This study was aimed at determining the phospholipase and haemolysin activity of Candida isolates in Malaysia. A total of 37 Candida clinical isolates representing seven species, Candida albicans (12), Candida tropicalis (8), Candida glabrata (4), Candida parapsilosis (1), Candida krusei (4), Candida orthopsilosis (1) and Candida rugosa (7) were tested. In vitro phospholipase activity was determined by using egg yolk plate assay whereas in vitro haemolysin activity was tested by using blood plate assay on sheep blood Sabouraud's dextrose agar (SDA) enriched with glucose. Phospholipase activity was detected in 75% (9 out of 12) of the C. albicans isolates. Among the 25 non- C. albicans Candida isolates, phospholipase activity was detected in only 24% of these isolates. The phospholipase activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.002). Haemolysin activity was detected in 100% of the C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, and C. orthopsilosis isolates while 75% of the C. krusei isolates and 12.3% of the C. rugosa isolates showed haemolysin activity. The haemolytic activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.0001).The findings in this study indicate that C. albicans isolates in Malaysia may possess greater virulence potential than the non-albicans species.
Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.
Forty clinical isolates of Vibrio parahaemolyticus were studied for the production of the thermostable direct hemolysin (TDH), and the TDH-related hemolysin (TRH) including the respective encoding genes, tdh and trh. The presence of TDH and its encoding genes were found amongst 95% of the strains, whereas the TRH was absent amongst these isolates. Thirty-two isolates were found to be plasmid-free, whereas eight isolates possessed plasmids with sizes ranging from 2.4 > or = 23 kb. Using a DNA probe coding for the homologous region of the tdh and trh, it was found that the tdh genes were present on the chromosomal DNA.
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.
The haemolysins produced by Aeromonas species were detected and compared by two assay methods--a modified blood agar plate assay and the rabbit erythrocyte haemolysin method. Both assays showed a high level of agreement (86%). The titres of the rabbit erythrocyte haemolysin assay correlated with the haemolytic zone diameter of the ox blood agar assay. In addition the agar haemolysin assay had simple media requirements, was easy to perform and results were well defined.
Staphylococcus aureus is a major cause of nosocomial infections and secretes a diverse spectrum of virulence determinants as well as forms biofilm. The emergence of antibiotic-resistant S. aureus highlights the need for alternative forms of therapeutics other than conventional antibiotics. One route to meet this need is screening small molecule derivatives for potential anti-infective activity. Using a previously optimized C. elegans - S. aureus small molecule screen, we identified a benzimidazole derivative, UM-C162, which rescued nematodes from a S. aureus infection. UM-C162 prevented the formation of biofilm in a dose-dependent manner without interfering with bacterial viability. To examine the effect of UM-C162 on the expression of S. aureus virulence genes, a genome-wide transcriptome analysis was performed on UM-C162-treated pathogen. Our data indicated that the genes associated with biofilm formation, particularly those involved in bacterial attachment, were suppressed in UM-C162-treated bacteria. Additionally, a set of genes encoding vital S. aureus virulence factors were also down-regulated in the presence of UM-C162. Further biochemical analysis validated that UM-C162-mediated disruption of S. aureus hemolysins, proteases and clumping factors production. Collectively, our findings propose that UM-C162 is a promising compound that can be further developed as an anti-virulence agent to control S. aureus infections.
Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever.
Resistance to the bacteria-derived insecticides spinosad (Conserve), abamectin (Vertimec), Bacillus thuringiensis var kurstaki (Btk) (Dipel), B thuringiensis var aizawai (Bta) (Xentari), B thuringiensis crystal endotoxins Cry1Ac and Cry1Ca, and to the synthetic insecticide fipronil was estimated in a freshly-collected field population (CH1 strain) of Plutella xylostella (L) from the Cameron Highlands, Malaysia. Laboratory bioassays at G1 indicated significant levels of resistance to spinosad, abamectin, Cry1Ac, Btk, Cry1Ca, fipronil and Bta when compared with a laboratory insecticide-susceptible population. Logit regression analysis of F1 reciprocal crosses indicated that resistance to spinosad in the CH1 population was inherited as a co-dominant trait. At the highest dose of spinosad tested, resistance was close to completely recessive, while at the lowest dose it was incompletely dominant. A direct test of monogenic inheritance based on a back-cross of F1 progeny with CH1 suggested that resistance to spinosad was controlled by a single locus.
