Romney M. Humphries

The Cpx envelope stress response both facilitates and inhibits elaboration of the enteropathogenic Escherichia coli bundle-forming pilus.

The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope‐spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle‐forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co‐ordinates the repression of cell‐surface structures during periods of envelope stress.

N-acetyllactosamine-induced retraction of bundle-forming pili regulates virulence-associated gene expression in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi‐step process culminating in the intimate adherence of the organisms to the hosts intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle‐forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin‐like properties, which enables BFP to initially tether EPEC to N‐acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc‐bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc‐BSA‐induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.

Sticky situation: localized adherence of enteropathogenic Escherichia coli to the small intestine epithelium

Enteropathogenic Escherichia coli (EPEC) primarily cause gastrointestinal illness in neonates. They accomplish this by a complex coordinated multistage strategy, whereby the organisms colonize the epithelial lining of the small intestine. This process can be divided into four stages: first, localized, nonintimate adherence; second, type III secretion-mediated injection of effector proteins, third effacement of microvilli and, finally, intimate adherence. In this article, we review the history and current state of knowledge, as well as present potential future directions for further investigating the fascinating processes by which EPEC and related organisms colonize the human intestine and cause disease.

From alpha to beta : identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin

Bundle‐forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, α and β, based on their amino acid sequences. Alpha bundlins are also N‐acetyllactosamine‐ (LacNAc) specific lectins and bind to HEp‐2 cells, whereas β bundlins do not display these characteristics. The four surface‐exposed regions of amino acid sequence heterogeneity between α and β bundlin were therefore investigated as potential LacNAc‐specific carbohydrate‐binding domains in a bundlin. Mutation of one of these domains, 137‐GENNI‐141, in α1 bundlin to that of β bundlin (136‐SPDST‐140) resulted in BFP that no longer bound to LacNAc or HEp‐2 cells. Conversely, mutating the β3 bundlin gene to encode the α bundlin sequence at this domain resulted in the gain of HEp‐2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI→GENNT altered the α1 bundlin carbohydrate‐binding specificity from LacNAc to the Lewis X glycan sequence.

Interactions of Enteropathogenic Escherichia coli with Pediatric and Adult Intestinal Biopsy Specimens during Early Adherence

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea almost exclusively in young children. The basis for this age discrimination has never been determined, but it may be related to host cell receptors. During infection, EPEC strains express type IV bundle-forming pili composed of repeating subunits of the protein called bundlin. The very first interaction between EPEC and in vitro-cultured epithelial cells is mediated by the binding of α-bundlin to a carbohydrate receptor that contains, at a minimum, the N-acetyllactosamine (LacNAc) glycan sequence. However, bundlins expressed from the β-bundlin allele do not bind LacNAc glycan sequences. Herein, we investigated whether EPEC strains use α-bundlin to mediate early adherence to human intestinal biopsy specimens cultured in vitro by assessing the ability of isogenic EPEC mutants expressing either the α1- or β1-bundlin allele or a bundlin-deficient EPEC strain to bind to these specimens. Furthermore, we directly compared the abilities of a wild-type EPEC strain to bind to the epithelial surfaces of both human adult and pediatric biopsy specimens. Our results demonstrate that β-bundlin does not act as an adhesin during early EPEC adherence to adult duodenal biopsy specimens. The results also indicate that EPEC binds equally well to adult and pediatric biopsy specimens in an early adherence assay. This result is supported by the finding that the early adherence of EPEC to both adult and pediatric biopsy specimens was inhibited by LacNAc neoglycoconjugates, suggesting that organisms expressing α-bundlin-type bundle-forming pili initially bind to related glycan receptors in both age groups.

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection.

CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests

ABSTRACT Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

Impact of 21st Century Cures Act on Breakpoints and Commercial Antimicrobial Susceptibility Test Systems: Progress and Pitfalls

ABSTRACT Antimicrobial resistance is the most pressing medical challenge of the past decade. At the front line are clinical laboratories, which are responsible for accurately reporting antimicrobial susceptibility test (AST) results to clinicians and public health authorities. The ability of the laboratory to detect resistance has been hampered by several factors. In 2016, the 21st Century Cures Act was signed into law, marking an important step toward resolving many regulatory dilemmas that hampered development and updates to commercial AST systems (cASTs). We describe the pathway and history of U.S. regulation of cASTs and outline both the rewards and unmet needs possible from the 21st Century Cures Act.

Practical methods for effective vancomycin-resistant enterococci (VRE) surveillance: experience in a liver transplant surgical intensive care unit

OBJECTIVEnWe evaluated the utility of vancomycin-resistant Enterococcus (VRE) surveillance by varying 2 parameters: admission versus weekly surveillance and perirectal swabbing versus stool sampling.nnnDESIGNnProspective, patient-level surveillance program of incident VRE colonization.nnnSETTINGnLiver transplant surgical intensive care unit (SICU) of a tertiary-care referral medical center with a high prevalence of VRE.PatientsAll patients admitted to the SICU from June to August 2015.nnnMETHODSnWe conducted a point-prevalence estimate followed by admission and weekly surveillance by perirectal swabbing and/or stool sampling. Incident colonization was defined as a negative screen followed by positive surveillance. VRE was detected by culture on Remel Spectra VRE chromogenic agar. Microbiologically-confirmed VRE bloodstream infections (BSIs) were tracked for 2 months. Statistical analyses were calculated using the McNemar test, the Fisher exact test, the t test, and the χ2 test.nnnRESULTSnIn total, 91 patients underwent VRE surveillance testing. The point prevalence of VRE colonization was 60.9%; VRE prevalence on admission was 30.1%. Weekly surveillance identified an additional 7 of 28 patients (25.0%) with incident colonization. VRE BSIs were more common in VRE-colonized patients than in noncolonized patients (8 of 43 vs 2 of 48; P=.028). In a direct comparison, perirectal swabs were more sensitive than stool samples in detecting VRE (64 of 67 vs 56 of 67; P=.023). Compliance with perirectal swabbing was 89% (201 of 226) compared to 56% (127 of 226) for stool collection (P≤0.001).nnnCONCLUSIONSnWe recommend weekly VRE surveillance over admission-only screening in high-burden units such as liver transplant SICUs. Perirectal swabs had greater collection compliance and sensitivity than stool samples, making them the preferred methodology. Further work may have implications for antimicrobial stewardship and infection control.