Abstract：Group B Streptococcus (GBS; Streptococcus agalactiae) is an important cause of sepsis and meningitis. Nine GBS serotypes, based on capsular polysaccharide (CPS) antigens, have been described. Their distribution varies worldwide and needs to be monitored to understand the epidemiology of GBS disease and inform the development of vaccines. In this study, we sequenced cpsH of GBS serotype II (cpsHII) and compared it with that of the other eight serotypes to identify serotype-specific regions. We then developed a DNA microarray based on the cpsH gene and used it to test 88 GBS isolates—9 serotype reference strains and 79 clinical isolates—and 7 other bacterial and fungal species which are commonly present in the vagina flora. The microarray was shown to be specific and reproducible. This is the first report of a microarray which can identify the nine GBS serotypes. The use of a microarray has advantages over traditional serotyping methods and will be of practical value in both reference and diagnostic laboratories.
Abstract：Streptococcus pneumoniae is a major worldwide human pathogen. This investigation has developed a reliable and accurate DNA microarray method for inter-species differentiation of S. pneumoniae and intra-species differentiation of the 23 groups of S. pneumoniae including serotypes represented in the 23-valent pneumococcal vaccine and the other 20 closely related serotypes. In addition to 16S rDNA probes, serotype- or serogroup-specific probes targeting the capsular polysaccharide synthesis (cps) genes, wzy or capA were generated. We adopted a two-step multiplex PCR to improve the sensitivity of detection to a level of 105 cfu/ml in pure culture or 50 ng DNA. A total of 169 isolates (from China, Australia, Canada and New Zealand) including 147 belonging to 23-valent vaccine and closely related serotypes of S. pneumoniae, 11 belonging to other serotypes and 11 of different species commonly isolated from respiratory tract were tested to verify the method. The DNA microarray method developed provides a sensitive means to rapidly identify the members of the most common S. pneumoniae serotypes in patients and to monitor their distribution in different patient groups and geographic locations. Such information is needed for disease surveillance and to monitor vaccine efficacy.
Abstract：Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotypespecific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 104 CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.
Abstract：Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 103 CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.
Abstract：The 16S–23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types—ITSnone (without tRNA genes), ITSglu [with a tRNAGlu (UUC) gene], and ITSile_ala [with tRNAIle (GAU) and tRNAAla (UGC) genes]—were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITSglu and ITSile_ala. The presence of ITSnone in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITSnone, from 0.967 to 1.000 for ITSglu, and from 0.968 to 1.000 for ITSile_ala. Interspecies sequence identities range from 0.775 to 0.989 for ITSnone, from 0.798 to 0.997 for ITSglu, and from 0.712 to 0.985 for ITSile_ala. Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.
6. 16S-23S rDNA internal transcribed spacer regions in four Proteus species. Journal of Microbiological Methods 2009, 77(1):109-118. (IF: 2.000)
Abstract：Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITSGlu (containing tRNAGlu (UUC) gene) and ITSIle+Ala (containing tRNAIle (GAU) and tRNAAla (UGC) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.
Abstract：Pathogen detection is critical to the process of generating and testing powdered infant formula (PIF). An obstacle associated with PIF microbial surveillance is that most current procedures are time-consuming and labor-intensive. We have developed a rapid, DNA microarray-based detection technique to identify 10 different pathogenic bacteria associated with PIF contamination based on the 16S–23S rRNA gene internal transcribed spacer (ITS) sequences and wzy (O antigen polymerase) gene. Using this procedure, Enterobacter sakazakii, Salmonella species, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Acinetobacter baumannii, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli O157 were identified. One hundred eighty-five strains were used to validate the microarray assay (including 134 target pathogen strains and 51 closely related bacteria). Twenty-seven probes reproducibly detected multiple pathogens with high specificity and sensitivity (0.100 ng genomic DNA or 104 CFU/ml). Twenty-one real PIF samples were tested by the microarray with 100% accuracy. The data presented reveal that the designed oligonucleotide microarray is a promising method for basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance.
Abstract：Shigella is the cause of shigellosis or bacillary dysentery, the occurrence of which is estimated to be 165 million cases per year worldwide, resulting in 1.1 million deaths. Rapid and reliable assays for detecting and identifying Shigella in food, environmental and clinical samples are therefore necessary. Shigella species are traditionally identified by their O antigens. This study developed a DNA microarray targeting O-serotype-specific genes to detect all 34 distinct O-antigen forms of Shigella, including Shigella boydii types 1–18, Shigella dysenteriae types 1–13, Shigella flexneri types 1–5 and 6, and Shigella sonnei. A total of 282 strains were used to test the specificity of the microarray, including 186 Shigella and Escherichia coli representative strains, 86 Shigella clinical isolates and ten strains of other bacterial species that are commonly isolated from food or clinical stool specimens. The oligonucleotide probes were printed on the microarray in concentrations from 1 to 100 µM, and 10µM proved to be the optimal probe concentration. The detection sensitivity for each serotype was 50 ng genomic DNA or 1 c.f.u. in 25 g milk powder sample following a 6 h enrichment in broth. The microarray is specific, sensitive and reproducible, and, to our knowledge, is the first report of a microarray for serotyping all O-antigen forms of Shigella.