Vibrio parahaemolyticus is one of the most widely recognized pathogenic Vibrio species due to numerous outbreaks and its’ wide occurrence in marine environment. In this study, 32 isolates of V. parahaemolyticus isolated from cockles were tested for sensitivity to 16 antibiotics and the presence of plasmids. All the isolates were multi-resistance, defined as resistant to atleast three different antibiotics with multiple antibiotic resistance indexes ranging from 0.31 to 0.69, indicating the isolates originate from high risk sources of contamination where antibiotics are often used. In the plasmid profiling test, only 15 isolates (47%) harbored plasmid DNA, which ranged in size from 2.7 to 56.2 kb, separating the isolates into 14 plasmid profiles. Hence, food contaminated with antibiotic resistant V. parahaemolyticus could be a major threat to public health due to the distinct possibility that they can be a significant reservoir of genes encoding antibiotic resistance determinants that can be transferred intra or interspecies. As in many developing countries, raw food hygiene and antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study and thus our data provide a current baseline profile of antimicrobial resistance and plasmid of V. parahaemolyticusfrom cockles in Padang, Indonesia.
Vibrio species isolated from four different sampling stations in the west coast of Peninsular Malaysia were screened for their antimicrobial resistance and plasmid profiles. A total of 138 isolates belonging to 15 different species were identified. Vibrio campbellii, V. parahaemolyticus, V. harveyi, and V. tubiashii were found to predominance species at all stations. High incidence of erythromycin, ampicillin, and mecillinam resistance was observed among the Vibrio isolates. In contrast, resistance against aztreonam, cefepime, streptomycin, sulfamethoxazole, and sulfonamides was low. All the Vibrio isolates in this study were found to be susceptible to imipenem, norfloxacin, ofloxacin, chloramphenicol, trimethoprim/sulfamethoxazole, and oxytetracycline. Ninety-five percent of the Vibrio isolates were resistant to one or more different classes of antibiotic, and 20 different resistance antibiograms were identified. Thirty-two distinct plasmid profiles with molecular weight ranging from 2.2 to 24.8 kb were detected among the resistance isolates. This study showed that multidrug-resistant Vibrio spp. were common in the aquatic environments of west coast of Peninsular Malaysia.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants in developing countries. A total of 38 EPEC isolates, obtained from diarrhea patients of Hospital Miri, Sarawak, were investigated through plasmid profile, antibiotic resistance and randomly amplified polymorphic DNA (RAPD) analysis. From the 8 types of antibiotics used, all isolates were 100% resistant to furoxime, cephalothin and sulphamethoxazole and showed high multiple antibiotic resistant (MAR) indexes, ranging from 0.5 to 1.0. In plasmid profiling, 22 isolates (58%) showed the presence of one or more plasmids in the range 1.0 to 30.9 mDa. The dendrogram obtained from the results of the RAPD-PCR discriminated the isolates into 30 single isolates and 3 clusters at the level of 40% similarity. The EPEC isolates were highly diverse, as shown by their differing plasmid profiles, antibiotic resistance patterns and RAPD profiles.
Prior to the implementation of Haemophilus influenzae type b vaccination worldwide, H. influenzae has been one of the main causative agents of community acquired pneumonia and meningitis in children. Due to the lack of information on the characteristics of the H. influenzae isolates that have previously been collected in Malaysia, the H. influenzae were assessed of their microbial susceptibility to commonly used antibiotics. Emphasis was made on strains that were resistance to co-trimoxazole (SXT) and their mode of transfer of the antibiotic resistance determinants were examined. A collection of 34 H. influenzae isolates was serotyped and antimicrobial susceptibility tests were performed to 11 antibiotics. To the isolates that were found to be resistant to co-trimoxazole, minimum inhibition concentration (MIC) to SXT was performed using Etest while agar dilution method was used to measure the individual MICs of trimethoprim (TMP) and sulfamethoxazole (SUL). These isolates were also examined for presence of plasmid by PCR and isolation method. Conjugal transfers of SXT-resistant genes to SXT-susceptible hosts were performed to determine their rate of transfer. Result showed that 20.6% of the total number of isolates was serotype B while the remaining was non-typeable. Antimicrobial susceptibility profile of all the isolates revealed that 58.8% was resistant to at least one antibiotic. Majority of these isolates were equally resistant to ampicillin and tetracycline (29.4% each), followed by resistance to SXT (26.5%). From nine isolates that were found to be SXT-resistant, five contained plasmid/s. Conjugal transfer experiment showed that these five isolates with plasmid transferred SXT-resistance determinants at a higher frequency than those without. From these observations, it is postulated that plasmid is not involved in the transfer of SXT-resistance genes but presence of plasmid facilitates their transfer. The information obtained from this study provides some basic knowledge on the antimicrobial susceptibility pattern of the H. influenzae isolates and their mode of transfer of SXT-resistance genes.
