Quorum sensing enables bacteria to control the gene expression in response to the cell density. It regulates a variety of bacterial physiological functions such as biofilm formation, bioluminescence, virulence factors and swarming which has been shown contribute to bacterial pathogenesis. The use of quorum sensing inhibitor would be of particular interest in treating bacterial pathogenicity and infections. In this work, we have tested caffeine as quorum sensing inhibitor by using Chromobacterium violaceum CV026 as a biosensor. We verified that caffeine did not degrade the N-acyl homoserine lactones tested. In this work, it is shown that caffeine could inhibit N-acyl homoserine lactone production and swarming of a human opportunistic pathogen, namely Pseudomonas aeruginosa PA01. To the best of our knowledge, this is the first documentation providing evidence on the presence of anti-quorum sensing activity in caffeine. Our work will allow caffeine to be explored as anti-infective drugs.
This study aimed to test the sterility of new unused orthodontic buccal tubes received from manufacturers. Four different types of buccal tubes were used straight from the manufactures package without any additional sterilizing step. Of these buccal tubes tested, three genera of bacteria, implicated as opportunistic pathogens, namely Micrococcus luteus, Staphylococcus haemolyticus and Acinetobacter calcoaceticus were recovered from these buccal tubes. Our data showing microbial contamination on buccal tubes highlights the need of sterilization before clinical use. We also suggest that manufacturers should list the sterility state of orthodontic buccal tubes on their packaging or instructions stating the need for sterilization.
We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium.
Cedecea neteri is a very rare human pathogen. We have isolated a strain of C. neteri SSMD04 from pickled mackerel sashimi identified using molecular and phenotypics approaches. Using the biosensor Chromobacterium violaceum CV026, we have demonstrated the presence of short chain N-acyl-homoserine lactone (AHL) type quorum sensing (QS) activity in C. neteri SSMD04. Triple quadrupole LC/MS analysis revealed that C. neteri SSMD04 produced short chain N-butyryl-homoserine lactone (C4-HSL). With the available genome information of C. neteri SSMD04, we went on to analyse and identified a pair of luxI/R homologues in this genome that share the highest similarity with croI/R homologues from Citrobacter rodentium. The AHL synthase, which we named cneI(636 bp), was found in the genome sequences of C. neteri SSMD04. At a distance of 8bp from cneI is a sequence encoding a hypothetical protein, potentially the cognate receptor, a luxR homologue which we named it as cneR. Analysis of this protein amino acid sequence reveals two signature domains, the autoinducer-binding domain and the C-terminal effector which is typical characteristic of luxR. In addition, we found that this genome harboured an orphan luxR that is most closely related to easR in Enterobacter asburiae. To our knowledge, this is the first report on the AHL production activity in C. neteri, and the discovery of its luxI/R homologues, the orphan receptor and its whole genome sequence.
Tropical carnivorous plant, Nepenthes, locally known as "monkey cup", utilises its pitcher as a passive trap to capture insects. It then secretes enzymes into the pitcher fluid to digest the insects for nutrients acquisition. However, little is known about the microbiota and their activity in its pitcher fluid. Eighteen bacteria phyla were detected from the metagenome study in the Nepenthes pitcher fluid. Proteobacteria, Bacteroidetes and Actinobacteria are the dominant phyla in the Nepenthes pitcher fluid. We also performed culturomics approach by isolating 18 bacteria from the Nepenthes pitcher fluid. Most of the bacterial isolates possess chitinolytic, proteolytic, amylolytic, and cellulolytic and xylanolytic activities. Fifteen putative chitinase genes were identified from the whole genome analysis on the genomes of the 18 bacteria isolated from Nepenthes pitcher fluid and expressed for chitinase assay. Of these, six clones possessed chitinase activity. In conclusion, our metagenome result shows that the Nepenthes pitcher fluid contains vast bacterial diversity and the culturomic studies confirmed the presence of biocatalytic bacteria within the Nepenthes pitcher juice which may act in symbiosis for the turn over of insects trapped in the Nepenthes pitcher fluid.
Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.
A novel chemically defined medium, named KG medium, supplemented with N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), an acylhomoserine lactone (AHL) used as signalling molecules in Gram-negative bacterial cell-to-cell communication, as the sole source of carbon and nitrogen, was designed and successfully used for the enrichment and isolation of AHL-degrading bacteria. A 3-oxo-C6-HSL-degrading bacterium, 13sw7, was isolated from sewage after six enrichment transfers in the 3-oxo-C6-HSL-containing KG medium. On the basis of the almost complete 16S ribosomal DNA sequence, isolate 13sw7 was clustered with unculturable beta-proteobacteria. This study indicates that the AHL-containing KG medium is effective in isolating AHL-degrading bacteria, including those previously considered unculturable, from environmental sources. To the best of our knowledge, this is the first documentation of the isolation of an AHL-degrading proteobacterium from sewage.
