The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% ofE. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produce attaching and effacing intestinal lesions, such as RDEC-1 (an EPEC of weanling rabbits) and enterohemorrhagic E. coli. The predicted amino acid sequence derived from the nucleotide sequence of eae shows significant homology to that of the invasin of Yersinia pseudotuberculosis.Enteropathogenic Escherichia coli (EPEC) are an important cause of infant diarrhea in the developing world (1-5). Diarrhea caused by EPEC can be severe, as evidenced by a 30% fatality rate in a recent nursery outbreak (5). Once a serious cause of "summer diarrhea" and nursery outbreaks in industrialized countries, diarrhea due to EPEC now occurs less frequently in these areas, although outbreaks in nurseries and day-care centers are reported occasionally (6, 7).Although EPEC were the first E. coli to be recognized as a diarrheal pathogen, the elucidation of EPEC virulence factors has lagged behind that of enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC). Unlike that of ETEC and EHEC, the pathogenesis of EPEC does not appear to involve a toxin and no fimbrial colonization factors have been described. A major advance in the understanding of EPEC pathogenesis was the demonstration that EPEC possess a high molecular weight plasmid which is required for full virulence in volunteers (8) and is associated with the ability to adhere to HEp-2 epidermoid carcinoma cells in a pattern described as localized adherence (9, 10). This adherence phenotype is a characteristic of E. coli of the major EPEC serotypes (11) and is detectable with a DNA probe derived from one such plasmid called the EAF probe (EPEC adherence factor).Perhaps the most important feature of EPEC pathogenesis is the ability of EPEC to produce characteristic histopathological intestinal lesions in humans or experimental animal models. This lesion has been described by Moon et al. (12) as an "attaching and effacing" (A/E) lesion and is characterized by the intimate adherence of bacteria to the enterocyte, dissolution of the brush border at the site of bacterial attachment, and disruption of the cellular cytoskeleton. Within the enterocyte, high concentrations of filamentous actin are present at the site of bacterial attachment and the enterocyte membrane is frequently seen cupping the bacteria, often forming a pedestal-like structure. The production of this lesion can occur in the absence of the EAF plasmid, as evidenced by the observation that A/E lesions are produced by EAF plasmid-cured derivatives of EPEC isolates in experimental an...

Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery1,2, spreading efficiently via low-dose fecal-oral transmission3,4. Historically, S. sonnei has been predominantly responsible for dysentery in developed countries, but is now emerging as a problem in the developing world, apparently replacing the more diverse S. flexneri in areas undergoing economic development and improvements in water quality4-6. Classical approaches have shown S. sonnei is genetically conserved and clonal7. We report here whole-genome sequencing of 132 globally-distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and has diversified into several distinct lineages with unique characteristics. Our analysis suggests the majority of this diversification occurred in Europe, followed by more recent establishment of local pathogen populations in other continents predominantly due to the pandemic spread of a single, rapidly-evolving, multidrug resistant lineage.

We assessed the ability of Streptococcus pneumoniae mutants deficient in either choline binding protein A (CbpA), pneumolysin (Pln), pyruvate oxidase (SpxB), autolysin (LytA), pneumococcal surface protein A, or neuraminidase A (NanA) to replicate in distinct anatomical sites and translocate from one site to the next. Intranasal, intratracheal, and intravenous models of disease were assessed in 4-week-old BALB/cJ mice by quantitation of bacterial titers in the relevant organs. Mice were also observed by use of real-time bioluminescent imaging (BLI). BLI allowed visualization of the bacteria in sites not tested by sampling. All mutants were created in D39 Xen7, a fully virulent derivative of capsular type 2 strain D39 that contains an optimized luxABCDE cassette. NanA, SpxB, and, to a lesser extent, CbpA contributed to prolonged nasopharyngeal colonization, whereas CbpA and NanA contributed to the transition to the lower respiratory tract. Once lung infection was established, Pln, SpxB, and LytA contributed to bacterial replication in the lungs and translocation to the bloodstream. In the bloodstream, only Pln and LytA were required for high-titer replication, whereas CbpA was required for invasion of the cerebrospinal fluid. We conclude that transitions between body sites require virulence determinants distinct from those involved in organ-specific replication.

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We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (S. aureus r ‫؍‬ 0.98; P. aeruginosa r ‫؍‬ 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 3 to 10 5 CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the lux constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.Microbial adhesion and biofilm formation on medical implants is a common occurrence and represents a serious medical problem. Since biofilm microorganisms are difficult to eradicate with antibiotic therapy, chronic, recurrent infections often develop. With the increased use of prosthetic biomedical implants, chronic nosocomial infections have become prevalent in recent years (9,41). Bacterial colonization of indwelling devices can be a prelude to both systemic infection and malfunction of the device.A variety of techniques, such as direct microscopic enumeration, total viable count, metabolically active dyes, radiochemistry, and fluorescence, have been used to investigate microbial biofilms (1,4,8,14,17,18,23,29,33,38). While some of these methods are useful for in vitro studies, they have not proved ideal for the investigation of biofilms in experimental infection models. The difficulty in analyzing biofilms in vivo lies in the lack of tools that allow noninvasive longitudinal study design. Assays developed to date, both direct and indirect, are timeconsuming and laborious and involve the extraction of bacteria from support surfaces. To better understand and control biofilms on medical devices, rapid, direct, nondestructive, realtime quantitative monitoring methods that are adaptable to the clinical situation are needed. These assays may be used to develop new preventive and therapeutic methods to combat biofilm related infections.To ...