Multidrug-resistant (MDR) Acinetobacter baumannii has increasingly emerged as an important nosocomial pathogen. The aim of this study was to determine the resistance profiles and genetic diversity in A. baumannii clinical isolates in a tertiary medical center in Malaysia. The minimum inhibitory concentrations of carbapenems (imipenem and meropenem), cephalosporins (ceftazidime and cefepime), and ciprofloxacin were determined by E-test. PCR and sequencing were carried out for the detection of antibiotic resistance genes and mutations. Clonal relatedness among A. baumannii isolates was determined by REP-PCR. Sequence-based typing of OXA-51 and multilocus sequence typing were performed. One hundred twenty-five of 162 (77.2%) A. baumannii isolates had MDR phenotype. From the 162 A. baumannii isolates, 20 strain types were identified and majority of A. baumannii isolates (66%, n = 107) were classified as strain type 1 and were positive for ISAba1-blaOXA-23and ISAba1-blaADCand had mutations in both gyrA and parC genes at positions, 83 and 80, resulting in serine-to-leucine conversion. REP-PCR analysis showed 129 REP types that generated 31 clones with a 90% similarity cutoff value. OXA-66 variant of the blaOXA-51-likegenes was predominantly detected among our A. baumannii clinical isolates belonging to ST195 (found in six clones: 1, 8, 9, 19, 27, and 30) and ST208 (found in clone 21). The study helps us in understanding the genetic diversity of A. baumannii isolates in our setting and confirms that international clone II is the most widely distributed clone in Universiti Kebangsaan Malaysia Medical Centre, Malaysia.
Fifteen independent E. coli strains of avian, bovine and porcine origin in Peninsular Malaysia were tested for antibiotic resistance and conjugative R plasmids. Eight (53%) isolates were found to be antibiotic resistant. Among them, 37.5% were mono-resistant and 62.5% were resistant to three or more antibiotics, i.e., multi-resistant. All of them were resistant to Tc and sensitive to Gm and Nx. Three of the eight antibiotic resistant strains were able to transfer all or part of their resistance to an E. coli K12 recipient by conjugation. The transfer frequencies of Km, Sm and Tc resistance of the three donors varied between 4.5 X 10(-8) to 6.8 X 10(-7). Analysis of the plasmid profiles of all the three donors and their respective transconjugants after agarose gel electrophoresis provided conclusive evidence that the transferable resistance traits were plasmid-mediated.
Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV) subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2%) and MCF-10A (0.5%) human breast cancer cells. Newly hatched specific-pathogen-free (SPF) chicks were inoculated once by oral gavage with 10(9) colony-forming unit (CFU) of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.
Between 1992 and 1994, 253 tetracycline-resistant Neisseria gonorrhoeae (TRNG) strains were isolated and characterized by auxotype and serogroup (A/S) classes to study TRNG prevalence in different years. TRNG accounted for 28.1, 42.5, and 51.9% of the strains isolated in 1992, 1993, and 1994, respectively, showing a significant increase in each successive year (chi square = 26.7, P < 0.001). There was no significant increase in penicillinase-producing TRNG, which accounted for 53.1, 53.8, and 63.2% of the TRNG isolates. The 253 TRNG isolates belonged to 53 A/S classes. Eighteen A/S classes not observed in 1992 were detected in 1993, and 11 A/S classes not observed in 1992 and 1993 were isolated in 1994, indicating dissemination of the tetracycline resistance gene among the N. gonorrhoeae strains in Malaysia. Its emergence and subsequent rapid spread are alarming. The plasmid is capable of self-transfer (S.A. Morse, S.R. Johnson, J.W. Biddle, and M.C. Roberts, J. Infect. Dis. 155:819-822, 1987), allowing further dissemination of tetracycline resistance.
Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.
