ABSTRACT
Type III secretion system (TTSS) clusters contain virulent genes of both animal and plant Gram-negative bacteria. The full length (933 bp) gene of bipD, a member of TTSS cluster of Burkholderia pseudomallei, was isolated by PCR amplification from B. pseudomallei DNA and cloned into pGEX 4T-1. GST-BipD fusion protein and BipD protein obtained by removal of GST using thrombin were employed to detect the presence of B. pseudomallei antibodies in sera obtained from melioidosis and non-melioidosis patients by immunoblotting. Sensitivity and specificity of BipD protein was 100% and 91.1%, respectively, whereas that of fusion protein was 77.8% and 90.0%, respectively.
Subject(s)
Amino Acid Sequence , Antibodies, Bacterial/blood , Bacterial Proteins/genetics , Burkholderia pseudomallei/genetics , Case-Control Studies , DNA Primers , Gene Fusion , Genes, Bacterial , Humans , Melioidosis/genetics , Molecular Sequence Data , Multigene Family , Polymerase Chain Reaction , Sensitivity and Specificity , Thailand , Virulence/geneticsABSTRACT
One hundred and eighty-three strains of gram-negative bacteria were isolated from 177 bacteremic patients during a 6-month period in 2004 at a teaching hospital in southern Thailand. Extended-spectrum beta-lactamases (ESBL) production was detected in K. pneumoniae, E. coli, and E. cloacae, at rates of 16/36 (44.4%), 3/59 (5.1%), and 2/13 (15.4%), respectively. All but one of the screened positive strains were also positive by both the combination disk method and the E-test. All of the K. pneumoniae strains that were resistant to ceftazidime by disk diffusion were demonstrated to be positive for ESBL production by both the combination disk and E-test methods. Most of the ESBL positive strains had a high MIC (more than 32 microg/ml) to ceftazidime. However, all the ESBL positive strains were sensitive to imipenem.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteremia/epidemiology , Colony Count, Microbial , Gram-Negative Bacteria/classification , Hospitals, Teaching , Humans , Microbial Sensitivity Tests , Thailand/epidemiology , beta-Lactam Resistance , beta-Lactamases/metabolismABSTRACT
The lambdaZAP II expressed genomic library of B. pseudomallei was screened with pooled melioidosis serum preabsorbed with E. coli host cell. The positive clones were detected by using protein A-CDP-star chemiluminescence. All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis. The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology. At least six in vivo expressed genes were identified by this approach. Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (type III secretion protein gene). Another two were genes coded for conserved hypothetical protein. The last two isolated genes were groEL (a chaperonine protein gene), and a gene encoding transmembrane protein.