SummaryEscherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn) Factor XIa, and kallikrein of the coagulation and the inflammation systems respectively. Additionally, C1-INH competes with Factor B for binding to C3b to inhibit the activation of the alternative complement pathway (Jiang et al., 2001). Circulating C1-INH has a M r of 105 kDa, with post-translational glycosylations accounting for nearly half of its mass. Most glycosylations occur in the N-terminal 100 amino acids that are unique to this serpin. Desialylation does not affect the in vitro inhibitory activity of C1-INH, but reduces its circulating half-life in rabbits from >24 h to 3-5 min (Minta, 1981). C1-INH interacts with its target proteases to form large SDS-insoluble complexes that are subsequently removed from circulation (reviewed in Caliezi et al., 2000). Also, data suggest that surfaceassociated C1-INH protects cells from proinflammatory events at their surface (Schmaier et al., 1989;Schmaier et al., 1993;Caliezi et al., 2000).In this article, we describe the identification and characterization of a zinc metalloprotease produced by E. coli O157:H7 that cleaves C1-INH. This protein, termed StcE, is encoded by a gene on pO157, secreted via the etp type II secretion pathway, is positively regulated by Ler, and is highly specific for its substrate. Results Lysates of E. coli carrying pO157 aggregate cultured human T cell linesTo find novel E. coli O157:H7 virulence factors, we looked for cytopathic effects in Jurkat cells, a human T cell lymphoma line, treated with lysates of STEC strains EDL933 and EDL933cu, E. coli K-12 strain C600, C600 carrying pO157 (WAM2035), enteropathogenic E. coli strain E2348/69, and the mouse pathogen Citrobacter rodentium strain DBS100. Lysates of wild-type and transformed E. coli containing pO157 aggregated Jurkat cells (Fig. 1A); lysates of E. coli without pO157 (Fig. 1B) or other bacteria able to induce the A/E phenotype, such as E2348/69 and DBS100, did not. Lysates of EDL933, but not EDL933cu, aggregated MOLT-4 cells ( Fig. 1C and D) but not HL-60, U937, or Raji cells (data not shown), suggesting T cell-lineage specificity for this effect. Identification and cloning of stcETo localize the gene(s) on pO157 responsible for this unusual phenotype, we subjected pO157 to mutagenesis using a minitransposon. The location of the transposon insertion of one mutant whose lysate was unable to aggregate Jurkat cells (WAM2553) was determined to be position 23 772 of pO157 (position based on GenBank accession no. #AF074613). The ORF into which the transposon inserted was designated L7031 (tagA) (Burland et al., 1998), and is...

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.

Infectious diseases caused by bacterial pathogens are a worldwide burden. Serious bacterial infection-related complications, such as sepsis, affect over a million people every year with mortality rates ranging from 30% to 50%. Crucial clinical microbiology laboratory responsibilities associated with patient management and treatment include isolating and identifying the causative bacterium and performing antibiotic susceptibility tests (ASTs), which are labor-intensive, complex, imprecise, and slow (taking days, depending on the growth rate of the pathogen). Considering the life-threatening condition of a septic patient and the increasing prevalence of antibiotic-resistant bacteria in hospitals, rapid and automated diagnostic tools are needed. This review summarizes the existing commercial AST methods and discusses some of the promising emerging AST tools that will empower humans to win the evolutionary war between microbial genes and human wits.

We used real-time polymerase chain reaction (PCR) targeting the cdc2 gene and direct fluorescent microscopy examination (DFME) to evaluate the prevalence of Pneumocystis jirovecii among immunocompetent patients without clinical pulmonary infection and immunosuppressed patients evaluated for opportunistic pulmonary infections. Among 102 bronchoalveolar lavage samples collected from immunocompetent patients without infection, none tested positive for P. jirovecii by either DFME or real-time PCR despite the presence of other comorbidities. Among patients with suspected pulmonary infection and tested with either assay, real-time PCR produced a higher number of positive results compared to DFME and increased P. jirovecii detection by 7% when added to DFME-negative samples. Real-time PCR may have increased sensitivity for P. jirovecii detection over DFME and decrease the risk of sample contamination compared to conventional and nested PCR. The use of single-copy gene targets (e.g., cdc2) may lower the rate of "colonization" detection and confer a high predictive value for Pneumocystis pneumonia.