The quorum sensing (QS) system has been used by many opportunistic pathogenic bacteria to coordinate their virulence determinants in relation to cell-population density. As antibiotic-resistant bacteria are on the rise, interference with QS has been regarded as a novel way to control bacterial infections. As such, many plant-based natural products have been widely explored for their therapeutic roles. These natural products may contain anti-QS compounds that could block QS signals generation or transmission to combat QS pathogens. In this study, we report the anti-QS activities of four different Chinese herbal plant extracts: Poria cum Radix pini, Angelica dahurica, Rhizoma cibotii and Schizonepeta tenuifolia, on Pseudomonas aeruginosa PAO1. All the plants extracted using hexane, chloroform and methanol were tested and found to impair swarming motility and pyocyanin production in P.aeruginosa PAO1, particularly by Poria cum Radix pini. In addition, all the plant extracts also inhibited violacein production in C.violaceum CV026 up to 50% while bioluminescence activities were reduced in lux-based E. coli biosensors, pSB401 and pSB1075, up to about 57%. These anti-QS properties of the four medicinal plants are the first documentation that demonstrates a potential approach to attenuate pathogens’ virulence determinants.
To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572(T) (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570(T) (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535(T) (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens.
A large number of Vibrio sp. thrive in the marine environment and they are notable to cause food borne infection associated with undercooked seafood. In this study, we report the whole genome sequence of Vibrio sinaloensis T47 which was isolated from coastal marine water in Morib Beach, Hulu Selangor. The genome is made up of approximately 4.59 Mbp with 80 contigs and 46% G+C content. From the annotated genome, genes associated with quorum sensing (QS) were identified. This research provides a genetic basis for better understanding of QS pathway which contributes to the physiological traits of strain T47 to thrive in the marine environment.
In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.
The quality of traditional Chinese soy sauce is determined by microbial communities and their inter-related metabolic roles in the fermentation tank. In this study, traditional Chinese soy sauce brine samples were obtained periodically to monitor the transitions of the microbial population and functional properties during the 6 months of fermentation process. Whole genome shotgun method revealed that the fermentation brine was dominated by the bacterial genus Weissella and later dominated by the fungal genus Candida. Metabolic reconstruction of the metagenome sequences demonstrated a characteristic profile of heterotrophic fermentation of proteins and carbohydrates. This was supported by the detection of ethanol with stable decrease of pH values. To the best of our knowledge, this is the first study that explores the temporal changes in microbial successions over a period of 6 months, through metagenome shotgun sequencing in traditional Chinese soy sauce fermentation and the biological processes therein.
We report the draft genome sequence of Staphylococcus sp. strain AL1, which degrades quorum-sensing molecules (namely, N-acyl homoserine lactones). To the best of our knowledge, this is the first documentation that reports the whole genome sequence and quorum-quenching activity of Staphylococcus sp. strain AL1.
Roseomonas sp. strain B5 was isolated from Malaysian tropical soil that showed N-acylhomoserine lactone degradation. This is the first genome announcement of a member from the genus of Roseomonas and the first report on the quorum-quenching activity of Roseomonas spp.
Kocuria rhizophila is a ubiquitous bacterium which is well known for its industrial value. Here, we present the draft genome of Kocuria rhizophila strain TPW45 which was isolated from Sungai Gabai, Selangor, Malaysia. The assembled genome comprised of 46 contigs and the estimated genome size is 2.7 Mb. Based on the RAST annotation, a gene cluster responsible for aromatic compound degradation was identified in this strain.
Streptomyces pluripotens MUSC 137 was isolated from mangrove soil obtained from Tanjung Lumpur, Pahang, Malaysia. We investigated the phylogenetic, genomic, biochemical, and phenotypic characteristics of this strain. Uniquely adapted microorganisms from mangrove habitats have previously yielded compounds of biopharmaceutical interest. In order to examine the bioactivities possessed by the strain, fermentation extract was prepared through solvent extraction method prior to bioactivities screenings. Antioxidant activity was examined via DPPH assay while the cytotoxic effect was assessed by means of examining the activity of the extract against selected human cancer cell lines, namely colon cancer cells (HCT-116, Caco-2, SW480, and HT-29), breast cancer cell (MCF-7), lung cancer cell (A549), prostate cancer cell (DU145), and cervical cancer cell (Ca Ski). The results revealed MUSC 137 possesses significant antioxidant activity and demonstrates cytotoxic effect against several cancer cell lines tested. The results indicated MCF-7 cells were most susceptible to the extract with the lowest IC50 (61.33 ± 17.10 μg/mL), followed by HCT-116 and A549. Additionally, selective index (SI) showed that MUSC 137 extract was less toxic against normal cell lines when compared to MCF-7 and HCT-116 cells. The extract was further subjected to chemical analysis using GC-MS and revealed the presence of deferoxamine and pyrrolizidines related compounds which may account for the antioxidant and cytotoxic properties observed.
