and fresh weight, proline, glycine betaine, chlorophyll content as well as the superoxide dismutase (SOD) and glutathione reductase (GR) activities of oats under cold stress conditions. Ochrobactrum thiophenivorans with all promising plant growth activities under cold stress could be used as an environmental friendly strategy for mitigation of low temperature stress. To the best of our knowledge, Ochrobactrum thiophenivorans has been reported for the first time as P-solubilizer and as bioinoculants in oats for cold stress mitigation.
The opportunistic pathogen Exophiala dermatitidis has been frequently isolated from tropical regions of the world. However, there is no report of environmental isolation of Exophiala spp. from Malaysia. The information regarding the ecology of this microbe is important for a better understanding of the opportunism. This study aims to conduct a survey of natural distribution of Exophiala spp. in Malaysia. Forty-seven strains of Exophiala-like was isolated by using selective media. These isolates from the fields were molecularly identified based on the ITS region. The biochemical activity of these microbes was tested by conducting various tests, i.e. DNase test, proteinase activity, and urea hydrolysis. Overall, 22 strains of E. dermatitidis were successfully obtained and identified from burnt tree bark, oil dripped soil sample, hot spring biofilm, railway track stones, tar road contaminated with petrol hydrocarbon, drain and deep mud of Sungai Pinang besides the new discovery from pigeon droppings. A single strain of E. heteromorpha was identified from tar road contaminated with petrol hydrocarbon. Genotypes of the isolated E. dermatitidis were identified by the neighbor-joining tree and grouped into Genotype A, A2 and B. The existence of new Genotype A4 was confirmed by a similar cladogram position in both neighbor-joining and maximum likelihood tree. The survival of E. dermatitidis in the hydrocarbon contaminated environment was studied by supplying engine oil and observing the growth pattern. The results of this study suggest that the opportunistic Exophiala spp. was isolated from nutrient limited and harsh conditions in the natural environment.
Methicillin-resistant Staphylococcus aureus (MRSA) infections have become one of the most threatening multidrug-resistant pathogens. Thus, an ongoing search for anti-MRSA compounds remains an urgent need to effectively treating MRSA infections. Phomopsidione, a novel antibiotic isolated from Diaporthe fraxini, has previously demonstrated potent anti-candidal activity. The present study aimed to investigate the effects of phomopsidione on the viability, virulence, and metabolites profile of MRSA. MRSA was sensitive to phomopsidione in a concentration-dependent manner. Phomopsidione exhibited minimum inhibitory concentration and minimum bactericidal concentration of 62.5 and 500.00 µg/mL against MRSA on broth microdilution assay. The compound showed significant reduction in virulence factors production including extracellular polymeric substances quantification, catalase, and lipase. An untargeted metabolomics analysis using liquid chromatography-high resolution mass spectrometry revealed a significant difference in the metabolites profile of MRSA with 13 putatively identified discriminant metabolites. The present study suggested the potential of phomopsidione as a promising anti-MRSA agent.
The draft genome here presents the sequence of Bacillus subtilis UMX-103. The bacterial strain was isolated from hydrocarbon-contaminated soil from Terengganu, Malaysia. The whole genome of the bacterium was sequenced using Illumina HiSeq 2000 sequencing platform. The genome was assembled using de novo approach. The genome size of UMX-103 is 4,234,627 bp with 4399 genes comprising 4301 protein-coding genes and 98 RNA genes. The analysis of assembled genes revealed the presence of 25 genes involved in biosurfactant production, where 14 of the genes are related to biosynthesis and 11 of the genes are in the regulation of biosurfactant productions. This draft genome will provide insights into the genetic bases of its biosurfactant-producing capabilities.
The presence of two cry-like genes first identified in Clostridium bifermentans subsp. malaysia CH18 was investigated in Clostridium species including 12 subspecies of Clostridium bifermentans, 13 strains of other members of Clostridia genus, and 13 different subspecies of Bacillus thuringiensis. Oligonucleotides designed to amplify the two toxin genes, cmb71 and cmb72, were used. We found that these genes are present in 80% of the Clostridium bifermentans strains tested and in 8% of the other Clostridium and Bacillus thuringiensis strains.
A novel polyhydroxyalkanoate (PHA)-producing bacterium, Jeongeupia sp. USM3 (JCM 19920) was isolated from the limestone soil at Gua Tempurung, Perak, Malaysia. This is the first report on the complete genome sequence for the genus Jeongeupia. This genome consists of a circular chromosome with a size of 3,788,814 bp and contains 3557 genes. Two PHA synthase (phaC) genes encoding for the key enzyme in the polymerization of PHA monomers and other PHA-associated genes were identified from the genome. Phylogenetic analysis of the PhaC protein sequences has revealed that both PhaC1 and PhaC2 of Jeongeupia sp. USM3 are categorized as Class I PHA synthases with 56% similarity to each other. Both of the PHA synthase genes of this isolate were cloned and heterologously expressed in a PHA mutant strain Cupriavidus necator PHB-4. The ability of the transformants to accumulate PHA showed that both PhaC1 and PhaC2 were functional.