Preliminary evaluation of the positively and negatively charge effects of tetra-substituted porphyrins on photoinactivation of rapidly growing mycobacteria.

This manuscript reports, at the first time, the photoinactivation evaluation of tetra-cationic and anionic porphyrins as photosensitizers (PS) for the photodynamic inactivation (PDI) of rapidly growing mycobacteria strains. Two different charged porphyrin groups were obtained commercially. PDI experiments in the strains Mycobacterium massiliense e Mycobacterium fortuitum conducted with adequate concentration (without aggregation) of photosensitizer under white light at a fluence rate of 50 mW/cm2 over 90 min showed that the most effective PS caused a 100 times reduction in the concentration of viable mycobacteria. The present results show that porphyrin with positively charge are more efficient PS than anionic porphyrin (negatively charged) against M. massiliense e M. fortuitum. It is also clear that the effectiveness of the molecule as PS for PDI studies with mycobacteria is strongly related with the porphyrin peripheral charge, and consequently their solubility in physiological media. Cationic PSs might be promising anti-mycobacteria PDI agents with potential applications in medical clinical cases and bioremediation.

A rapid, standardized protein extraction method using adaptive focused acoustics for identification of mycobacteria by MALDI-ToF MS.

Mycobacterial identification using MALDI-ToF MS (MALDI) has been hindered by inadequate extraction methods. Adaptive Focused Acoustics™ uses concentrated ultrasonic energy to achieve cellular disruption. Using this technology, we developed a rapid mycobacterial inactivation/protein extraction method for MALDI-based identification. Agreement for identification to the species level versus conventional identification was stratified by log confidence cut-offs of ≥2.0, ≥ 1.8, or ≥1.7. A total of 182 mycobacterial isolates were tested. Complete inactivation of all species/strains was achieved after 2min. Using a log confidence cut-off of ≥2.0, overall agreement for the commercial method (CM) was 41.7% versus 66.7% for the novel method (NM). For the CM, agreement increased to 66.7% and 83.3% using log confidence cut-offs of ≥1.8 and ≥1.7, respectively; for the NM, agreement was 100% for both cut-offs with all isolates. With no alteration to the existing database, overall agreement for the NM was 83.4%, largely due to low scores for clinical isolates of M. chelonae and M. mucogenicum. Addition of spectra from a single clinical strain of each species to the existing database increased overall agreement to 93.1%.

Comparison of heat inactivation and cell disruption protocols for identification of mycobacteria from solid culture media by use of vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Two novel protocols for inactivation and extraction were developed and used to identify 107 Mycobacterium clinical isolates, including Mycobacterium tuberculosis complex, from solid cultures using Vitek matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The protocol using heat inactivation with sonication and cell disruption with glass beads resulted in 82.2% and 88.8% species and genus level identifications, respectively.

Potentiation of high hydrostatic pressure inactivation of Mycobacterium by combination with physical and chemical conditions.

Mycobacterium abscessus is an important hospital-acquired pathogen involved in infections associated with medical, surgical, and biopharmaceutical materials. In this work, we investigated the pressure-induced inactivation of two strains [2544 and American Type Culture Collection (ATCC) 19977] of M. abscessus in combination with different temperatures and pH conditions. For strain 2544, exposure to 250 MPa for 90 min did not significantly inactivate the bacteria at 20 °C, whereas at -15 °C, there was complete inactivation. Exposure to 250 MPa at ≥60 °C caused rapid inactivation, with no viable bacteria after 45 min. With 45 min of exposure, there were no viable bacteria at any temperature when a higher pressure (350 MPa) was used. Extremes of pH (4 or 9) also markedly enhanced the pressure-induced inactivation of bacteria at 250 MPa, with complete inactivation after 45 min. In comparison, exposure of this strain to the disinfecting agent glutaraldehyde (0.5 %) resulted in total inactivation within 5 min. Strain 19977 was more sensitive to high pressure but less sensitive to glutaraldehyde than strain 2544. These results indicate that high hydrostatic pressure in combination with other physical parameters may be useful in reducing the mycobacterial contamination of medical materials and pharmaceuticals that are sensitive to autoclaving.

Using computational fluid dynamics modeling to evaluate the design of hospital ultraviolet germicidal irradiation systems for inactivating airborne mycobacteria.

This research was conducted to evaluate the design of hospital ultraviolet germicidal irradiation (UVGI) systems and to assess their effectiveness for inactivating airborne mycobacteria. A computational fluid dynamics (CFD) model was developed and tested by simulating previous experiments measuring the effectiveness of a lab-based UVGI system. Model testing showed reasonable agreement with experimental measurements. The model captured trends similar to the experiments: Effectiveness of an upper-room UVGI system is higher when there is no ventilation compared with when there is ventilation, and wintertime ventilation conditions can markedly decrease the performance of an upper-room UVGI system. The CFD model was then applied to evaluate the design of three hospital patient rooms. A patient and an exam room with upper-room UVGI systems, and a patient room with an exhaust duct system were studied. Results showed that one of the UVGI systems was not very effective, due to the very efficient ventilation design. The other two configurations were reasonably to very effective at inactivating airborne mycobacteria. The most effective application was the one in which the room air-exchange rate was very low. CFD modeling can be useful for assessing whether hospital UVGI installations and ventilation systems are effective for infection control.

A Sir2-like protein participates in mycobacterial NHEJ.

In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP) screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.

Inactivation of environmental mycobacteria by free chlorine and UV.