The present study aimed to screen plants for bioactive compounds with potential antibacterial activities. In our efforts to evaluate plants from Borneo, we isolated and elucidated the structures of four natural products from the bioactive fraction of a chloroform extract of Goniothalamus longistipetes using various chromatographic and spectroscopic techniques. The bioactive compounds were identified as a known styryllactone, (+)-altholactone ((2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (1), a new styryllactone, (2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (2) as well as a new alkaloid, 2,6-dimethoxyisonicotinaldehyde (3) and a new alkenyl-5-hydroxyl-phenyl benzoic acid (4). 1 and 4 showed broad-spectrum anti-bacterial activities against Gram-positive and Gram-negative bacteria as well as acid-fast model selected for this study. Compound 2 only demonstrated activities against Gram-positive bacteria whilst 3 displayed selective inhibitory activities against Gram-positive bacterial strains. Additionally, their mechanisms of anti-bacterial action were also investigated. Using Mycobacterium smegmatis as a fast-growing model of tubercle bacilli, compounds 1, 2 and 4 demonstrated inhibitory activities against whole-cell drug efflux and biofilm formation; two key intrinsic mechanisms of antibiotic resistance. Interestingly, the amphiphilic compound 4 exhibited inhibitory activity against the conjugation of plasmid pKM101 in Escherichia coli using a plate conjugation assay. Plasmid conjugation is a mechanism by which Gram-positive and Gram-negative-bacteria acquire drug resistance and virulence. These results indicated that bioactive compounds isolated from Goniothalamus longistipetes can be potential candidates as 'hits' for further optimisation.
This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 μg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.
The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. We constructed a plasmid vector pSVac that harbors a 255-bp single-repeat sequence of the cell wall-binding protein region of the AcmA protein. The recombinant plasmid was transformed into Escherichia coli and expression of the gene fragment was driven by the T7 promoter of the plasmid. SDS-PAGE showed the presence of the putative AcmA' fragment and this was confirmed by Western blot analysis. The protein was isolated and purified using a His-tag affinity column. When mixed with a culture of L. lactis MG1363, ELISA and immunofluorescence assays showed that the cell wall-binding fragment was anchored onto the outer surface of the bacteria. This indicated that the AcmA' repeat unit retained the active site for binding onto the cell wall surface of the L. lactis cells. Stability assays showed that the fusion proteins (AcmA/A1, AcmA/A3) were stably docked onto the surface for at least 5 days. The AcmA' fragment was also shown to be able to strongly bind onto the cell surface of naturally occurring lactococcal strains and Lactobacillus and, with less strength, the cell surface of Bacillus sphericus. The new system designed for cell surface display of recombinant proteins on L. lactis was evaluated for the expression and display of A1 and A3 regions of the VP1 protein of enterovirus 71 (EV71). The A1 and A3 regions of the VP1 protein of EV71 were cloned upstream to the cell wall-binding domains of AcmA protein and successfully expressed as AcmA/A1 and AcmA/A3. Whole-cell ELISA showed the successful display of VP1 protein epitopes of EV71 on the surface of L. lactis. The success of the anchoring system developed in this study for docking the A1 and A3 epitopes of VP1 onto the surface of L. lactis cells opens up the possibilities of peptide and protein display for not only Lactococcus but also for other gram-positive bacteria. This novel way of displaying epitopes on the cell surface of L. lactis and other related organisms should be very useful in the delivery of vaccines and other useful proteins.
Cellulose acetate phthalate (CAP) microcapsules were formulated to deliver plasmid DNA (pDNA) to the intestines. The microcapsules were characterized and were found to have an average diameter of 44.33 ± 30.22 μm, and were observed to be spherical with smooth surface. The method to extract pDNA from CAP was modified to study the release profile of the pDNA. The encapsulated pDNA was found to be stable. Exposure to the acidic and basic pH conditions, which simulates the pH environment in the stomach and the intestines, showed that the release occurred in a stable manner in the former, whereas it was robust in the latter. The loading capacity and encapsulation efficiency of the microcapsules were low but the CAP recovery yield was high which indicates that the microcapsules were efficiently formed but the loading of pDNA can be improved. In vitro transfection study in 293FT cells showed that there was a significant percentage of green-fluorescent-protein-positive cells as a result of efficient transfection from CAP-encapsulated pDNA. Biodistribution studies in BALB/c mice indicate that DNA was released at the stomach and intestinal regions. CAP microcapsules loaded with pDNA, as described in this study, may be useful for potential gene delivery to the intestines for prophylactic or therapeutic measures for gastrointestinal diseases.
