Adhesion heterogeneity of individual bacterial cells in an axenic culture studied by atomic force microscopy.

The evaluation of bacterial adhesive properties at a single-cell level is critical for under standing the role of phenotypic heterogeneity in bacterial attachment and community formation. Bacterial population exhibits a wide variety of adhesive properties at the single-cell level, suggesting that bacterial adhesion is a rather complex process and some bacteria are prone to phenotypic heterogeneity. This heterogeneity was more pronounced for Escherichia coli, where two subpopulations were detected. Subpopulations exhibiting higher adhesion forces may be better adapted to colonize a new surface, especially during sudden changes in environmental conditions. Escherichia coli was characterized by a higher adhesion force, a stronger ability to form biofilm and larger heterogeneity index calculated in comparison with Bacillus subtilis. Higher adhesion forces are associated with a more efficient attachment of bacteria observed in an adhesion assay and might provide a basis for successful colonization, survival and multiplications in changing environment. The atomic force microscopy provides a platform for investigation of the adhesion heterogeneity of individual cells within a population, which may be expected to underpin further elucidation of the adaptive significance of phenotypic heterogeneity in a natural environment.

Dynamics, nanomechanics and signal transduction in reelin repeats.

Reelin is a large glycoprotein controlling brain development and cell adhesion. It regulates the positioning of neurons, as well as neurotransmission and memory formation. Perturbations in reelin signaling are linked to psychiatric disorders. Reelin participates in signal transduction by binding to the lipoprotein receptors VLDLR and ApoER2 through its central region. This part is rich in repeating BNR-EGF-BNR modules. We used standard molecular dynamics, steered molecular dynamics, and perturbation response scanning computational methods to characterize unique dynamical properties of reelin modules involved in signaling. Each module has specific sensors and effectors arranged in a similar topology. In the modules studied, disulfide bridges play a protective role, probably making both selective binding and protease activity of reelin possible. Results of single reelin molecule stretching by atomic force microscopy provide the first data on the mechanical stability of individual reelin domains. The forces required for partial unfolding of the modules studied are below 60 pN.

Effect of ampicillin on adhesive properties of bacteria examined by atomic force microscopy.

Discovery of new antibacterial agents requires the development of novel techniques for bacteria surface characterization after treatment with antibiotics. In this study, we investigate the effect of ampicillin at MICs levels on adhesive properties of Gram-positive and Gram-negative bacteria, using atomic force microscopy (AFM). Our results revealed that the treatment leads to changes of bacterial surface properties, especially cell surface roughness. A nanomechanical alteration of the cells led to an increase of adhesive forces and rupture lengths. Changes in adhesive properties are determined not only by the modification of physicochemical cell properties but also by an increase in roughness, leading to an increase of the contact area with a cantilever tip. We discovered that the contribution of non-specific physicochemical interactions in the bacteria attachment to a substrate is not negligible and was significantly influenced by the presence of antibiotic. Ampicillin caused much greater change in the adhesion properties of Bacillus subtilis than Escherichia coli due to the mode of action of ß-lactam antibiotic. Adhesion measurements may by a new way to investigate subtle changes of the bacterial surface properties caused by antibiotic, especially those targeting the bacterial cell wall. In contrast to nanoindentation assays, they provide information on adhesive properties of the bacteria surface.

Nanopuller-open data acquisition platform for AFM force spectroscopy experiments.

Atomic Force Microscope (AFM) is a widely used tool in force spectroscopy studies. Presently, this instrument is accessible from numerous vendors, albeit commercial solutions are expensive and almost always hardware and software closed. Approaches for open setups were published, as with modern low cost and readily available piezoelectric actuators, data acquisition interfaces and optoelectronic components building such force spectroscopy AFM is relatively easy. However, suitable software to control such laboratory made instrument was not released. Developing it in the lab requires significant time and effort. Our Nanopuller software described in this paper is intended to eliminate this obstacle. With only minimum adjustments this program can be used to control and acquire data with any suitable National Instruments universal digital/analog interface and piezoelectric actuator analog controller, giving significant freedom and flexibility in designing force spectroscopy experiment. Since the full code, written in a graphical LabVIEW environment is available, our Nanopuller can be easily customized. In this paper we describe the program and test its performance in controlling different setups. Successful and accurate force curve acquisition for standard samples (single molecules of I27O reference titin polyprotein and DNA as well as red blood cells) is shown.

Mechanical transition in a highly stretched and torsionally constrained DNA.

We show results of our high force (up to 1.8 nN) atomic force microscopy force spectroscopy measurements of a double stranded DNA. We have found that the force spectra of torsionally constrained molecules display a small plateau occurring at a force of approximately 1 nN. This transition is absent in molecules with rotational freedom. Based on all-atom molecular dynamics simulations, we suggest that this plateau is a result of reducing the diameter of a double helix through extreme stretching. The simulation suggests that the molecule is forced into a form resembling an underwound P-DNA, with bases protruding outside of the backbones. These results broaden our understanding of the fundamental aspects of DNA nanomechanics.

