Periplaneta americana Arginine Kinase as a Major Cockroach Allergen among Thai Patients with Major Cockroach Allergies

Periplaneta americana is the predominant cockroach (CR) species and a major source of indoor allergens in Thailand. Nevertheless, data on the nature and molecular characteristics of its allergenic components are rare. We conducted this study to identify and characterize the P. americana allergenic protein. A random heptapeptide phage display library and monoclonal antibody (MAb) specific to a the P. americana component previously shown to be an allergenic molecule were used to identify the MAb-bound mimotope and its phylogenic distribution. Two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, peptide mass fingerprinting, and BLAST search were used to identify the P. americana protein containing the MAb-specific epitope. We studied the allergenicity of the native protein using sera of CR-allergic Thai patients in immunoassays. The mimotope peptide that bound to the MAb specific to P. americana was LTPCRNK. The peptide has an 83–100% identity with proteins of Anopheles gambiae, notch homolog scalloped wings of Lucilia cuprina, delta protein of Apis mellifera; neu5Ac synthase and tyrosine phosphatase of Drosophila melanogaster, and a putative protein of Drosophila pseudoobscura. This finding implies that the mimotope-containing molecule of P. americana is a pan-insect protein. The MAb-bound protein of P. americana was shown to be arginine kinase that reacted to IgE in the sera of all of the CR-allergic Thai patients by immunoblotting, implying its high allergenicity. In conclusion, our results revealed that P. americana arginine kinase is a pan-insect protein and a major CR allergen for CR-allergic Thai patients

Staphylococcus spp. associated with subclinical bovine mastitis in central and northeast provinces of Thailand

Background Staphylococcus spp. are major cause of bovine mastitis (BM) worldwide leading to economic damage to dairy farms and public health threat. Recently, a newly emerged Staphylococcus argenteus has been found as a human and animal pathogen. Molecular characteristics, virulence and antibiotic resistant phenotypes of bacteria causing BM in Thailand are rare. This study aimed to investigated Staphylococcus spp. associated with subclinical bovine mastitis (SCM) in Thailand. Methods Milk samples were collected from 224 cows of 52 dairy herds in four central and northeast provinces. Total somatic cell counts (SCC) and California mastitis test (CMT) were used to identify SCM cows. Milk samples were cultured for Staphylococcus spp. Coagulase-positive isolates were subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Organisms suspected as S. argenteus were verified by detecting nonribosomal peptide synthetase gene. All isolates were checked for antibiograms and the presence of various virulence genes. Results From the 224 milk samples of 224 cows, 132 (59%) were positive for SCM by SCC and CMT and 229 staphylococcal isolates were recovered. They were 32 coagulase-positive (24 S. aureus and eight S. argenteus) and 197 coagulase-negative. PFGE of the S. aureus and S. argenteus revealed 11 clusters and a non-typeable pattern. MLST of representatives of the 11 PFGE clusters, three PFGE non-typeable S. aureus isolates from different locations and S. argenteus showed 12 sequence types. The eight S. argenteus isolates belonged to ST1223 (three isolates), ST2250 (two isolates), and ST2793 (two isolates). The antimicrobial tests identified 11 (46%) methicillin-resistant S. aureus and 25 (13%) methicillin-resistant coagulase-negative isolates, while seven S. argenteus were methicillin-susceptible and one isolate was methicillin-resistant. All of the 229 isolates were multiply resistant to other antibiotics. The most prevalent virulence genes of the 24 S. aureus isolates were clfA, coa and spa (X and IgG-binding region) (100%), hla (96%), pvl (96%) and sec (79%). Six S. argenteus isolates carried one enterotoxin gene each and other virulence genes including coa, clfA, hla/hlb, spa, tsst and pvl, indicating their pathogenic potential. Conclusion and perspective This is the first report on the S. argenteus from cow milk samples with SCM. Data on the molecular characteristics, virulence genes and antibiograms of the Staphylococcus spp. obtained from the present study showed a wide spread and increasing trend of methicillin-resistance and multiple resistance to other antibiotics. This suggests that the “One Health” practice should be nurtured, not only at the dairy farm level, but also at the national or even the international levels through cooperation of different sectors (dairy farmers, veterinarians, medical and public health personnel and scientists) in order to effectively combat and control the spread of these pathogens

A novel bacteriocin from Enterococcus faecalis 478 exhibits a potent activity against vancomycin-resistant enterococci.

