Repetitive sequence-based PCR (rep-PCR) is a distinctive typing approach that is used to
differentiate between bacterial strains. This method is also useful for studying bacterial diversity
from different sources. In this study, four rep-PCR which are enterobacterial repetitive intergenic
consensus PCR (ERIC-PCR), BOX-PCR, repetitive extragenic palindromic PCR (REP-PCR)
and polytrinucleotide (GTG)5-PCR were evaluated for differentiation of eighteen Escherichia
coli isolates to correct source based on part of intestine and age. These isolates were recovered
earlier from ileal and caecal mucosal contents of chickens at different age. The purpose of this
study was to investigate the efficacy of four rep-PCR methods and composite of rep-PCR
patterns to differentiate E. coli isolates to original sources of part of intestines and age based on
the D index (discriminatory power determined based on Simpson’s index of diversity calculated
at similarity coefficient of 90%). The (GTG)5-PCR had the highest D index (0.9804) for part of
intestine and age factors. The similar D index was observed in the composite of rep-PCR
patterns. The lowest D index was observed in ERIC- and BOX-PCR at 0.9020 and 0.8039 for
part of intestine and age factors, respectively. (GTG)5-PCR was also the most discriminative rep-
PCR observed due to its ability to cluster 14I 3E and 14I 2X isolates, and 14C 1E and 14C 3E
isolates correctly in part of intestine and age factors. It was concluded that (GTG)5-PCR is a
promising tool for discriminating E. coli isolates extracted from chicken intestines.
The immunodominant region of hepatitis B virus (HBV) located in the viral small surface antigen (S-HBsAg) elicits virus-neutralizing and protective antibodies. In order to develop an easy and inexpensive method to produce this region without the need for extensive purification, amino acid residues 111-156 of S-HBsAg were fused to the C-terminal end of the 10B capsid protein of T7 phage. Western blotting and ELISA confirmed the expression of the recombinant protein on the surface of the phage particles. The recombinant phage exhibited the antigenic and immunogenic characteristics of HBsAg, illustrating its potential as an immunological reagent and vaccine.
The surface antigen (HBsAg) of hepatitis B virus (HBV) is highly conformational and generally evokes protective humoral immune response in human. A disulfide constrained random heptapeptide library displayed on the coat protein III of filamentous bacteriophage M13 was employed to select specific ligands that interact with HBsAg subtype ad. Fusion phages carrying the amino acid sequence ETGAKPH and other related sequences were isolated. The binding site of peptide ETGAKPH was located on the immunodominant region of HBsAg. An equilibrium binding assay in solution showed that the phage binds tightly to HBsAg with a relative dissociation constant (KDrel) of 2.9+/-0.9 nM. The phage bearing this peptide has the potential to be used as a diagnostic reagent and two assays for detecting HBsAg in blood samples are described.
Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model.
Latex production from Hevea brasiliensis rubber tree is the second most important commodity in Malaysia, but this industry is threatened by the white root rot disease (WRD) caused by Rigidoporus microporus that leads to considerable latex yield loss and tree death. This study aimed to characterize and compare the virulence of five R. microporus isolates obtained from infected rubber trees located at different states in Malaysia. These isolates were subjected to morphological and molecular characterization for species confirmation and pathogenicity test for the determination of virulence level. BLAST search showed that the ITS sequences of all the pathogen isolates were 99% identical to R. microporus isolate SEG (accession number: MG199553) from Malaysia. The pathogenicity test of R. microporus isolates conducted in a nursery with 24 seedlings per isolate showed that isolate RL21 from Sarawak has developed the most severe above- and below-ground symptoms of WRD on the rubber clone RRIM600 as host. Six months after being infected with R. microporus, RL21 was evaluated with the highest average of disease severity index of 80.52% for above- and below-ground symptoms, followed by RL22 (68.65%), RL20 (66.04%), RL26 (54.38%), and RL25 (43.13%). The in vitro growth condition tests showed that isolate RL21 of R. microporus has optimum growth at 25-30 °C, with the preference of weakly acidic to neutral environments (pH 6-7). This study revealed that different virulence levels are possessed among different R. microporus isolates even though they were isolated from the same host species under the same climate region. Taken together, field evaluation through visual observation and laboratory assays have led to screening of the most virulent isolate. Determination of the most virulent isolate in the present study is vital and shall be taken into consideration for the selection of suitable pathogen isolate in the development of more effective control measures in combating tenacious R. microporus.