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Polystyrene waste pollutes the environment and poses a significant health risk to humans, animals, and marine ecology. This study aims to evaluate the effectiveness of degradation on expanded (EPS) and extruded (XPS) polystyrene with different diets using superworms (Zophobas atratus larvae) obtained in Malaysia. The growth and development of the larvae after consumption of EPS and XPS and the gut microbial community changes in response to high polystyrene consumption diets were also identified. The oatmeal, wheat bran, and cornmeal were used as supplement diets and showed significantly enhanced EPS and XPS consumption and degradation compared to sole diet treatment. Gel permeation chromatography was carried out using egested frass of Z. atratus larvae to characterize depolymerization of EPS and XPS, indicating a significant reduction in the average molecular weight and average molecular weight. The highest reduction occurred in the presence of oatmeal. Proton nuclear magnetic resonance and Fourier transform infrared spectroscopy analyses indicated functional group changes and chemical modification occurred with depolymerization and partial oxidation of EPS and XPS. The larvae length increased, while the number of instars and duration of larvae became shorter with the addition of supplement diets. Oatmeal is predominantly effective among other supplements in assisting Z. atratus larvae with EPS and XPS degradation. The results of this study support the ubiquity of polystyrene biodegradation in Z. atratus and the next-generation sequencing studies. Kluyvera sp., Klebsiella sp., and Enterobacter sp. were found to be strongly associated with degrading EPS and XPS polystyrene with oatmeal as a supplemental diet.

Background: Bacterial leaf blight (BLB) disease caused 80% of disease incidence in paddy in Kedah and Selangor states of Malaysia. The pathogenic bacterium, Xanthomonas oryzae pv. oryzae (Xoo), is ...

The rise in in the number of drug-resistant bacteria that can resist almost all kinds of antibiotics is due to the overuse of these antibiotics (e.g., carbapenems). Thus, there is a need to find an alternative to antibiotic treatment such as the use of phages. In this study, phage UPM1705 was isolated from a polluted lake which can lyse its host Klebsiella pneumoniae ATCC BAA-1705. Based on morphological appearance from transmission electron microscopy, UPM1705 belongs to Caudovirales (Myoviridae). UPM1705 can reach a titer of 107 PFU/ml based on the double-layer method. It has a burst size of 298 PFU/bacteria cell and a latent period of 80 min, a rise period of 75 min, and adsorption time of 20 min based on a one-step growth curve assay using an MOI of 0.02. It was stable from 4°C to 80°C and retained its functionality at pH between 4 to 11, with pH of 7 being the optimum pH for the phage growth. The efficiency of UPM1705 was tested via a turbidity assay at MOI of 0.02, 0.2, and 2. UPM1705 was able to clear the turbidity of the host bacteria culture at all of these three MOIs. Thus, UPM1705 has the potential to be used for phage therapy.

Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/ XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/ XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.

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.

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.

Background Probiotics are the most recognised alternative to antibiotic growth promoters (AGPs) for poultry production. They adhere to the intestinal epithelial cells to exert their beneficial effects on the host. However, this is often restricted by indigenous gut microbes that compete for adhesion sites and nutrients. Therefore, phage application has been proposed to reduce the competition of probiotics with gut microbiota. The main objective of this study was to investigate the effects of phages on the gut microbiota and their impact on probiotic efficacy in chickens. Results Four best Escherichia coli lytic phages were selected and characterised. The in vivo trial was conducted to investigate the effects of dietary treatments of phage cocktail at 0.1% (1ɸ) and 0.2% (2ɸ), 0.1% PrimaLac ® probiotic (P) and their combinations (1ɸP, 2ɸP) on growth performance and gut microbiota of chickens. The results from the in vivo trial showed that chickens in the 1ɸP group had significantly (P < 0.05) better body weight (BW), body weight gain (BWG), and feed conversion ratio (FCR), lower serum triglycerides and higher ileal villus height than the control. Chickens in the 1ɸP group also had higher (P < 0.05) ileal villus height, and lower crypt depth than those in the probiotic group. The gut microbiota profiles revealed that 1ɸP supplementation significantly (P < 0.05) reduced E. coli population, increased (P < 0.001) short chain fatty acids (SCFAs) producers, and improved (P < 0.001) gene expressions that are related to carbohydrate and amino acid metabolisms, and nutrient uptake. Conclusions The results from this study showed that 1ɸP treatment could modulate the gut microbiota and enhance growth performance in chickens. This finding could be considered as a potential alternative to AGPs for poultry.

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 a 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.

A modified phage-enzyme link immunosorbent assay (phage-ELISA) and a phage dot-blot assay specific for hepatitis B surface antigen (HBsAg) and its antibody were developed by using phage display technology. The phage-ELISA and phage dot-blot assays enabled to detect HBsAg and anti-HBsAg in human sera, and compatible to commercial detection kit. The fusion phages were immobilized onto microtiter plate wells and nitrocellulose membrane sheets, then blocked with 10% milk diluent, and added with human serum at dilution of 1:5000. The absorbance at 405 nm was determined once the colour changes formed. The same human serum also applied on the commercial diagnostic kit for comparison. The statistical analysis was carried out using ANOVA and T Test (LSD) for variable comparison between phage-ELISA and phage dot-blot assays. Based on these studies, the phage-ELISA was found to be more sensitive compared to phage dot-blot assay as the detection of HBsAg in human sera was about 80% as compared to 51.7% by using phage dot-blot assay. Meanwhile, the sensitivity for detection of anti-HBsAg by using phage-ELISA was slightly higher which showed about 83.3%. However, the sensitivity of the assay was dropped almost half when using phage dot-blot assay. Therefore, they are practical to be used as a reliable alternative way for the detection HBsAg and its antibody in human sera.

Background Phage display is an alternative method for constructing and selecting antibodies with desired specificity towards an antigen. Objectives To construct a library of single chain variable fragment (ScFv) towards hepatitis B core antigen (HBcAg). To isolate a ScFv phage clone that interacts with HBcAg and to develop a phage-ELISA for detecting the antigen. Study design Mice were inoculated with HBcAg and RNA was extracted from their spleen cells. The genes encoding heavy (V H ) and light (V L ) chains were amplified, linked via PCR and cloned into a phagemid vector. Phage particles displaying ScFv were panned against HBcAg and a selected clone was characterized and employed as a diagnostic reagent for detecting HBcAg in serum samples. Results A phage clone that interacts with HBcAg was selected from the antibody library. The binding of the phage to HBcAg was inhibited by a cyclic peptide bearing the WSFFSNI sequence. A phage-ELISA was established using the recombinant phage and as low as 10 ng of HBcAg can be detected by the assay. Conclusion The ScFv displayed on the surface of filamentous phage is an alternative choice for diagnosis of HBcAg in serum samples.

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.