The red-light emitting diode irradiation increases proliferation of human bone marrow mesenchymal stem cells preserving their immunophenotype.

PURPOSE: 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. MATERIALS AND METHODS: The hBM-MSCs were exposed to the LED light at 630 nm, 4 J/cm2, and power densities of 7, 17, or 30 mW/cm2. To assess the cell proliferation rate in the sham-irradiated and irradiated samples the cells metabolic activity and DNA content were determined. The number of apoptotic and necrotic cells in the samples was also evaluated. The expression of the crucial surface antigens of the hBM-MSCs up to nine days after irradiation at 4 J/cm2 and 17 mW/cm2 was monitored with flow cytometry. Additionally, the potential of hBM-MSCs for induced differentiation was measured. RESULTS: When the metabolic activity was assayed, the significant increase in the cell proliferation rate by 31 and 50% after the irradiation with 4 J/cm2 and 17 mW/cm2, respectively, was observed at day five and nine when compared to the sham-irradiated cells (p < .05). Similarly, DNA content within the irradiated hBM-MSCs increased by 31 and 41% at day five and nine after the irradiation with 4 J/cm2 and 17 mW/cm2 in comparison to the sham-irradiated cells. LED irradiation did not change the expression of the crucial surface antigens of the hBM-MSCs up to nine days after irradiation at 4 J/cm2 and 17 mW/cm2. At the same experimental conditions, the hBM-MSCs maintain in vitro their capability for multipotential differentiation into osteoblasts, adipocytes, and chondrocytes. CONCLUSION: Therefore, LED irradiation at a wavelength of 630 nm, energy density 4 J/cm2, and power density 17 mW/cm2 can effectively increase the number of viable hBM-MSCs in vitro.

Utility of antimicrobial susceptibility testing of multiple Haemophilus influenzae isolates from throat swabs of children with adenoid hypertrophy.

Eleven out of 40 children with adenoiditis were colonized with multiple genotypes of Haemophilus influenzae. Heterogeneous antibiotic susceptibility to ampicillin and cotrimoxazole was observed in 6 children. A multiple-colony methodology may potentially help to find the resistant strains of H. influenzae in patients who do not respond to the antibiotic treatment.

Carriage of antibiotic-resistant Haemophilus influenzae strains in children undergoing adenotonsillectomy.

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.

Microorganisms in metalworking fluids: current issues in research and management.

The microbial contamination of water miscible metalworking fluids (MWFs) is a serious problem in metal industry. A good maintenance of MWF re-circulation systems can extend the lifetime of coolants and ensure the quality of the tools produced. In MWFs, as in the other water-based environments, microorganisms usually live in the form of biofilms, the communities of bacteria and fungi attached to the surface of sumps, metal parts and also to each other. Biofilms exhibit very high resistance to biocides. The effect of biocides that are used as additives to MWFs to control the growth of the bacterial and fungal microbiomes (microorganisms characteristic to the individual coolant system) have become the subject of research only in recent years. There are also only sparse reports on the impact of biocides on microorganisms growing in biofilms in MWF installations. Fast growing mycobacteria are important members of these biofilm communities. Their presence has recently been linked with the occurrence of cases of hypersensitivity pneumonitis, a serious respiratory disorder in the metal industry employees. The new, relatively fast and inexpensive techniques to assess the species diversity within MWF microbiomes and their population size should be developed in order to control the microorganisms' proliferation in MWFs and to diminish the occupational exposure to harmful bioaerosols in metal industry.

Evaluation of applicability of the Sartorius Airport MD8 sampler for detection of Bacillus endospores in indoor air.

This study was designed to evaluate the measuring range and lowest limit of detection of Bacillus endospores in the ambient room air when the Sartorius MD8 sampler, and two different culture methods for bacterial enumeration were used. Different concentrations of bioaerosol were generated inside the test chamber filled with either the high-efficiency particulate air (HEPA)-filtered air or with the ambient room air. The detection of endospores in the HEPA-filtered air was achievable: (1) when they were aerosolized at a concentration above 7.56 × 10(3) CFU/m(3) and analyzed with spread plate method, and (2) when they were aerosolized at a concentration above 4.00 × 10(2) CFU/m(3) and analyzed with pour plate method. The detection of endospores in the ambient room air was possible: (1) when they were aerosolized at a concentration above 9.1 × 10(3) CFU/m(3) and analyzed with spread plate method, and (2) when they were aerosolized at a concentration above 5.6 × 10(2) CFU/m(3) and analyzed with pour plate method. The microorganisms present in the ambient room air interfere with precise quantification of Bacillus endospores when their concentration is relatively low. The results of this study may be helpful in critical assessment of the results obtained from monitoring the air for bacterial endospores.

Comparison of fluorescence spectroscopy and FTIR in differentiation of plant pollens.

Spectroscopic techniques are under investigation on possibility of differentiation of airborne particles. This paper describes pollen discrimination among others bio-particles in laboratory conditions. Pollen samples were characterized with UV-Vis fluorescence, drift and KBr pellet techniques of infrared spectroscopy. Principal Component Analysis of UV-Vis fluorescence and FTIR spectra revealed that pollens can be distinguished from other bio-materials with use of these methods. Both methods resulted in similar classification capability. Combined FTIR and fluorescence data analysis did not improve the discrimination between pollen allergens and other airborne biological materials.

Classification of the biological material with use of FTIR spectroscopy and statistical analysis.

