Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli , and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach. These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease.

The formation of attaching and effacing (A/E) lesions on intestinal epithelial cells is an essential step in the pathogenesis of human enteropathogenic and enterohemorrhagicEscherichia coliand of the mouse pathogenCitrobacter rodentium. The genes required for the development of the A/E phenotype are located within a pathogenicity island known as thelocus ofenterocyteeffacement (LEE). The LEE-encoded transcriptional regulators Ler, an H-NS-like protein, and GrlA, a member of a novel family of transcriptional activators, positively control the expression of the genes located in the LEE and their corresponding virulence. In this study, we usedC. rodentiumas a model to study the mechanisms controlling the expression of Ler and GrlA. By deletion analysis of thelerandgrlRAregulatory regions and complementation experiments, negative and positivecis-acting regulatory motifs were identified that are essential for the regulation of both genes. This analysis confirmed that GrlA is required for the activation ofler, but it also showed that Ler is required for the expression ofgrlRA, revealing a novel regulatory loop controlling the optimal expression of virulence genes in A/E pathogens. Furthermore, our results indicate that Ler and GrlA induce the expression of each other by, at least in part, counteracting the repression mediated by H-NS. However, whereas GrlA is still required for the optimal expression oflereven in the absence of H-NS, Ler is not needed for the expression ofgrlRAin the absence of H-NS. This type of transcriptional positive regulatory loop represents a novel mechanism in pathogenic bacteria that is likely required to maintain an appropriate spatiotemporal transcriptional response during infection.

The occurrence, serotype diversity, and antimicrobial resistance of Salmonella bacteria in commercial ground beef at retail establishments were investigated. Salmonella was isolated from 135 (56.7%) of 238 ground beef samples collected at the same number of butcher's shops located in three municipalities of Jalisco State, Mexico, during an 11-month period. The isolation frequency differed by municipality (P < 0.05) and was higher (P < 0.05) during the warm season (68.5%) than during the cold season (43.2%). Overall, 25 serotypes and 8 serogroups were identified among 135 Salmonella isolates; predominant were Salmonella group B (9.6%), Salmonella Anatum (8.9%), Salmonella Agona (6.7%), Salmonella Infantis (6.7%), and Salmonella Typhimurium (5.9%). All Salmonella isolates were tested for susceptibility to 11 antimicrobial drugs of human and veterinary use. Resistance to tetracycline was the most commonly observed (40.7%), followed by resistance to streptomycin (35.6%), trimethoprim-sulfamethoxazole (20.7%), and nalidixic acid (19.3%). Thirty-seven Salmonella isolates (27.4%) were multidrug resistant, and the majority corresponded to Salmonella Group B, Salmonella Anatum, and Salmonella Typhimurium. Three Salmonella isolates were resistant to seven different antimicrobials. The frequency of Salmonella in ground beef samples (56.7%) was higher than that observed in our previous investigation on beef carcasses (15.4%) at small abattoirs in the same region of Mexico. This may be a result of increasing contamination at these two points of the raw-beef production chain or may be an effect of the grinding process that facilitates a more-homogeneous pathogen distribution in the product. Poor hygiene, temperature abuse, and practices allowing cross-contamination during ground beef fabrication at these retail establishments increase the consumer's exposure to Salmonella.

The prevalence and antimicrobial resistance of Salmonella serotypes on beef carcasses from four small abattoirs in Jalisco State, Mexico, were investigated during a 10-month period. Following U.S. Department of Agriculture Food Safety and Inspection Service protocols, Salmonella was isolated from 78 (15.4%) beef carcasses (n = 505) after the final carcass water wash. Isolation frequency differed by establishment (P < 0.05) and was higher (P < 0.05) during the wet season (May through September) for all establishments. Thirteen Salmonella serotypes and four serogroups (partially serotyped isolates) were identified. The most prevalent were Salmonella enterica Give (24.4%), Salmonella Typhimurium (17.9%), and Salmonella Group B (14.1%). Antimicrobial susceptibility was tested against 11 drugs, and results indicated that 46.2% of the isolates were resistant to tetracycline, 42.3% were resistant to streptomycin, 23.1% were resistant to chloramphenicol, 21.8% were resistant to trimethoprim-sulfamethoxazole, and 19.2% were resistant to gentamicin. No resistance to ceftriaxone or ciprofloxacin was observed, and 33% of the isolates were resistant to three or more antimicrobials. Although Salmonella Give was the most prevalent serotype, 95% of the isolates of this serotype were susceptible to all antimicrobials tested. Antimicrobial resistance was more common in Salmonella Typhimurium, and 93% (13 of 14) of the isolates of this serotype were resistant to at least five antimicrobials. The frequency of multidrug-resistant Salmonella isolates differed among establishments (P < 0.05) and may be related to the origin of the cattle presented for harvesting. These findings highlight the need for control measures to reduce Salmonella prevalence on beef carcasses in small abattoirs in Mexico and for strategies to ensure the cautious use of antimicrobials in animal production to prevent and control the spread of antimicrobial-resistant foodborne pathogens.

Salmonellais one of the major foodborne pathogens worldwide. The antimicrobial resistance (AMR) of this foodborne pathogen has raised a great concern in recent years. Studies on the frequency and characterization ofSalmonellaserotypes can help to improve our knowledge on the epidemiology of this pathogen. The purpose of this study was to compare the serotypes, AMR and genetic profiles ofSalmonellaisolates recovered from raw beef throughout the beef production chain and from human feces associated with clinical cases of salmonellosis. The serotype, AMR and pulsed-field gel electrophoresis profile of 243Salmonella entericaisolates recovered from beef carcasses (n = 78), ground beef (n = 135), and human feces from clinical cases of salmonellosis (n = 30) were compared. Forty-three differentSalmonellaserotypes were identified and regardless of the source, the top five corresponded to Typhimurium, Give, Group B (partially serotyped), Infantis and Anatum. Twelve serotypes from beef carcasses were also found in ground beef, showing their presence throughout the beef production chain.SalmonellaTyphimurium, Infantis, Anatum and Montevideo were the only serotypes identified in all sample types. Resistance to tetracyclines was the most frequent (41.2%) followed by resistance to aminoglycosides (37%), folate pathway inhibitors (21%), quinolones (20.2%), phenicols (17.1%), penicillins (15.6%) and cephems (7%). Multidrug resistance was observed in 28.8% of the isolates, and those from human feces showed resistance to a larger number of antimicrobials. Great concern arises from the resistance and reduced susceptibility observed to quinolones and cephalosporins because these drugs are the first line of treatment for invasiveSalmonellainfections. Twenty-seven distinct pulse-types were detected among 238 isolates. Clustering analysis for the most frequent serotypes identified groups of isolates with similar AMR profiles. Multidrug resistance spreading throughout the food production chain should be continually monitored and its importance emphasized.