A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB,entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection offemA was used as an internal positive control. The multiplex PCR assay combined the primers for sea tosee and femA in one set and those foreta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.

A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx 1 , stx 2 , stx 2c , stx 2d , stx 2e , stx 2f , EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.

The Walkerton (Ontario, Canada) outbreak of waterborne Escherichia coli O157:H7 and Campylobacter jejuni was quite limited in both space and time, making it a good model for exploring the utility of different typing and subtyping methods for the characterization of relationships among isolates of these organisms. We have extended previous work with these organisms through analysis by the Oxford multilocus sequence typing (MLST) and the flagellin short variable region (fla-SVR) sequencing methods. Additional isolates not epidemiologically related to the Walkerton outbreak have also been included. Both sequencing methods identified and differentiated between Walkerton outbreak strains 1 and 2. When these strains were compared with isolates that were not part of the outbreak, the information produced by the fla-SVR method more often correlated with epidemiological findings than that produced by MLST, though both methods were required for optimal discrimination. The MLST data were more relevant in terms of the overall population structure of the organisms. Both mutation and recombination appeared to be responsible for generating diversity among the isolates tested.

No abstract

Identification and typing of bacteria occupy a large fraction of time and work in clinical microbiology laboratories. With the certification of some MS platforms in recent years, more applications and tests of MS‐based diagnosis methods for bacteria identification and typing have been created, not only on well‐accepted MALDI‐TOF‐MS‐based fingerprint matches, but also on solving the insufficiencies of MALDI‐TOF‐MS‐based platforms and advancing the technology to areas such as targeted MS identification and typing of bacteria, bacterial toxin identification, antibiotics susceptibility/resistance tests, and MS‐based diagnostic method development on unique bacteria such as Clostridium and Mycobacteria. This review summarizes the recent development in MS platforms and applications in bacteria identification and typing of common pathogenic bacteria.

Sulfonic acid functionalized mesoporous SBA-15 materials were prepared using the co-condensation and grafting methods, respectively, and their catalytic performance in the dehydration of xylose to furfural was examined. SBA-15-SO 3 H(C) prepared by the co-condensation method showed 92-95% xylose conversion and 74% furfural selectivity, and 68-70% furfural yield under the given reaction conditions. The deactivation and regeneration of the SBA-15-SO 3 H(C) catalyst for the dehydration of xylose was also investigated. The results indicate that the used and regeneration catalysts retained the SBA-15 mesoporous structure, and the S content of SBA-15-SO 3 H(C) almost did not change. The deactivation of the catalysts is proposed to be associated with the accumulation of byproducts, which is caused by the loss reaction of furfural. After regeneration by H 2 O 2 , the catalytic activity of the catalyst almost recovered.