Rapid detection and discrimination of dangerous biological materials such as bacteria and their spores has become a security aim of considerable importance. Various analytical methods, including FTIR spectroscopy combined with statistical analysis have been used to identify vegetative bacteria, bacterial spores and background interferants. The present work discusses the application of FTIR technique performed in reflectance mode using Horizontal Attenuated Total Reflectance accessory (HATR) to the discrimination of biological materials. In comparison with transmission technique the HATR is more rapid and do not require the sample destruction, simultaneously giving similar absorbance bands. HATR-FTIR results combined with statistical analysis PCA and HCA demonstrate that this combination provides novel and accurate microbial identification technique.

Proteolytic activity of Pseudomonas aeruginosa isolates with TTSS-mediated cytotoxicity and invasiveness to host cells.

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.

Genetic similarity of Campylobacter isolates in humans, food, and water sources in central Poland.

Campylobacter spp. is an important cause of gastroenteritis in humans throughout the world. However, sources of these infections are often difficult to identify. Therefore, this study aimed at analyzing the genetic relatedness of Campylobacter isolates from environmental and food samples as well as stool specimens of diarrheal patients obtained in a single geographic region in Poland. Only a few Campylobacter jejuni isolates (4/18, 22%) could be assigned to one cluster, whereas the majority were unrelated. In contrast, the majority of Campylobacter coli strains (25/35, 71%) belonged to three pulsed-field gel electrophoresis (PFGE) clusters containing isolates of various origins (i.e., water samples, chicken carcasses, and humans). Isolates belonging to the clusters showed also similar antibiotic resistance patterns and similar genotypes with respect to the occurrence of the virB11 and iam genes. This suggests that Campylobacter strains may circulate between humans, poultry, and recreational water sources in the rural region in central Poland.

Use of a foam spatula for sampling surfaces after bioaerosol deposition.

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 x 10(4) CFU and 8.09 x 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.

Diverse type III secretion phenotypes among Pseudomonas aeruginosa strains upon infection of murine macrophage-like and endothelial cell lines.

The interaction of over 100 isolates of Pseudomonas aeruginosa representing different genotypes of type III secretion system (TTSS) with RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial (PME) cells were studied. The strains were isolated from clinical materials and from stool specimens of healthy carriers and were analyzed by pulsed field gel electrophoresis (PFGE) to characterize their heterogeneity. In order to differentiate TTSS genotypes of P. aeruginosa isolates, the distribution of the following genes: exoU, exoS, pcrV, exoT, and exoY was assessed by multiplex and duplex PCR assays. The cytotoxicity and invasiveness of the P. aeruginosa isolates were determined. P. aeruginosa isolates showed a discrepancy in their ability to induce cytotoxicity and to invade mammalian cells. Up to four phenotypes among the isolates were observed and the most diverse interactions of the isolates were noticed with PME cells. The reduction of the viability of the cells, infected by P. aeruginosa isolates of the same clone, was associated with the ability of these strains to secrete the TTSS effectors: ExoU or ExoS. The results of this study also suggest that healthy people can be the carriers of cytotoxic strains of this dangerous pathogen.

Application of multiplex PCR for monitoring colonization of pig tonsils by Yersinia enterocolitica, including biotype 1A, and Yersinia pseudotuberculosis.

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n=6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n=31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid-bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.

Secretion of proteases by Pseudomonas aeruginosa biofilms exposed to ciprofloxacin.

Pseudomonas aeruginosa proteases are considered important virulence factors which damage host tissues and interfere with host antibacterial defense mechanisms. P. aeruginosa biofilm cells are not completely killed by antibacterials, and therefore this study addresses the question whether ciprofloxacin attenuates the virulence of biofilm communities by abolishing their secretion of proteases. The surviving cells of the colony biofilms studied, despite their cyclical exposure to four doses of ciprofloxacin at bactericidal concentrations (one dose a day), still secreted active proteases to the environment surrounding the biofilms. The biofilm cells secreted elastase B (LasB) over the duration of the experiments as confirmed by Western immunoblot analysis. The colony biofilms did not secrete LasA-a protease with staphylolytic activity. The same profiles on zymogram gels with gelatin were observed for the proteases secreted by both ciprofloxacin-exposed and unexposed (control) biofilms. Total proteolytic activities of the colony biofilms studied were significantly reduced after exposure to ciprofloxacin at bactericidal concentrations-after 96 h of exposure they dropped to 38% for the strain intermediate resistant to ciprofloxacin and to 65% for the strain highly resistant to the antibiotic, relative to the control biofilms. The surviving cells of the colony biofilms after their release into a fresh medium displayed transient increased resistance to ciprofloxacin compared to their planktonic counterparts.

[Prevalence of Escherichia coli strains in the canteens]. / Wystepowanie Escherichia coli w srodowisku punktów zbiorowego zywienia.

The prevalence of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli strains in stool specimens from asymptomatic human carriers working in the canteens and also in the kitchen and sanitary facilities was evaluated. The E. coli genes coding for the following virulence markers: intimin (eae), enterohaemolysin (hlyA), and verotoxins type I and II (stx1 and stx2) were sought by multiplex PCR assay. E. coli isolates were obtained from 144 stool specimens, 295 swabs taken from kitchen hardware and surrounding facilities, and from 33 meat specimens. Only 66 (8.5%) of total 777 E. coli isolates belonged to O44, O18, O25, O127, O55, O114, O125, and O142 serogroups, the prevalent serogroups in Poland. None of the strains was classified as serogroup O157. The serogroups O44 and O18 were present most often among all typeable strains and their incidence was 51.5% and 25.8% respectively. Among 363 isolates assayed for the presence of the genes encoding virulence markers only 10 isolates (2.8%) carried eae gene. None of the isolates possessing eae gene belonged to the serogroups tested. The hlyA, stx1 and stx2 genes were absent in all E. coli isolates tested.