High consumer demand for shellfish has led to the need for large-scale, reliable shellfish supply through aquaculture or shellfish farming. However, bacterial infections which can spread rapidly among shellfish poses a major threat to this industry. Shellfish farmers therefore often resort to extensive use of antibiotics, both prophylactically and therapeutically, in order to protect their stocks. The extensive use of antibiotics in aquaculture has been postulated to represent a major contributing factor in the rising incidence of antimicrobial resistant pathogenic bacteria in shellfish. This study aimed to investigate the incidence of pathogenic Vibrio parahaemolyticus and determine the antibiotic resistance profile as well as to perform plasmid curing in order to determine the antibiotic resistance mediation. Based on colony morphology, all 450 samples tested were positive for Vibrio sp; however, tox-R assay showed that only 44.4% (200/450) of these were V. parahaemolyticus. Out of these 200 samples, 6.5% (13/200) were trh-positive while none were tdh-positive. Antibiotic resistance was determined for all V. parahaemolyticus identified against 14 commonly used antibiotics and the multiple antibiotic resistance index (MAR) was calculated. The isolates demonstrated high resistance to several antibiotics tested- including second and third-line antibiotics- with 88% resistant to ampicillin, 81% to amikacin,70.5% to kanamycin, 73% to cefotaxime, and 51.5% to ceftazidime. The MAR index ranged from 0.00 to 0.79 with the majority of samples having an index of 0.36 (resistant to five antibiotics). Among the 13 trh-positive strains, almost 70% (9/13) demonstrated resistance to 4 or more antibiotics. Plasmid profiling for all V. parahaemolyticus isolates revealed that 86.5% (173/200) contained plasmids - ranging from 1 to 7 plasmids with DNA band sizes ranging from 1.2 kb to greater than 10 kb. 6/13 of the pathogenic V. pathogenic strains contained plasmid. After plasmid curing, the plasmid containing pathogenic strains isolated in our study have chromosomally mediated ampicillin resistance while the remaining resistance phenotypes are plasmid mediated. Overall, our results indicate that while the incidence of pathogenic V. parahaemolyticus in shellfish in Selangor still appears to be at relatively reassuring levels, antibiotic resistance is a real concern and warrants ongoing surveillance.
A Streptomyces strain, MUM256 was isolated from Tanjung Lumpur mangrove soil in Malaysia. Characterization of the strain showed that it has properties consistent with those of the members of the genus Streptomyces. In order to explore the potential bioactivities, extract of the fermented broth culture of MUM256 was prepared with organic solvent extraction method. DPPH and SOD activity were utilized to examine the antioxidant capacity and the results have revealed the potency of MUM256 in superoxide anion scavenging activity in dose-dependent manner. The cytotoxicity of MUM256 extract was determined using cell viability assay against 8 different panels of human cancer cell lines. Among all the tested cancer cells, HCT116 was the most sensitive toward the extract treatment. At the highest concentration of tested extract, the result showed 2.3-, 2.0-, and 1.8-folds higher inhibitory effect against HCT116, HT29, and Caco-2 respectively when compared to normal cell line. This result has demonstrated that MUM256 extract was selectively cytotoxic toward colon cancer cell lines. In order to determine the constituents responsible for its bioactivities, the extract was then subjected to chemical analysis using GC-MS. The analysis resulted in the identification of chemical constituents including phenolic and pyrrolopyrazine compounds which may responsible for antioxidant and anticancer activities observed. Based on the findings of this study, the presence of bioactive constituents in MUM256 extract could be a potential source for the development of antioxidative and chemopreventive agents.
Dickeya sp. strain 2B12 was isolated from a freshwater lake in Malaysia. Here, we report the draft genome sequence of Dickeya sp. 2B12 sequenced by the Illumina MiSeq platform. With the genome sequence available, this genome sequence will be useful for the study of quorum-sensing activity in this isolate.