An erythromycin resistance plasmid, pAJ01 was isolated from Loctococcus lactis isolate C5 that was isolated from a healthy two-week-old chicken cecum. A 4 kb plasmid was transformed into plasmidless L. lactis MG1363 before a restriction endonuclease map was constructed. It was then fused with pUC19 to form pAJ02, which can replicate in Escherichia coli XLI-Blue as well as L. lactis MG1363. The plasmid was stably maintained in Lactococcus for more than 100 generations.
Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs)-producing Enterobacteriaceae have been increasingly reported and imposing significant threat to public. Livestock production industry might be the important source for clinically important ESBL-producing Enterobacteriaceae. This study aims to investigate the resistance profile, phenotypic ESBL production, beta-lactamase genes, virulence factors, and plasmid replicon types among 59 Enterobacteriaceae strains isolated from poultry faecal samples in Malaysia's commercial poultry farm. There were 38.7% and 32.3% of Escherichia coli resistant to cefotaxime and cefoxitin, respectively, while Klebsiellaspp. demonstrated resistance rate of 52.6% to both mentioned antimicrobials. Majority of the E. coli isolates carried blaTEM and blaCMY-2 group. blaSHV was the most prevalent gene detected in Klebsiellaspp., followed by blaDHA and blaTEM. Resistance to extended spectrum cephalosporin in our isolates was primarily mediated by plasmid mediated AmpC beta-lactamase such as CMY-2 group and DHA enzyme. The CTX-M genes were found in two ESBL-producing E. coli. IncF, IncI1, and IncN plasmids were most frequently detected in E. coli and Klebsiellaspp. The virulence factor, including EAST1 and pAA were identified at low frequency. This study highlights the poultry as a reservoir of resistance and virulence determinants and prevalence of plasmids in Enterobacteriaceae might drive their dissemination.
A cryptic plasmid, pMWHK1 recovered from an Antarctic bacterium Pedobacter cryoconitis BG5 was sequenced and characterised. The plasmid is a circular 6206bp molecule with eight putative open reading frames designated as orf1, orf2, orf3, orf4, orf5, orf6, orf7 and orf8. All the putative open reading frames of pMWHK1 are found to be actively transcribed. Proteins encoded by orf2 and orf4 are predicted to be responsible for the mobilization and replication of the plasmid respectively. orf4 shares 55% and 61% identities with the theta-type Rep proteins from two strains of Riemerella anatipestifer. This suggests that pMWHK1 could be a member of the theta-type replicating plasmid. The origin of replication is located within the AT-rich region upstream of orf4. orf5 and orf6 encode bacterial toxin-antitoxin proteins predicted to maintain plasmid stability. orf3 encodes an entry exclusion protein that is hypothetically involved in reducing the frequency of DNA transfer through conjugation. orf1, orf7 and orf8 encode proteins with unknown functions. Plasmid, pMWHK1 is stably maintained in P. cryoconitis BG5 at 20°C.
In recent years, there has been considerable interest in simple sequence repeats (SSRs) particularly as molecular markers with applications in many different fields. We have carried out an effort to identify and analyse SSRs in the genome of the Asian seabass, Lates calcarifer by random sequencing. Genomic DNA was isolated from the muscle tissue of L. calcarifer, sheared by nebulisation and ligated into plasmid vector. Recombinant clones were selected randomly from the genomic libraries constructed. Subsequently, plasmid DNA was extracted and subjected to one-pass sequencing. A total of 4175 random sequences, also known as genome survey sequences (GSSs), with a total length of 1.7 Mb was generated. Screening of the whole L. calcarifer GSS data set allowed for the identification of a total of 151 perfect (100% similarity) SSRs. These SSR consensus patterns spread over a wide range of size (1 to 226 bp). The most frequent consensus pattern is dinucleotide, which represents 60% of all SSRs identified. The dinucleotides (AC)n, (AT)n and (AG)n were also found to occur frequently in the L. calcarifer genome. Sequence comparison between L. calcarifer and other fish species showed variation in repeat content, indicating the different ways in which repeats may evolve in the genome of these species. Data generated from this random sequencing of the L. calcarifer genome should serve as a valuable resource for further studies of this organism.
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 total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.
Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.