Motility provides specific adhesion patterns and improves Listeria monocytogenes invasion into human HEp-2 cells.

Listeria monocytogenes is motile at 22°C and non-motile at 37°C. In contrast, expression of L. monocytogenes virulence factors is low at 22°C and up-regulated at 37°C. Here, we studied a character of L. monocytogenes near surface swimming (NSS) motility and its effects on adhesion patterns and invasion into epithelial cells. L. monocytogenes and its saprophytic counterpart L. innocua both grown at 22°C showed similar NSS characteristics including individual velocities, trajectory lengths, residence times, and an asymmetric distribution of velocity directions. Similar NSS patterns correlated with similar adhesion patterns. Motile bacteria, including both pathogenic and saprophytic species, showed a preference for adhering to the periphery of epithelial HEp-2 cells. In contrast, non-motile bacteria were evenly distributed across the cell surface, including areas over the nucleus. However, the uneven distribution of motile bacteria did not enhance the invasion into HEp-2 cells unless virulence factor production was up-regulated by the transient shift of the culture to 37°C. Motile L. monocytogenes grown overnight at 22°C and then shifted to 37°C for 2 h expressed invasion factors at the same level and invaded human cells up to five times more efficiently comparatively with non-motile bacteria grown overnight at 37°C. Taken together, obtained results demonstrated that (i) NSS motility and correspondent peripheral location over the cell surface did not depend on L. monocytogenes virulence traits; (ii) motility improved L. monocytogenes invasion into human HEp-2 cells within a few hours after the transition from the ambient temperature to the human body temperature.

Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model.

Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

Microcolonies: a novel morphological form of pathogenic Mycoplasma spp.

Introduction. The Mollicutes class unites cell wall lacking bacteria many of which are membrane parasites and opportunistic bacteria.Aim. This study describes a novel morphological form found in the five species belonging to the bacterial class Mollicutes, and referred to as microcolonies (MCs).Methodology. MCs were obtained as described below and characterized with bacteriological and immunological methods, and microscopy.Results. In contrast to typical colonies (TCs), MCs are characterized by tiny propeller-shaped colonies formed by rod-like cells tightly packed in parallel rows. These colonies were observed within routinely cultivated cultures of type strains 7-12 days post-plating. Rod-like cells were visualized using a scanning electron microscope within TCs with a 'fried-egg-like' appearance. MCs were not observed to revert to TCs. MCs were resistant to antibiotics and other treatments effective against TCs. Pure MC cultures were generated in vitro by treatment of Mycoplasma cultures with hyperimmune serum, antibiotics or argon non-thermal plasma. MCs of Mycoplasma hominis strain H-34 were characterized in detail to confirm that they belonged to that species. MCs tested positive via PCR with M. hominis-specific primers, direct fluorescence and epifluorescence tests, and Western blotting with the camel-derived nanobody aMh-FcG2a, which is specific to the MH3620 transporter protein. Meanwhile, MCs behaved differently in standard bacteriological tests. Pure MC cultures were also isolated directly from clinical samples of the serum, synovial liquid and urine of patients within flammatory urogenital tract diseases, asthma or arthritis. In total, 79 independent MC cultures were isolated from clinical samples including M. hominis (n=70), Mycoplasma pneumoniae (n=2), Mycoplasma fermentans (n=2) and Mycoplasma spp. (n=5).Conclusion. MCs play an unknown role in infection pathology and display prominent antibiotic resistance, making them a challenge for the future studies on Mollicutes.

Topical treatment with the bacterium-derived c-Met agonist InlB321/15 accelerates healing in the abrasion wound mouse model.

Studies of factors affecting wound-healing rates are encouraged by a critical need for new treatments to manage an increasing burden of non-healing wounds. The InlB protein produced by the Gram-positive bacterium Listeria monocytogenes is an agonist of the tyrosine kinase receptor c-Met and a functional analog of the hepatocyte growth factor (HGF), which is a mammalian ligand of c-Met. The recombinant InlB321 protein, which is the c-Met-binding InlB domain (amino acids 31-321), was cloned from the L. monocytogenes serovar 4b clinical strain VIMHA015 and serovar 1/2a strain EGDe (InlB321/15 and InlB321/EGDe, respectively). Both InlB321 variants stimulated proliferation of endothelial HUVEC cells. InlB321/15 was more active in Erk1/2 phosphorylation assay, and more potent than InlB321/EGDe in the 2D-scratch wound-healing assay. Scratch closure reached 86%, 29% and 72% for InlB321/15, InlB321/EGDe and HGF, respectively, 72 h post-wounding (p < 0.05). Topically applied glycerol-mixed InlB321/15 (300 µg ml- 1) increased abrasion wound-healing rates in mice. The 50% wound closing time (CT50) was reduced by InlB321/15 (4.18 ± 0.91 days; CI: 3.05; 5.31) compared with control animals (5.51 ± 1.21 days; CI: 4.01; 7.01; p < 0.05). Taken together, obtained results suggested a potential of InlB321/15 as a means of accelerating wound healing.

Route of Injection Affects the Impact of InlB Internalin Domain Variants on Severity of Listeria monocytogenes Infection in Mice.

