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For effective clinical application of human bone marrow mesenchymal stem cells (hBM-MSCs), the enhancement of their proliferation in vitro together with maintaining the expression of their crucial surface antigens and differentiation potential is necessary. The present study aimed to investigate the effect of light-emitting diode (LED) irradiation on hBM-MSCs proliferation after two, five, or nine days post-irradiation.The hBM-MSCs were exposed to the LED light at 630 nm, 4 J/cmWhen the metabolic activity was assayed, the significant increase in the cell proliferation rate by 31 and 50% after the irradiation with 4 J/cmTherefore, LED irradiation at a wavelength of 630 nm, energy density 4 J/cm

The size and shape of biological particles are important parameters allowing discrimination between various species. We have studied several aerosols of biological origin such as pollens, bacterial spores and vegetative bacteria. All of them presented different morphology. Using optical size and shape analyser we found good correlation between light scattering properties and actual particle features determined by scanning electron and fluorescence microscopy. In this study, we demonstrated that HCA (Hierarchical Cluster Analysis) offers fast and continuous bioaerosol classification based on shape and size data matrices of aerosols. The HCA gives an unequivocal interpretation of particle size vs. asymmetry data. Therefore, it may provide high throughput and reliable screening and classification of bioaerosols using scattering characteristics. Keywords: bioaerosol classification, scattering, particle size and shape analysis, biological warfare agents’ detection, hierarchical cluster analysis (HCA)

Microbial contamination is commonly found in metalworking fluids (MWFs) used in metal manufacturing industry. The aim of this study was to determine whether the use of MWF containing a biocide reduces the growth and influences the biofilm-forming capacity of microorganisms from field coolants, thereby efficiently protecting MWFs from biodeterioration. Overall, 164 bacterial strains classified into 40 species were collected from 69 machines in ten plants visited. Among them, Comamonas testosteroni was the most numerous species, and Pseudomonas was the most abundant genus. The isolation of Mycobacterium immunogenum was done from MWF for the first time in Poland. The number of bacteria in MWFs and size of microbial populations in biofilms were not affected by the presence of biocides in the industrial samples. In MWFs contaminated with microorganisms with the highest biofilm-forming capacity, several species of Enterobacteriaceae were identified. The proper management of MWF delivery systems is required to control biofilm development even when MWFs containing biocides are used. Moreover, good hygiene practices are needed to prevent the metal working machines from biofilms. This case study can help in appropriate monitoring the MWF distribution systems vulnerable to microbial colonization in metal industry.

Autofluorescence (AF) is the natural emission of light by intrinsic fluorophores. Oxidized mitochondrial flavins, lipofuscin and reduced nicotinamideadenine dinucleotide phosphate (NAD(P)H) are the main sources of the autofluorescence in cells upon excitation with visible light. The aim of the study was to investigate changes in the metabolism of four cell lines by monitoring their autofluorescence with a microplate reader. Autofluorescence intensities of cells were collected at two wavelengths for the excitation and fluorescence emission: for endogenous NAD(P)H at 366/450 nm, for the oxidized flavoproteins and lipofuscin at 460/540 nm. Human mesenchymal stem cells (hMSC), epithelial cells from mammary gland (MCF 10A), breast ductal carcinoma (T-47D) prostate carcinoma (DU-145) were observed daily for 16 days. The level of NAD(P)H autofluorescence did not differ among the cell lines investigated. The significant increase in oxidized flavoproteins fluorescence intensity was recorded for hMSC and ranged from 140 to 175% of control. During 28 days differentiation process, the NAD(P)H, FAD and lipofuscin fluorescence intensities were recorded every day, and the redox ratio was then calculated. The redox ratio gradually decreased during the last eight days of osteogenesis and adipogenesis. Therefore, in our opinion the NAD(P)H, FAD, and lipofuscin fluorescence emission at the wavelengths selected are the optimal parameters to be collected during the differentiation process in order to monitor the metabolism of hMSC undergoing structural and morphological changes.

Human mesenchymal stem cells (hMSCs) with their vast differentiation potential are very useful for cell-based regenerative medicine. To achieve sufficient numbers of cells for tissue engineering, many different methods have been used to reach the effective increase of cell proliferation. Low-energy red light provided by light emitting diodes (LEDs) have been recently introduced as a method that promoted biomodulation and proliferation of hMSCs in vitro. The purpose of this study was to find the optimum stimulatory dosimetric parameters of LED (630 nm) irradiation on the hMSCs proliferation. The energy density was 2, 3, 4, 10, 20 J/cm2 and the power density used was 7, 17 or 30 mW/cm2. Human MSCs were irradiated with single or triple exposures daily at room temperature and the cell proliferation rate was evaluated during nine days after irradiation. The results showed that after irradiation 4 J/cm2 and 17 mW/cm2 at a single dose the proliferation rate of hMSCs increased on day 5 and 9 (13% and 7%, respectively) when compared to nonirradiated cells. However, triple LED irradiation under the same parameters resulted in the decline in the cell proliferation rate on day 5, but the proliferation rate was at the same level on day 9, when compared with the cell proliferation after irradiation with a single dose. The effect of a single dose irradiation with 4 J/cm2 and 17 mW/cm2 on the proliferation of cells was the highest when the cells were irradiated in phosphate-buffered saline (PBS) instead of MSCGM culture medium.

Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14+ cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14+ cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14+ cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14+ cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.

The improvements in the existing methods of rapid detection and biological pathogen surveillance are still needed. The new spectroscopic methods that rely on the unique structural features and intrinsic fluorescence of microorganisms are well fitted for monitoring the spread of airborne biological agents or their reagentless detection in the air, and these methods may bring a new quality to bioaerosols remote detection. This review describes the problem of the confidence in the environmental testing results that may affect clearance standard, sampling techniques, and the estimation of risk of human exposure to the low concentrations of harmful microorganisms during bioterrorist event or naturally occurring outbreaks. Higher recovery efficiency of dangerous biological agents from the air and contaminated surfaces would enable more reliable environmental human risk exposure assessment.

Haemophilus influenzae is one of the major pathogenic bacteria in upper respiratory tract of children. In this study, the presence of various H. influenzae genotypes were followed-up for at least 13 weeks, starting from one week before surgery. Forty-one children with chronic adenoid hypertrophy were prospectively enrolled to the study. The consecutive swabs of adenoid and tonsils, two before adenotonsillectomy and two after the surgery together with homogenates of adenotonsillar tissues and lysates of the CD14 + cells fraction were acquired from 34 children undergoing adenotonsillectomy. Up to ten isolates from each patient at each collection period were genotyped using a PFGE method and their capsular type and antibiotic susceptibility was determined. Of the 1001 isolates examined, we identified 325 isolates grouped into 16 persistent genotypes, which colonized throats for more than seven weeks and were not eliminated by the surgery. The other 506 isolates grouped into 48 transient genotypes that had been eliminated by the surgery. The resistance to ampicillin were found in 23.8% of the transient strains, and 4.7% of the newly acquired strains following the surgical intervention. In contrast, none of the persistent strains were resistant to ampicillin; however, these strains showed apparently higher level of resistance to co-trimoxazole when compared to transient strains. The transient and persistent strains did not significantly differ in bacterial viability in the biofilms formed in vitro. Some of the strains were identified in two or three different patients and were considered as the strains circulating in the region between 2010 and 2012.

This study was undertaken to examine if receptor recognizing saccharides may be involved in the adherence of Pseudomonas aeruginosa to collagen type I and type II. We performed an adherence inhibition assay: cells of individual P. aeruginosa isolates attached to immobilized collagen type I or type II in the presence of monosaccharides, which could serve as blockers of bacterial receptors. Bacterial binding to collagen type I molecules was inhibited to the highest degree by sugar composition D-galactose/D-mannose/N-acetylneuraminic acid (5:5:1), whereas attachment of P. aeruginosa to collagen type II was inhibited by composition d-glucose/D-galactose (1:1). The same strains which were sensitive to inhibition of binding to collagen type II by both collagen types, were also sensitive to blocking by composition D-glucose/D-galactose. It suggests that saccharides play a role in adherence of P. aeruginosa to collagen type I and type II, and a common receptor for both types of collagen may be available on the surface of P. aeruginosa cells.

The present study had three goals: (i) to evaluate the relative quantities of aerosolized Bacillus atrophaeus spores deposited on the vertical, horizontal top, and horizontal bottom surfaces in a chamber; (ii) to assess the relative recoveries of the aerosolized spores from glass and stainless steel surfaces with a polyester swab and a macrofoam sponge wipe; and (iii) to estimate the relative recovery efficiencies of aerosolized B. atrophaeus spores and Pantoea agglomerans using a foam spatula at several different bacterial loads by aerosol distribution on glass surfaces. The majority of spores were collected from the bottom horizontal surface regardless of which swab type and extraction protocol were used. Swabbing with a macrofoam sponge wipe was more efficient in recovering spores from surfaces contaminated with high bioaerosol concentrations than swabbing with a polyester swab. B. atrophaeus spores and P. agglomerans culturable cells were detected on glass surfaces using foam spatulas when the theoretical surface bacterial loads were 2.88 × 10 4 CFU and 8.09 × 10 6 CFU per 100-cm 2 area, respectively. The median recovery efficiency from the surfaces using foam spatulas was equal to 9.9% for B. atrophaeus spores when the recovery was calculated relative to the theoretical surface spore load. Using a foam spatula permits reliable sampling of spores on the bioaerosol-exposed surfaces in a wide measuring range. The culturable P. agglomerans cells were recovered with a median efficiency of 0.001%, but staining the swab extracts with fluorescent dyes allowed us to observe that the viable cell numbers were higher by 1.83 log units than culturable organisms. However, additional work is needed to improve the analysis of the foam extracts in order to decrease the limit of detection of Bacillus spores and Gram-negative bacteria on contaminated surfaces.

Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU −/exoS + or exoU +/exoS −) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU −/exoS + isolates showed significant higher levels of the median elastolytic activity when compared to the exoU +/exoS − isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU + /exoS − genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.