While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA, aceK, mdh, icdA, manB, and spaN). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB, fimA, and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This "three-gene sequence typing scheme" allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:-is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.

Salmonella is the leading cause of known food-borne bacterial infections in the United States, with an incidence rate of approximately 15 cases per 100,000 people. The rise of antimicrobial-resistant Salmonella subtypes, including the appearance of subtypes resistant to ceftriaxone, represents a particular concern. Ceftriaxone is used to treat invasive cases of Salmonella in children and is closely related to ceftiofur, an antibiotic commonly used to treat diseases of cattle. In order to develop a better understanding of the evolution and transmission of ceftiofur resistance in Salmonella, we characterized ceftiofur-resistant and -sensitive Salmonella isolates from seven New York dairy farms. A total of 39 isolates from these seven farms were analyzed for evolutionary relatedness (by DNA sequencing of the Salmonella genes fimA, manB, and mdh), antibiotic resistance profiles, and the presence of bla CMY-2 , a beta-lactamase gene associated with resistance to cephalosporins. Our data indicate that (i) resistance to ceftriaxone and ceftiofur was highly correlated with the presence of bla CMY-2 ; (ii) ceftiofur-resistant Salmonella strains were geographically widespread, as shown by their isolation from farms located throughout New York State; (iii) ceftiofur-resistant Salmonella strains isolated from farms represent multiple distinct subtypes and evolutionary lineages, as determined by serotyping, DNA sequence typing, and antimicrobial-resistance profiles; and (iv) ceftiofur-resistant Salmonella strains evolved by multiple independent acquisitions of an identical bla CMY-2 allele and by clonal spread of ceftiofurresistant subtypes.

In Listeria monocytogenes the alternative sigma factor B plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the B regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ⌬sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was B dependent; 17 of these proteins were found to require the presence of B for full expression, while 21 were expressed at a higher level in the ⌬sigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for Listeria monocytogenes is a gram-positive facultative intracellular pathogen associated with a life-threatening disease in humans (43). Infections occur in immunocompromised individuals following ingestion of contaminated food. The success of L. monocytogenes as a human pathogen is determined in part by the presence of specialized virulence genes that allow it to multiply and spread within the host and in part by its ability to persist in harsh environmental conditions. It is now clear that there is a strong link between the virulence potential of L. monocytogenes and its ability to tolerate stress. Several studies have identified genes that play important roles in stress tolerance and virulence. For example, genes involved in osmotic stress tolerance (11, 37), acid tolerance (40), oxidative stress tolerance (17), and bile salt tolerance (36) have all been implicated in the virulence of L. monocytogenes. The link between stress tolerance and virulence is further underlined by the fact that several dedicated virulence genes are stress inducible (38,39).In all bacteria an effective response to stress requires rapid adaptation at the transcriptional level to changing environmental conditions. L. monocytogenes regulates the transcription of many genes with stress-related functions by employing an alternative sigma factor, B . Mutants lacking B display increased sensitivity to a range of stresses, such as osmotic pressure (4, 21, 42), acid (19,42,46), heat, ethanol, and oxidative stress (18), cold (4), and high hydrostatic pressure (44). Deletion of sigB also decreases the ability of L. monocytogenes to survive in the presence of bacteriocins and antibiotics (5).More recently, roles for B in regulating the expression of dedicated virulence genes have been described. A mutant lacking B expresses the internalin gene, inlA, at a reduced level, and this correlates with a defect in its ability to invade human epithelial cells (25). Recently, B was also shown to be solely responsible for transcription of the inlC2 and inlD internalin genes, while it coregulates expression of the inlA...

By integrating genotype information, microRNA transcript abundances and mRNA expression levels, Eric Schadt and colleagues provide insights into the genetic basis of microRNA gene expression and the role of microRNAs within the liver gene-regulatory network.