Nanomechanics of ß-rich proteins related to neuronal disorders studied by AFM, all-atom and coarse-grained MD methods.

Computer simulations of protein unfolding substantially help to interpret force-extension curves measured in single-molecule atomic force microscope (AFM) experiments. Standard all-atom (AA) molecular dynamics simulations (MD) give a good qualitative mechanical unfolding picture but predict values too large for the maximum AFM forces with the common pulling speeds adopted here. Fine tuned coarse-grain MD computations (CG MD) offer quantitative agreement with experimental forces. In this paper we address an important methodological aspect of MD modeling, namely the impact of numerical noise generated by random assignments of bead velocities on maximum forces (F(max)) calculated within the CG MD approach. Distributions of CG forces from 2000 MD runs for several model proteins rich in ß structures and having folds with increasing complexity are presented. It is shown that F(max) have nearly Gaussian distributions and that values of F(max) for each of those ß-structures may vary from 93.2 ± 28.9 pN (neurexin) to 198.3 ± 25.2 pN (fibronectin). The CG unfolding spectra are compared with AA steered MD data and with results of our AFM experiments for modules present in contactin, fibronectin and neurexin. The stability of these proteins is critical for the proper functioning of neuronal synaptic clefts. Our results confirm that CG modeling of a single molecule unfolding is a good auxiliary tool in nanomechanics but large sets of data have to be collected before reliable comparisons of protein mechanical stabilities are made.

Efficiency of the Cepheid Xpert vanA/vanB assay for screening of colonization with vancomycin-resistant enterococci during hospital outbreak.

This study aimed to assess the efficiency of the Cepheid Xpert vanA/vanB test for detecting vancomycin-resistant enterococci (VRE) colonization during a VanA Enterococcus faecium outbreak and to compare the Cepheid Xpert vanA/vanB (Cepheid, Sunnyvale, USA) test to a culture method with chromogenic medium chromID VRE agar (bioMérieux). The Cepheid Xpert vanA/vanB assay showed sensitivity 61.5%, specificity 79.2%, positive predictive value 61.5% and negative predictive value 79.2%. The results obtained in this study demonstrate that a positive result in the Cepheid Xpert vanA/vanB test for vanA enables the rapid (less than 1 h) presumptive, prior to culture, recognition of patients colonized with VRE. However, the Cepheid Xpert vanA/vanB assay cannot be the only test used to screen patients during an ongoing VRE outbreak, because additional culturing of all samples negative for both vanA and vanB or positive for vanB should be performed in order to confirm the carrier status of the patient.

The current status of invasive pneumococcal disease in Poland.

The objectives of this study were to assess the incidence of invasive pneumococcal disease (IPD) in Poland (2006-2009), where mass vaccination had not been implemented, and to determine the serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae isolates. The IPD incidence rates were highest among children under 2 years of age (3.39/100,000 in 2009) and children 2-5 years old (2.44/100,000). The most common serotypes were 14, 3, 1, 4, 19F, 23F, 6B, and 12F (61.7% of all isolates). In children aged less than 5 years, isolates of serotypes 14, 6B, and 19F were most prevalent (52.7% of the IPD cases). The PCV7, PCV10, and PCV13 covered 43.3%, 54.8%, and 68.8% of all IPD cases, and 68.7%, 76.3%, and 86.3% of cases involving children under 5 years of age. Penicillin resistance was found in 21.3% of the isolates responsible for meningitis and in 1.2% of isolates responsible for other invasive infections. Introduction of antipneumococcal conjugated vaccines into the national immunisation programme would likely lead to a significant reduction of IPD-associated morbidity among Polish children in particular, as well as in the population as a whole, especially in cases involving pneumococci with a decreased susceptibility to antibiotics.

Gelatinase-associated phenotypes and genotypes among clinical isolates of Enterococcus faecalis in Poland.

Enterococcus faecalis is an important nosocomial pathogen causing serious invasive infections. One of the virulence factors of this pathogen, gelatinase GelE, is a protease whose gene expression is regulated by the Fsr quorum sensing system. In this study, we used a well-characterized collection of 153 clinical E. faecalis isolates to investigate the distribution of genes involved in gelatinase expression. Although 140 isolates (91% of the group) harbored the gelE gene, only 81 isolates (53%) produced active gelatinase. The gelatinase-negative phenotype was found in several unrelated clones, and appeared to be caused by various genetic events. Isolates of the hospital-adapted clonal complex 2 (CC2) and of CC40 were uniformly gelatinase-positive, while all the CC87 isolates contained the 23.9 kb deletion encompassing most of the fsr locus and were gelatinase-negative. No significant differences among isolates of different clinical origin and gelatinase activity or presence of the fsr genes were found with the exception of isolates from cerebrospinal fluid, which were more often gelatinase-positive than colonizing isolates.