No abstract

No abstract

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10 4 cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future. Molecular & Cellular Proteomics 7: 448 -456, 2008.Currently the most popular methods for bacterial identification are based on microbiologic procedures, antibody recognition, and PCR amplification. Traditionally microbiologic methods are culture-based assays that examine the presence of bacterial species. These methods provide high sensitivity and specificity, but their efficiency is limited by the complexity of the procedures, including culture, selection, isolation, and morphologic and biochemical characterization, which usually take 48 h or longer. Serologic methods are presumptive and confined to the availability of antibodies and to bacteria that are included ahead in the assays. Molecular biology techniques, particularly PCR, have been regarded as non-culturebased methods with high efficiency and specificity (1). However, they are completely dependent on the known genetic sequences of the target bacteria.MS with its capability of de novo protein/peptide sequencing (such as electrospray ionization or MALDI-TOF MS for tandem MS/MS) or its high efficiency for proteome profiling (particularly MALDI-TOF MS) has been suggested as an alternative for microbial identification (2-7...

In recent years a significant increase in the incidence of Serratia marcescens infections was noted at the Chang Gung Memorial Hospital, Taoyuan

Infection by extended-spectrum ␤-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla SHV , bla CTX-M-3 -like, and bla CTX-M-14 -like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla SHV genes (bla SHV-1 , bla SHV-2 , bla SHV-2a , bla SHV-5 , bla SHV-11 , and bla SHV-12 ) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla SHV and bla CTX-M genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV-and CTX-M-type ESBLs. In contrast toEnterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla CTX-M-3 -like (CTX-M-15) and bla CTX-M-14 -like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

No abstract

No abstract

Background and Objective: The rare occurrence of anti-D-associated hemolytic disease of the newborn among Chinese is attributable in part to the existence of the weak D phenotype D_el among apparently RhD-negative individuals. While exciting advances in the molecular genetics of the Rh blood group have been noted in recent years, the genomic structure of the D_el phenotype has seldom been studied in the literature. We try to explore the genomic structure of the RhD gene among apparently Rh-negative Chinese in Taiwan in this study. Methods: Genomic DNA from 230 samples of apparently RhD-negative Chinese was studied using four polymerase chain reaction (PCR)-based RhD typing methods. These PCR methods amplified RHD and RHCE genes at exons 4, 5, 7 and 10. All nucleotides responsible for exofacial amino acid differences between RhD and RhCeEe peptides, including amino acids 169, 170, 172, 223, 226, 233, 238, 350, 353, and 354, were contained in these amplified DNA segments. Southern blot analysis using RHD cDNA fragments as probes was performed. Results: According to the serological study, 155 samples (67.4%) were genuinely RhD-negative and 75 samples (32.6%) were of the D_el phenotype. Successful amplifications for RHD sequences were possible in all 75 D_el samples using four PCR methods. Apparently, all D_el individuals carried an intact RHD gene. While 145 individuals of 155 genuinely Rh-negative (63.0% of apparently RhD-negative individuals) had total deletion of their RHD genes, 10 individuals (4.3% of apparently RhD-negative individuals) were shown to have a preserved 3′ noncoding region of the RHD exon 10 and a gross deletion of RHD exons 4–10. Conclusions: Three classes of RhD-negative polymorphisms among Chinese in Taiwan were observed. These included D_el with grossly intact RHD and weak RhD expression, genuinely RhD-negative with partial preservation of the RHD gene, and genuinely RhD-negative with total deletion of the RHD gene. A molecular study is warranted to clarify the mechanism responsible for the weak RHD gene expression in D_el individuals.