The facultative intracellular pathogen Listeria monocytogenes causes a severe food-borne infection in humans and animals. L. monocytogenes invasion factor InlB interacts with the tyrosine kinase c-Met via the N-terminal internalin domain. Previously, distinct variants of the InlB internalin domain (idInlB) have been described in L. monocytogenes field isolates. Three variants were used to restore full-length InlB expression in the L. monocytogenes strain EGDeΔinlB. Obtained isogenic L. monocytogenes strains were tested in the invasion assay and intravenous, intraperitoneal, and intragastric models of infection in mice. All idInlBs were functional, restored InlB activity as an invasion factor, and improved invasion of the parental strain EGDeΔinlB into human kidney HEK23 cells. Meanwhile, distinct idInlBs provided different mortality rates and bacterial loads in internal organs. When recombinant strains were compared, the variant designated idInlB14 decreased severity of disease caused by intravenous and intraperitoneal bacterial administration, whereas this variant improved intestine colonization and stimulated intragastric infection. Obtained results demonstrated that naturally occurring idInlBs differed in their impact on severity of L. monocytogenes infection in mice in dependence on the infection route.

Atmospheric pressure nonthermal plasmas for bacterial biofilm prevention and eradication.

Biofilms are three-dimensional structures formed by surface-attached microorganisms and their extracellular products. Biofilms formed by pathogenic microorganisms play an important role in human diseases. Higher resistance to antimicrobial agents and changes in microbial physiology make treating biofilm infections very complex. Atmospheric pressure nonthermal plasmas (NTPs) are a novel and powerful tool for antimicrobial treatment. The microbicidal activity of NTPs has an unspecific character due to the synergetic actions of bioactive components of the plasma torch, including charged particles, reactive species, and UV radiation. This review focuses on specific traits of biofilms, their role in human diseases, and those effects of NTP that are helpful for treating biofilm infections. The authors discuss NTP-based strategies for biofilm control, such as surface modifications to prevent bacterial adhesion, killing bacteria in biofilms, and biofilm destruction with NTPs. The unspecific character of microbicidal activity, proven polymer modification and destruction abilities, low toxicity for human tissues and absence of long-living toxic compounds make NTPs a very promising tool for biofilm prevention and control.

Non-thermal argon plasma is bactericidal for the intracellular bacterial pathogen Chlamydia trachomatis.

Non-thermal plasma (NTP) is a flow of partially ionized argon gas at an ambient macroscopic temperature and is microbicidal for bacteria, viruses and fungi. Viability of the Gram-negative obligate intracellular bacterial parasite Chlamydia trachomatis and its host cells was investigated after NTP treatment. NTP treatment of C. trachomatis extracellular elementary bodies (EBs) diminished the concentration of infectious bacteria by a factor of 9×10(4), as established by the parallel infection of murine fibroblast McCoy cells with treated and control EBs. NTP treatment of infected McCoy cells caused disruption of membrane-restricted vacuoles (inclusions), where C. trachomatis intracellular reticulate bodies (RBs) multiply, and a 2×10(6)-fold reduction in the concentration of infectious bacteria. When the samples were covered with magnesium fluoride glass to obstruct plasma particles and UV rays alone were applied, the bactericidal effect was reduced 1.4×10(1)-fold and 5×10(4)-fold for EBs and RBs, respectively. NTP treatment caused the viability of host McCoy cells to diminish by 19%. Therefore, the results obtained demonstrated that (i) both extracellular and intracellular forms of C. trachomatis are sensitive to NTP treatment; (ii) the reduction in concentration of infectious bacteria after NTP treatment of infected cells is superior to the reduction in viability of host cells; and (iii) the effect of NTP on intracellular bacteria does not depend on UV rays.

Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal model of infected wounds.

Non-thermal (low-temperature) physical plasma is under intensive study as an alternative approach to control superficial wound and skin infections when the effectiveness of chemical agents is weak due to natural pathogen or biofilm resistance. The purpose of this study was to test the individual susceptibility of pathogenic bacteria to non-thermal argon plasma and to measure the effectiveness of plasma treatments against bacteria in biofilms and on wound surfaces. Overall, Gram-negative bacteria were more susceptible to plasma treatment than Gram-positive bacteria. For the Gram-negative bacteria Pseudomonas aeruginosa, Burkholderia cenocepacia and Escherichia coli, there were no survivors among the initial 10(5) c.f.u. after a 5 min plasma treatment. The susceptibility of Gram-positive bacteria was species- and strain-specific. Streptococcus pyogenes was the most resistant with 17 % survival of the initial 10(5) c.f.u. after a 5 min plasma treatment. Staphylococcus aureus had a strain-dependent resistance with 0 and 10 % survival from 10(5) c.f.u. of the Sa 78 and ATCC 6538 strains, respectively. Staphylococcus epidermidis and Enterococcus faecium had medium resistance. Non-ionized argon gas was not bactericidal. Biofilms partly protected bacteria, with the efficiency of protection dependent on biofilm thickness. Bacteria in deeper biofilm layers survived better after the plasma treatment. A rat model of a superficial slash wound infected with P. aeruginosa and the plasma-sensitive Staphylococcus aureus strain Sa 78 was used to assess the efficiency of argon plasma treatment. A 10 min treatment significantly reduced bacterial loads on the wound surface. A 5-day course of daily plasma treatments eliminated P. aeruginosa from the plasma-treated animals 2 days earlier than from the control ones. A statistically significant increase in the rate of wound closure was observed in plasma-treated animals after the third day of the course. Wound healing in plasma-treated animals slowed down after the course had been completed. Overall, the results show considerable potential for non-thermal argon plasma in eliminating pathogenic bacteria from biofilms and wound surfaces.