Prevalence and mechanisms of resistance to carbapenems in Enterobacteriaceae

Objectives: To determine the point prevalence of carbapenem-non-susceptible Enterobacteriaceae (CNSE) and carbapenemase-producing Enterobacteriaceae (CPE) isolates among hospitalized patients in Belgium. Methods: Twenty-four hospital-based laboratories prospectively collected 200 non-duplicated Enterobacteriaceae isolates from clinical specimens of hospitalized patients over a 2 month period. All isolates were screened locally for decreased susceptibility to carbapenem drugs using a disc diffusion method according to CLSI interpretative criteria. CNSE strains were referred centrally for confirmation of carbapenemase by phenotypic and molecular testing. Results: From February to April 2012, 158 of the 4564 screened Enterobacteriaceae isolates were categorized as non-susceptible to carbapenems, resulting in a point prevalence of CNSE of 3.5% (95% CI: 2.9%–4.2%; range per centre: 0.5%–8.5%). Of the 125 referred CNSE isolates, 11 Klebsiella pneumoniae isolates [OXA-48 (n=7), KPC type (n=3) and NDM type (n=1)], 1 OXA-48-positive Escherichia coli isolate and 1 KPC-positive Klebsiella oxytoca isolate were detected in eight hospitals. None of the 72 carbapenem-non-susceptible Enterobacter spp. isolates were confirmed as CPE. The minimal estimated point prevalence of CPE isolates was 0.28% (13/ 4564; 95% CI: 0.13%–0.44%) overall (range per centre: 0%–1.5%). Conclusions: Despite the overall low prevalence of CNSE found in this study, the detection of CPE isolates in one-third of the participating centres raises concerns and highly suggests the spread and establishment of CPE in Belgian hospitals

Nanoscale probing of the mycobacterial cell wall

Studying the structure and functions of microbial cell walls is essential given the important role they play in various cellular processes and diseases, as well as their potential as drug targets. In this context, the objective of this thesis is to gain detailed molecular insight into the nanoscale properties of the medically-important organism Mycobacterium bovis BCG. To reach this goal, we developed advanced nanoscale and single-molecule atomic force microscopy (AFM) methods for analyzing microbial surfaces in physiological conditions (cell immobilization, tip functionalization, single-molecule detection and localization). Using AFM techniques, we: (i) probed the surface structure of mycobacteria before and after treatment with the antibiotic ethambutol, revealing that the drug causes major alterations of the cell wall, (ii) mapped the localization of single lipoarabinomannan molecules, demonstrating that these are not exposed on native mycobacteria, but hidden by an outermost layer of mycolic acids, (iii) measured the specific binding forces of the mycobacterial heparin binding adhesin (HBHA), that mediate homophilic HBHA-HBHA interactions, HBHA-actin interactions, and HBHA-receptor interactions, and (iv) analyzed the surface distribution and binding strength of mycobacterial fibronectin attachment proteins (FAPs), showing that these adhesins are widely exposed on the mycobacterial surface. Collectively, our data provide novel insight into the structure-function relationships of the mycobacterial cell wall. The nanoscale and single-molecule methods developed here complement traditional proteomic and molecular biological approaches for the functional analysis of surface-associated proteins, and may help in the search for novel anti-microbial drugs.(AGRO 3) -- UCL, 200

Direct measurement of Mycobacterium-fibronectin interactions.

Bacterial surface-associated proteins play essential roles in mediating pathogen-host interactions and represent privileged targets for anti-adhesion therapy. We used atomic force microscopy (AFM) to investigate, in vivo, the binding strength and surface distribution of fibronectin attachment proteins (FAPs) in Mycobacterium bovis bacillus Calmette-Guérin (BCG). We measured the specific binding forces of FAPs ( approximately 50 pN) and found that they increased with the loading rate, as observed earlier for other receptor-ligand systems. We also mapped the distribution of FAPs, revealing that the proteins are widely exposed on the mycobacterial surface. To demonstrate that the proteins are surface-associated, we showed that treatment of the cells with pullulanase, an enzyme possessing carbohydrate-degrading activities, or with protease, an enzyme that conducts proteolysis, led to a substantial reduction of the FAP surface density. A similar trend was also noted following treatment with ethambutol, an antibiotic which inhibits the synthesis of cell wall polysaccharides. The nanoscale analyses presented here complement traditional proteomic and molecular biology approaches for the functional analysis of surface-associated proteins, and may help in the search for novel anti-adhesive drugs

Probing molecular recognition sites on biosurfaces using AFM.