The emergence of multidrug-resistant enterococci (MDRE) and particularly vancomycin-resistant enterococci (VRE) is considered a serious health problem worldwide, causing the need for new antimicrobials. The aim of this study was to discover and characterize bacteriocin against clinical isolates of MDRE and VRE. Over 10,000 bacterial isolates from water, environment and clinical samples were screened. E. faecalis strain 478 isolated from human feces produced the highest antibacterial activity against several MDRE and VRE strains. The optimum condition for bacteriocin production was cultivation in MRS broth at 37°C, pH 5-6 for 16 hours. The bacteriocin-like substance produced from E. faecalis strain EF478 was stable at 60°C for at least 1 hour and retained its antimicrobial activity after storage at -20°C for 1 year, at 4°C for 6 months, and at 25°C for 2 months. A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) analysis showed that the amino acid sequences of the bacteriocin-like substance was similar to serine protease of E. faecalis, gi|488296663 (NCBI database), which has never been reported as a bacteriocin. This study reported a novel bacteriocin with high antibacterial activity against VRE and MDRE

DISSEMINATION OF CLASS I INTEGRON IN ACINETOBACTER BAUMANNII ISOLATED FROM VENTILATOR-ASSOCIATED PNEUMONIA PATIENTS AND THEIR ENVIRONMENT

Abstract. Multidrug resistant Acinetobacter baumannii has become the most common cause of health care-associated infections at Maharaj Nakhon Si Thammarat Hospital, Thailand. The objective of the study was to detect integrons using PCR-based method from 96 A. baumannii isolates from ventilator-associated pneumonia (VAP) patients and their environment. Antibiotic susceptibility was determined using a disk diffusion technique. Forty-six isolates exhibited integrase genes, with only class I and class II integron detected in 43 and 3 A. baumannii isolates, respectively. Twenty-seven of 52 clinical and 19 of 44 environmental isolates were integron-positive. Detection rate of integron-positive A. baumannii isolated from VAP patients increased from 25% to 83% over the 4 month study period. The majority (91%) of integron-positive A. baumannii showed resistance to 6 or more of 11 antibiotics tested and 72% of class I integronpositive isolates were imipenem-resistant. Thus, class I integron-positive A. baumannii had spread among the VAP patients and into hospital environment, the latter acting as reservoirs of potential pathogens possessing drug resistance genes

Human scFvs That Counteract Bioactivities of Staphylococcus aureus TSST-1

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vβ region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application

ROTAVIRUS INFECTION IN CHILDREN AND ADULTS WITH ACUTE GASTROENTERITIS IN THAILAND

Abstract. Rotaviruses are the most important cause of severe diarrhea in infants and young children, but rotavirus gastroenteritis in adults is uncommon. In this study, 260 stool samples collected in Thailand from January 2006 to February 2007 from patients of all ages with acute gastroenteritis, were tested for group A rotavirus and compared with rotavirus infections in children and adults. Rotavirus was detected in 42% of the patients' samples, but children (< 18 years old) have a significantly higher prevalence (57%) of rotavirus infection than adults (≥ 18 years old) (27%) (OR 3.55; 95% CI: 2.11-5.96; p < 0.001). The highest attack rate was found in the age group of < 2 years old (14%), followed by 2-4 years of age (9%), 18-59 years of age (8%), 5-17 years of age (6%) and ≥ 60 years of age (5%). The dominant genotype was G1P Rotaviruses G3, G9, and P[4] were found only in children and genotype P[6] was found in adults (75%) at a higher frequency than in children (25%) (p < 0.001). The number of rotavirus in children was 1.99x10 8 /ml and in adult patients was 7.32x10 6 / ml. The present study highlights the higher prevalence of rotavirus infection in children compared to adults and rotavirus genetic heterogeneity

Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis

AbstractTargeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.</jats:p