Study On The Genic Identification Of Pathogenic Bacteria Responsible For Bacteremia And Enteric Infection

Bacteremia and enteric infectious diseases are the common diseases in clinic. In the event of these, urgent patient management and antimicrobial therapy is needed otherwise progression to generalized sepsis with shock ensues especially in the status of reduced immunity. Rapid identification of clinical isolates with a high degree of specificity is desired for choice of antibiotic in clinical therapy. Conventional diagnostic method based on phenotypic characteristics, including colonial morphology and biochemical reactions, takes a long time and thus it would not facilitate earlier effective therapy. The development of microarray technology is revolutionizing research in medical microbiology and the management of infectious diseases. Microarrays are just beginning to be used widely in the clinical setting, in particular as a valuable tool for the diagnosis of infection. The aim of this project is to develop a robust, simple and cheap microarray for the detection of bacterial pathogens, suitable for use in diagnostic laboratories in China. Prevous investigators have usually chosen the 16S ribosomal RNA (rRNA) gene as a target. Recently, analysis of sequence data for large subunit (23 S rRNA) gene suggests that this region shows more variation between species of medical importance than 16S Rrnagene. For this reason, the development and validation of an oligonucleotide suspension array using 23S rRNA target gene for rapid identification of clinical strains responsible for bacteremia was performed. In the process, we found the target gene 23S rRNA is lack of discrimination between closely related species within Enterobacteriaceae. We consequently choose gyrB gene, which encodes the B-subunit of DNA gyrase, as a target for the classification and identification of enteropathogenic bacteria. In order to determine whether diversities in gyrB gene sequences could be used for discrimination of the clinical enteropathogenic bacteria involved Enterobacteriaceae and Vibrionaceae, a phylogenetic analysis was carried out. It could be helpful for designing oligonucleotide probes used in suspension array for enteropathogenic bacteria.Part I. Use of an Oligonucleotide Suspension Array for Laboratory Identification of Bacteria Responsible for BacteremiaOligonucleotide array analysis following universal PCR amplification has been commonly recognized as having many advantages for the rapid detection of multiple pathogens, and especially less common or unexpected bacteria. We undertook a study to investigate the suitability of suspension array system for identifying pathogenic bacteria implicated in bacteremia on the basis of partial 23S rRNA gene sequences.Thirty oligonucleotide probes were covalently bound to different bead sets and hybridized with PCR amplification products of a variable region of bacterial 23S ribosomal RNA gene of 91 strains representing 28 bacterial species responsible for bacteremia. DNA hybridization reactants on the surface of the beads were quantified by green fluorescence coupled to reporter molecule by the LiquiChip Reader.The majority of isolates used in this study could be identified through species-specific hybridization patterns, including Gram negative species: Aeromonas hydrophila, Burkholderia cepacia, Citrobacter freundii, Haemophilus influenzae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella typhimurium and Stenotrophomonas maltophilia, and Gram positive species: E. faecalis, Enterococcus faecium, L. monocy to genes, S. aureus and Staphylococcus saprophyticus. Three groups of strains could not be distinguished from one other, including Proteus mirabilis and Proteus vulgaris;six species of coagulase-negative.staphylococci (CoNS) and Staphylococcus epidermidis;and Streptococcus pneumoniae and Streptococcus Viridans group. Moreover, due to variation of hybridization pattern of isolates within a species, Enterobacter aerogenes, E. coli, K. pneumoniae and Serratia marcesens could not be reliablyidentified. More isolates need to be studied to determine the significance of these variations, and more probes must be designed to allow identification of a wider range of species and to reduce the risk of misidentification.The results provide a proof of concept for the use of suspension array system to identify a broad range of bacterial species, which holds great promise for microbial diagnostics in the routine laboratory.Part II. Classification and Identification of Enteropathogenic bacteria Basedon gyrB GeneIn China, Shigella, Salmonella, pathogenic Escherichia coli, Aeromonas hydrophila, and Vibrio parahaemolyticus are the major enteropathogens for enteric infectious diseases. Based on the results of part I , some Enterobacteriaceae species could not be easily distinguished using the target gene 23S rRNA. There is a growing interest in employing protein-encoding genes as alternative markers such as gyrB gene which is suitable to identify the enteric bacteria. GyrB gene, a single-copy gene which encodes the B-subunit of DNA gyrase (a topoisomerase type II), is distributed ubiquitously among bacterial species. Therefore, in order to determine if diversities in gyrB gene sequences could be used for discrimination of the clinical enteropathogenic bacteria prevalent in China and design specific oligonucleotide probes for- the bacterial identification, PCR amplification and sequencing were applied and we obtained partial gyrB sequences of 101 strains, belonging to 8 genera and 14 species, including Aeromonas (caviae, hydrophila, Veronnii), Escherichia coli, Enterobacter cloacae, Salmonella choleraesuis subspecies choleraesuis {Choleraesuis, Enterititis, Paratyphi A, Paratyph B, Typhi, Typhimurium), Shigella (boydii, dysenteria, flexneri, sonnei), Plesiomonas shigelloides, Yersinia enterocolitica, and Vibrio (cholerae, parahaemolyticus). The phylogenetic relationships of all these species described as above were investigated using the sequence of gyrB at nucleotide and amino acid level. Furthermore, the phenotype trees were also created on the basis of 38 biochemical characteristics using the methods of numerical taxonomy.Three gene trees generated with neighbor-joining, maximum parsimony and maximum likelihood methods revealed almost identical branching patterns except for Yersinia Enterocolitica and Plesiomonas shigelloides which were grouped with the family Enterobacteriaceae or Vibrionaceae. The strains of each species formed their own monophyletic group except the Shigella and E. coli and some Aeromonas species, moreover these groups were bifurcated with high bootstrap values. The genetic relationship based on GyrB protein sequenceswas similar to that of the gyrB gene sequences. GyrB sequences from E. coli, E. cloacae, Salmonella, Shigella and Y. Enterocolitica formed a monophyletic group with a significant high bootstrap value. P. shigelloides was grouped with Enterobacteriaceae strains. In the phenotype trees obtained by neighbor-joining and UPGMA (Unweighted-Pair Group Method with Arithmetic means) methods, two groups of Vibrionaceae and Enterobacteriaceae were also formed with the exception of P. shigelloides which was integrated with Enterobacteriaceae.Phylogenetic trees and phenotype trees provides full supports for each other. GyrB gehe is a useful molecular target for species identification of these common clinical enteropathogenic bacteria involved two family Vibrionaceae and Enterobacteriaceae.