Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.
The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.
The emergence of antibiotic resistance among multidrug-resistant (MDR) microbes is of growing concern, and threatens public health globally. A total of 129 Escherichia coli isolates were recovered from lowland aqueous environments near hospitals and medical service centers in the vicinity of Kuala Lumpur, Malaysia. Among the eleven antibacterial agents tested, the isolates were highly resistant to trimethoprim-sulfamethoxazole (83.7%) and nalidixic acid (71.3%) and moderately resistant to ampicillin and chloramphenicol (66.7%), tetracycline (65.1%), fosfomycin (57.4%), cefotaxime (57.4%), and ciprofloxacin (57.4%), while low resistance levels were found with aminoglycosides (kanamycin, 22.5%; gentamicin, 21.7%). The presence of relevant resistance determinants was evaluated, and the genotypic resistance determinants were as follows: sulfonamides (sulI, sulII, and sulIII), trimethoprim (dfrA1 and dfrA5), quinolones (qnrS), β-lactams (ampC and blaCTX-M), chloramphenicol (cmlA1 and cat2), tetracycline (tetA and tetM), fosfomycin (fosA and fosA3), and aminoglycosides (aphA1 and aacC2). Our data suggest that multidrug-resistant E. coli strains are ubiquitous in the aquatic systems of tropical countries and indicate that hospital wastewater may contribute to this phenomenon.
This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.
Group C Streptococcus dysgalactiae (GCSD) is a pathogen of farmed fish. Almost all GCSD isolates from Asian countries, including Japan, Taiwan, Malaysia, and China, have a serum opacity factor (SOF-FD). Although the SOF-FD sequences in different GCSD isolates are identical, different opacification activities are observed. Three types of variations were observed in the upstream sequence of the sof-FD gene in GCSD isolates with different SOF-FD activities. Type 1 was characterized by insertion of an IS981-like element into the upstream region of the sof-FD gene. In Type 2, an IS981-like element was inserted into the upstream region in a direction opposite to that in Type 1. In Type 3, no IS element was inserted. Type 1 was predominant among Japanese isolates (129 of 133). Isolates from other Asian countries were generally Type 3 (13 of 16). Except for 1 strain, Type 1 strains exhibited opacification activities with optical densities (ODs)>0.6, while Type 2 and Type 3 strains have low opacification activities (ODs >0.2). Only Type 1 strains have putative -10 and -35 promoter regions upstream of the sof-FD gene, and the expression level of the sof-FD gene was higher in Type 1 strains than in Type 2 and Type 3 strains.
A biosurfactant-producing and hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa USM-AR2, was used to assist conventional distillation. Batch cultivation in a bioreactor gave a biomass of 9.4 g L(-1) and rhamnolipid concentration of 2.4 g L(-1) achieved after 72 h. Biosurfactant activity (rhamnolipid) was detected by the orcinol assay, emulsification index and drop collapse test. Pretreatment of crude oil TK-1 and AG-2 with a culture of P. aeruginosa USM-AR2 that contains rhamnolipid was proven to facilitate the distillation process by reducing the duration without reducing the quality of petroleum distillate. It showed a potential in reducing the duration of the distillation process, with at least 2- to 3-fold decreases in distillation time. This is supported by GC-MS analysis of the distillate where there was no difference between compounds detected in distillate obtained from treated or untreated crude oil. Calorimetric tests showed the calorie value of the distillate remained the same with or without treatment. These two factors confirmed that the quality of the distillate was not compromised and the incubation process by the microbial culture did not over-degrade the oil. The rhamnolipid produced by this culture was the main factor that enhanced the distillation performance, which is related to the emulsification of hydrocarbon chains in the crude oil. This biotreatment may play an important role to improve the existing conventional refinery and distillation process. Reducing the distillation times by pretreating the crude oil with a natural biosynthetic product translates to energy and cost savings in producing petroleum products.
Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.
The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.
The taxonomic position of three actinomycete strains isolated from Malaysian soil was established by using a polyphasic approach. The isolates formed chains composed of four spores on the tip of sporophores branching from the aerial mycelium, and their chemotaxonomic properties were common to those of members of the family Streptosporangiaceae. These phenotypic properties as well as a phylogenetic analysis based on 16S rRNA gene sequences indicated that they should be classified in the genus Microtetraspora. The three isolates showed a unique pattern of cultural, physiological and biochemical properties that distinguished them from previously described species of the genus Microtetraspora. The isolates showed more than 72% DNA relatedness to each other, but only 58% or less relatedness to any previously described species. On the basis of the data presented, a new species of the genus Microtetraspora, Microtetraspora malaysiensis, is proposed. The type strain of the new species is strain H47-7(T) (=JCM 11278(T)=DSM 44579(T)).
In a previous study, we isolated Leifsonia sp. strain SIU, a new bacterium from agricultured soil. The bacterium was tested for its ability to degrade caffeine. The isolate was encapsulated in gellan gum and its ability to degrade caffeine was compared with the free cells. The optimal caffeine degradation was attained at a gellan gum concentration of 0.75% (w/v), a bead size of 4 mm diameter, and 250 beads per 100 mL of medium. At a caffeine concentration of 0.1 g/L, immobilised cells of the strain SIU degraded caffeine within 9 h, which is faster when compared to the case of free cells, in which it took 12 h to degrade. The immobilised cells degraded caffeine completely within 39 and 78 h at 0.5 and 1.0 g/L, while the free cells took 72 and 148 h at 0.5 and 1.0 g/L, respectively. At higher caffeine concentrations, immobilised cells exhibited a higher caffeine degradation rate. At concentrations of 1.5 and 2.0 g/L, caffeine-degrading activities of both immobilised and free cells were inhibited. The immobilised cells showed no loss in caffeine-degrading activity after being used repeatedly for nine 24-h cycles. The effect of heavy metals on immobilised cells was also tested. This study showed an increase in caffeine degradation efficiency when the cells were encapsulated in gellan gum.
A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.
Prazosin (PRZ), a drug used to treat hypertensive patients, is an emergent contaminant in water systems. PRZ is an alpha-adrenergic receptor blocker used to treat anxiety, and is believed to reach the environment through human excretion, irresponsible disposal of unused medicine, and waste products from manufacturing plants. The purpose of this research was to isolate and characterize potential microbes for PRZ biodegradation and to identify the degradation pathway. After screening, isolated strain STP3 showed a capability for PRZ degradation and was chosen for further analysis. Resting cell assays with PRZ were conducted to identify the intermediate metabolites formed from biodegradation by Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) analysis. Two metabolites degraded from PRZ by STP3 were successfully found, and as these metabolites are derived from the main structure of PRZ, their presence proved PRZ degradation. Draft genome sequencing analysis of STP3 was performed to identify potential enzymes for PRZ biodegradation based on the metabolites found.
A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 x 10(4) cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.