Effective surface area on rough substrates for bacterial adhesion is examined by analyzing the solid area fraction of surfaces, where the bacterial medium is in contact with the solid surface.

To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a SalmoneUla-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonela organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100lo compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC)eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coliO157:H7 when ≥103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

Results suggested that vaccination against PCV2 was effective at reducing mortality rate and improving growth performance among pigs in a herd infected with PCV2.