Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity.

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15-0.6 µM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 µM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 µg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

Airborne Spread of Methicillin Resistant Staphylococcus aureus From a Swine Farm.

Spread of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) to farmworkers has been recognized as a risk when working in LA-MRSA positive stables, due to LA-MRSA being present on airborne dust particles. Based on this, airborne spread of LA-MRSA through stable vents is a concern that is addressed in this study. The aim of the investigation was to quantify the airborne spread of LA-MRSA from a MRSA positive swine farm. In order to achieve this, a method for sampling large volumes of air was applied. The results were compared to meteorological data and bacteriological investigation of samples from the air inside the swine barn, soil outside the farm, and nasal samples from the individuals participating in the sampling process. MRSA was detected up to 300 m (the maximal measuring distance) from the swine farm in the air but only at low levels at distances above 50 meters (0.085 CFU/m3 at a distance of 50 m in the wind plume). MRSA was detected in sock samples obtained at the soil surfaces up to 400 m (the maximal measuring distance) from the farm building. The proportion of MRSA positive soil samples decreased from ~80 to 30% with increasing distance from the farm. A total of 25 human nasal samples were sampled after the farm visits after the participants had stayed in the surroundings of the farm for an average of 10.5 h. When leaving the farm, only two of the samples (8%) were LA-MRSA-positive both obtained from one individual who was the one who had sampled the ventilation shafts. In conclusion, airborne spread of MRSA from swine farms does not seem to be an important route for human contamination for individuals staying a whole working day outside a swine farm.

Polymorphisms of the T cell receptor CD3delta and CD3epsilon chains affect anti-CD3 antibody binding and T cell activation.

T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms result in amino acid substitutions in the ectodomains of both the CD3delta and CD3epsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3delta and epsilon ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for stimulation of T cells derived from gene-modified mice originating from 129 ES cell lines.

Microfluidic dissolved oxygen gradient generator biochip as a useful tool in bacterial biofilm studies.

A microfluidic chip for generation of gradients of dissolved oxygen was designed, fabricated and tested. The novel way of active oxygen depletion through a gas permeable membrane was applied. Numerical simulations for generation of O(2) gradients were correlated with measured oxygen concentrations. The developed microsystem was used to study growth patterns of the bacterium Pseudomonas aeruginosa in medium with different oxygen concentrations. The results showed that attachment of Pseudomonas aeruginosa to the substrate changed with oxygen concentration. This demonstrates that the device can be used for studies requiring controlled oxygen levels and for future studies of microaerobic and anaerobic conditions.

Cellular dynamics in the draining lymph nodes during sensitization and elicitation phases of contact hypersensitivity.

BACKGROUND: The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody-induced depletion of specific subtypes of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations. OBJECTIVES: To develop a method for studying the collective cellular dynamics of immune cells in the draining lymph nodes during CHS in intact animals. PATIENTS/METHODS: Mice were sensitized and/or challenged with 2,4-dinitrofluorobenzene or oxazolone. Using multi-parameter flow cytometry we determined the proliferation, activation state, and absolute number of helper T cells, cytotoxic T cells, B cells, and natural killer cells in the draining lymph nodes. RESULTS: The presented method can be applied to evaluate the effect of different contact allergens on various cell populations of the immune system. CONCLUSIONS: Our study support recent findings that several cell types seem to be involved in CHS.

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression.

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.