A field population of Plutella xylostella from Malaysia (SERD4) was divided into five sub-populations and four were selected (G2-G5) with the Bacillus thuringiensis insecticidal crystal (Cry) toxins Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da. Bioassay at G6 gave resistance ratios of 88, 5, 2 and 3 for Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da respectively compared with the unselected sub-population (UNSEL-SERD4). The Cry1Ac-selected population showed little cross-resistance to Cry1Ab, Cry1Ca and Cry1Da, (3-, 2- and 3-fold compared with UNSEL-SERD4), whereas the Cry1Ab-SEL sub-population showed marked cross-resistance to Cry1Ac (40-fold), much greater than Cry1Ab itself. In contrast, the Cry1Ca- and Cry1Da-SEL sub-population showed little if any cross-resistance to Cry1Ac and Cry1Ab. The mode of inheritance of resistance to Cry1Ac was examined in Cry1Ac-selected SERD4 by standard reciprocal crosses and back-crosses using a laboratory insecticide-susceptible population (ROTH). Logit regression analysis of F1 reciprocal crosses indicated that resistance to Cry1Ac was inherited as an incompletely dominant trait. At the highest dose of Cry1Ac tested, resistance was recessive, while at the lowest dose it was almost completely dominant. The F2 progeny from a back-cross of F1 progeny with ROTH were tested with a concentration of Cry1Ac that would kill 100% of ROTH. The mortality ranged between 50 and 95% in seven families of back-cross progeny, which indicated that more than one allele on separate loci were responsible for resistance to Cry1Ac.
Bacillus thuringiensis Berliner (Bt) crystal (Cry) toxins are expressed in various transgenic crops and are also used as sprays in integrated pest management and organic agricultural systems. The diamondback moth (Plutella xylostella L.) is a major worldwide pest of crucifer crops and one that has readily acquired field resistance to a broad range of insecticides.
The current study focused on the microscopic studies of a native Bacillus thuringiensis strain isolated from Malaysia, Bt-S84-13a, that produced an unusual crystal type. Primary detection of parasporal inclusions using a phase contrast microscope presented one to two small crystal proteins in the sporulating cells of Bt-S84-13a. Compound light microscopic examination of autolysed Bt-S84-13a cells stained with 0.133% Coomassie Brilliant Blue showed two types of crystal morphology: small crystals independent of spores and spore-associated crystals. Surface structure analysis with a scanning electron microscope revealed spherical-like, coarse and wrinkled-looking crystal in Bt-S84-13a. A close-up observation of the crystal morphology using a transmission electron microscope also demonstrated two parasporal inclusions in Bt-S84-13a. One inclusion was deposited against the forespore and was in a shape of incomplete rectangular. Another smaller inclusion was developed within the exosporium and was rectangular in shape. However, the latter inclusion was found lack in another bacterial cell which was still in the early stages of sporulation. This unique crystal morphology may imply some biological potential in Bt-S84-13a.
The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.
The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets.
Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.
Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.
INTRODUCTION: Uropathogenic virulence factors have been identified by comparing the prevalence of these among urinary tract isolates and environmental strains. The uropathogenic-specific protein (USP) gene is present on the pathogenicity island (PAI) of uropathogenic Escherichia coli (UPEC) and, depending on its two diverse gene types and the sequential patterns of three open reading frame units (orfUs) following it, there is a method to characterize UPEC epidemiologically called PAIusp subtyping.
METHODOLOGY: A total of 162 UPEC isolates from Sabah, Malaysia, were tested for the presence of the usp gene and the sequential patterns of three orfUs following it using polymerase chain reaction (PCR). In addition, by means of triplex PCR, the prevalence of the usp gene was compared with other two VFs of UPEC, namely alpha hemolysin (α-hly) and cytotoxic necrotizing factor (cnf-1) genes encoding two toxins.
RESULTS: The results showed that the usp gene was found in 78.40% of UPEC isolates, indicating that its prevalence was comparable to that found in a previous study in Japan. The two or three orfUs were also associated with the usp gene in this study. All the PAIusp subtypes observed in Japan were present in this study, while subtype IIa was the most common in both studies. The usp gene was observed in a higher percentage of isolates when compared with α-hly and cnf-1 genes.
CONCLUSIONS: The findings in Japan and Sabah, East Malaysia, were similar, indicating that PAIusp subtyping is applicable to the characterization of UPEC strains epidemiologically elsewhere in the world.