In recent years, advances in PCR techniques have aided in the rapid and accurate detection of common respiratory pathogens from patient specimens. Multiplex PCR can identify and differentiate a large panel of viral and bacterial targets simultaneously. Published studies have shown that multiplex PCR panels are more rapid and more sensitive methods of virus detection than cultures or antigen detection (1, 2). One such method, the FilmArray respiratory panel (FARP) (BioFire Diagnostics, Inc.), is a multiplex, nested PCR technique that can detect 17 common respiratory viruses and 3 bacterial targets in a single reaction in just over 1 h (3). Published studies have shown that for both immunocompetent and immunocompromised patients, the FARP identifies significantly more viral pathogens in both bronchoalveolar lavage (BAL) fluid and nasopharyngeal (NP) samples than viral cultures and direct fluorescent antibody staining and that the FARP is among the most sensitive of the available multiplex assays (1, 4-10). In addition, the FARP has a low hands-on time and very fast turnaround time. Since the FARP is associated with a significant cost to the laboratory and the patient, its judicious use is necessary.Choosing the least invasive, highest yield, and most cost-effective investigations in a stepwise manner has always been central to the practice of medicine. Patients who present with symptoms of a respiratory tract infection often undergo testing for respiratory viruses. The initial testing at our center may involve collection of an NP sample for influenza A and B and respiratory syncytial viruses. The comprehensive FARP is obtained for patients with complex conditions or those who are immunocompromised and have symptoms of upper or lower respiratory tract infections. In the presence of concurrent pulmonary infiltrates, fever, and hypoxia, patients (especially if immunocompromised) may then undergo a bronchoscopy with BAL with repeat FARP testing on the BAL sample. To date, no studies have compared the yield of FARP on BAL samples with the yield on NP samples. Whether additional microbiologic information is obtained from FARP testing on a BAL sample after testing on a NP swab is not known. This retrospective case-control study evaluates the concordance between FARP testing on NP and BAL samples.(Part of this research was presented as a poster at the American Thoracic Society International Conference, Denver, CO, 15 to 20 May 2015.) MATERIALS AND METHODSWe retrospectively reviewed the electronic medical records of all patients evaluated at the Mayo Clinic in Arizona between 1 June 2013, and 31 May 2014, who had FARP testing on both NP and BAL samples. All patients who were included had a BAL sample FARP (BAL FARP) performed within 7 days after an NP swab FARP (NP FARP) during the same hospitalization or illness episode. FARP results obtained on tracheal aspirates were excluded.Patient electronic medical records were reviewed, and the following information was obtained: demographics (age and sex) and the presence of immun...

Timely determination of antimicrobial susceptibility for a bacterial infection enables precision prescription, shortens treatment time, and helps minimize the spread of antibiotic resistant infections. Current antimicrobial susceptibility testing (AST) methods often take several days and thus impede these clinical and health benefits. Here, we present an AST method by imaging freely moving bacterial cells in urine in real time and analyzing the videos with a deep learning algorithm. The deep learning algorithm determines if an antibiotic inhibits a bacterial cell by learning multiple phenotypic features of the cell without the need for defining and quantifying each feature. We apply the method to urinary tract infection, a common infection that affects millions of people, to determine the minimum inhibitory concentration of pathogens from both bacteria spiked urine and clinical infected urine samples for different antibiotics within 30 min and validate the results with the gold standard broth macrodilution method. The deep learning video microscopy-based AST holds great potential to contribute to the solution of increasing drug-resistant infections.

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of food-borne outbreaks of diarrhea (15). Disease caused by STEC is characterized by abdominal pain and bloody diarrhea, and 5 to 15% of those individuals infected with serotype O157:H7 develop hemolytic uremic syndrome (HUS), a potentially life-threatening condition consisting of hemolytic anemia, thrombocytopenia, and kidney failure caused primarily by Stx (8). STEC may carry genes for one or both types of Stx, Stx1 and Stx2 (17).Although STEC strains are a diverse group of pathogens, up to the present, the most common serotype in the United States has been O157:H7. A common association is that of E. coli O157:H7 contaminating ground beef (3, 7), but recent large outbreaks have involved a variety of other foods, including leafy greens (6, 29). The diversity of potentially contaminated food means that patients may acquire STEC infection from many foodstuffs, far beyond the stereotypical risk of undercooked ground beef. The common denominator of tainted food products seems to be direct or indirect contamination from bovine feces. To best detect infected patients and potential outbreaks, clinical laboratories must have tools to quickly and accurately detect STEC in stool specimens. Culture on sorbitol MacConkey agar is an inexpensive, effective, and widely used method based on lack of sorbitol fermentation by E. coli O157:H7. Several drawbacks limit...

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4°C to 55°C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23°C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37°C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k cat /K m data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an antiinflammatory agent by localizing C1-INH to cell membranes.Enterohemorrhagic E. coli (EHEC) O157:H7 strains cause infections that result in diarrhea and hemorrhagic colitis, which can progress to hemolytic-uremic syndrome (17). These bacteria belong to a group of pathogens that form attaching and effacing (A/E) lesions, which are characterized by intimate adherence to the host cells and effacement of the microvilli (9, 17). The StcE protease produced by EHEC is secreted by the type II secretion system (15) that is encoded 3Ј of stcE on the pO157 virulence plasmid (2). Our laboratory has recently demonstrated that StcE contributes to intimate adherence of EHEC to host cells (12), a process essential for colonization of the terminal rectum of the bovine host (18). We have also shown that StcE cleaves C1 esterase inhibitor (C1-INH), a regulator of multiple inflammatory pathways (15), and localizes it to host cell surfaces (14).The current model for StcE activity (12) proposes that initially, StcE allows passage of EHEC through the oral cavity by cleaving the salivary glycoproteins that are responsible for bacterial aggregation. Similarly, in the colon, StcE cleaves the glycoproteins that protect the intestinal epithelial surface, allowing EHEC to come into close contact with host cell membranes, where components of the locus of enterocyte effacement mediate formation of the characteristic A/E lesions. Later during infection, Shiga toxins and other virulence factors compromise the intestinal epithelial and endothelial barriers, allowing blood with its complement effectors into the intestinal lumen (9, 17). At this point, StcE localizes C1-...