Knowledge of the molecular forces that drive receptor-ligand interactions is a key to gain a detailed understanding of cell adhesion events and to develop novel applications in biomaterials science. Until recently, there was no tool available for analyzing and mapping these forces on complex biosurfaces like cell surfaces. During the past decade, however, single-molecule atomic force microscopy (AFM) has opened exciting new opportunities for detecting and localizing molecular recognition forces on artificial biosurfaces and on living cells. In this review, we describe the general principles of the AFM technique, present procedures commonly used to prepare samples and tips, and discuss a number of applications that are relevant to the field of biomaterials

The NTA-His6 bond is strong enough for AFM single-molecular recognition studies.

There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies

Exploring the molecular forces within and between CbsA S-layer proteins using single molecule force spectroscopy.

We used single molecule atomic force microscopy (AFM) to gain insight into the molecular forces driving the folding and assembly of the S-layer protein CbsA. Force curves recorded between tips and supports modified with CbsA proteins showed sawtooth patterns with multiple force peaks of 58+/-26pN that we attribute to the unfolding of alpha-helices, in agreement with earlier secondary structure predictions. The average unfolding force increased with the pulling speed but was independent on the interaction time. Force curves obtained for CbsA peptides truncated in their C-terminal region showed similar periodic features, except that fewer force peaks were seen. Furthermore, the average unfolding force was 83+/-45pN, suggesting the domains were more stable. By contrast, cationic peptides truncated in their N-terminal region showed single force peaks of 366+/-149pN, presumably reflecting intermolecular electrostatic bridges rather than unfolding events. Interestingly, these large intermolecular forces increased not only with pulling speed but also with interaction time. We expect that the intra- and intermolecular forces measured here may play a significant role in controlling the stability and assembly of the CbsA protein

Single-molecule force spectroscopy of microbial cell envelope proteins

Most microbes possess a well-deined cell envelope, consisting of a plasma membrane and of a cell wall, that presumably evolved in the course of evolution by selection in response to environmental and ecological pressures.1 Because the envelope represents the boundary between the external environment and the cell, it plays several important roles, including determining cellular shape, growth and division, enabling the organisms to resist turgor pressure, acting as molecular sieves, interacting with drugs and mediating molecular recognition and cellular interactions

Single-molecule force spectroscopy of microbial cell envelope proteins

Most microbes possess a well-deined cell envelope, consisting of a plasma membrane and of a cell wall, that presumably evolved in the course of evolution by selection in response to environmental and ecological pressures.1 Because the envelope represents the boundary between the external environment and the cell, it plays several important roles, including determining cellular shape, growth and division, enabling the organisms to resist turgor pressure, acting as molecular sieves, interacting with drugs and mediating molecular recognition and cellular interactions

Fishing single molecules on live cells

A crucial challenge in cell biology is to understand how cell surface-associated molecules are organized, and how they interact with their environment. Clarification of these issues is central to our understanding of the functions of cell surfaces (e.g. cell adhesion), and of their implication in biomedicine (e.g. pathogen interactions). The past years have witnessed rapid progress in our use of atomic force microscopy (AFM) to map the distribution of single polysaccharides and proteins on live cells, and to measure their molecular interactions. These nanoscale analyses complement traditional glycomic and proteomic approaches for the functional analysis of surface-associated molecules, and may help in the search for novel drugs. (C) 2009 Elsevier Ltd. All rights reserved