SUMMARY This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create highavidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.heterobifunctional ligand ͉ multivalency ͉ Shiga toxin

Shiga toxins (Stx1 and Stx2) are responsible for initiating haemolytic uraemic syndrome, a serious extraintestinal complication caused by enterohaemorrhagic Escherichia coli O157 : H7 infection in humans. Shiga toxins are classical AB 5 -type exotoxins, consisting of a globotriaosylceramide (Gb 3 )-binding B subunit pentamer and an enzymic A subunit. It is demonstrated in this study that Stx2 binds to human neutrophils by a non-classical mechanism that is independent of Gb 3 . In contrast, the investigation revealed that Stx2 binds to murine neutrophils by the classical Gb 3 -dependent mechanism. Moreover, whereas the human serum amyloid P (HuSAP) component inhibited Stx2 binding to murine neutrophils, HuSAP increased Stx2 binding to human neutrophils by 84.2 % (P¡0.002, Student's t-test). These observations may explain why HuSAP protects mice from the lethal effects of Stx2, whereas there is no indication that HuSAP plays a similar protective role in humans infected by E. coli O157 : H7.

SummarySynthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAcspecific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K assoc), to synthetic LacNAc-Benzene and LacNAc-O(CH2)8CONH2 glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express a bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the a bundlin monomer. Collectively, these results suggest that a bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of a bundlin-expressing EPEC strains to host intestinal epithelial cells.