The resistance of environmental mycobacteria (EM) against chlorine and ultraviolet (UV) was evaluated for determination of the Ct value and UV dose to inactivate EM. Chlorine disinfection experiments were done on Mycobacterium fortuitum in oxidant demand-free buffered water at the worst condition (pH 8.5, 4 degrees C) and normal condition (pH 7.0, 20 degrees C). The Ct value for 3log inactivation of M. fortuitum was 600 times greater than that of Escherichia coli. UV experiments were performed for various species of Mycobacterium avium, M. fortuitum, Mycobacterium intracellulare, and Mycobacterium lentiflavum. A UV collimated beam device was used for irradiation of four species suspended in phosphate buffered saline with doses of 5, 10, 20, 50, and 100mJ/cm(2). UV sensitivity of mycobacteria was species-specific. More than 3log of M. avium, M. intracellulare, and M. lentiflavum could be inactivated at 20mJ/cm(2), whereas M. fortuitum was so resistant that 3log inactivation required a dose of more than 50mJ/cm(2). Mycobacteria were found 2-10 times more resistant to UV than E. coli for 3log inactivation. There was no significant difference in the inactivation of mycobacteria with either low-pressure or medium-pressure UV irradiation.

Effectiveness of ultrasound for the destruction of Mycobacterium sp. strain (6PY1).

Ultrasound is widely used to disinfect drinking water and wastewater due to its strong physical and chemical effects on microorganisms. The aim of this study was to investigate the effect of ultrasound on the destruction of Mycobacterium strain 6PY1. Ultrasound waves (20 kHz or 612 kHz) were used to treat aqueous suspensions of Mycobacterium at different volumes, initial bacterial concentrations, and power densities. At the same power density and the same exposure time, sonication at high frequency resulted in a lower destruction of Mycobacterium sp. 6PY1 (35.5%) than sonication at low frequency (93%). The percentage of removal was not significantly affected by the volume of the irradiated suspension (150-300 ml) or the initial cell concentration (2.15 x 10(-3)-1.4 x 10(-2)mg protein L(-1)). At low frequency, the removal percentage of Mycobacterium sp. 6PY1 increased with increasing the power density, with a constant level reached after a certain power density. At high frequency, the removal percentage of Mycobacterium sp. 6PY1 increased with increasing the power density. The mechanism of cell killing was investigated by examining the effects of OH() radical scavengers such as sodium carbonate. At high frequency the presence of sodium carbonate suppressed the removal process. However, at low frequency the removal process was not affected, thus indicating that OH() radicals have a negligible role in this case. The latter result was supported by ten time's H(2)O(2) production at high frequency greater than that at low frequency.

Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks.

Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.

UV air cleaners and upper-room air ultraviolet germicidal irradiation for controlling airborne bacteria and fungal spores.

In-room air cleaners (ACs) and upper-room air ultraviolet germicidal irradiation (UVGI) are engineering control technologies that can help reduce the concentrations of airborne bacteria and fungal spores in the indoor environment. This study investigated six different types of ACs and quantified their ability to remove and/or inactivate airborne bacteria and fungal spores. Four of the air cleaners incorporated UV lamp(s) into their flow path. In addition, the efficacy of combining ACs with upper-room air UVGI was investigated. With the ventilation system providing zero or six air changes per hour, the air cleaners were tested separately or with the upper-room air UVGI system in operation in an 87-m3 test room. Active bacteria cells and fungal spores were aerosolized into the room such that their numbers and physiologic state were comparable both with and without air cleaning and upper-room air UVGI. In addition, the disinfection performance of a UV-C lamp internal to one of the ACs was evaluated by estimating the percentage of airborne bacteria cells and fungal spores captured on the air filter medium surface that were inactivated with UV exposure. Average airborne microbial clean air delivery rates (CADRm) varied between 26-981 m3 hr-1 depending on the AC, and between 1480-2370 m3 hr-1, when using air cleaners in combination with upper-room air UVGI. Culturing, direct microscopy, and optical particle counting revealed similar CADRm. The ACs performed similarly when challenged with three different microorganisms. Testing two of the ACs showed that no additional air cleaning was provided with the operation of an internal UV-C lamp; the internal UV-C lamps, however, inactivated 75% of fungal spores and 97% of bacteria cells captured in the air filter medium within 60 min.

Impact of environmental factors on efficacy of upper-room air ultraviolet germicidal irradiation for inactivating airborne mycobacteria.

This study evaluated the efficacy of an upper-room air ultraviolet germicidal irradiation (UVGI) system for inactivating airborne bacteria, which irradiates the upper part of a room while minimizing radiation exposure to persons in the lower part of the room. A full-scale test room (87 m3), fitted with a UVGI system consisting of 9 louvered wall and ceiling fixtures (504 W all lamps operating) was operated at 24 and 34 degrees C, between 25 and 90% relative humidity, and at three ventilation rates. Mycobacterium parafortuitum cells were aerosolized into the room such that their numbers and physiologic state were comparable both with and without the UVGI system operating. Airborne bacteria were collected in duplicate using liquid impingers and quantified with direct epifluorescent microscopy and standard culturing assay. Performance of the UVGI system degraded significantly when the relative humidity was increased from 50% to 75-90% RH, the horizontal UV fluence rate distribution was skewed to one side compared to being evenly dispersed, and the room air temperature was stratified from hot at the ceiling to cold at the floor. The inactivation rate increased linearly with effective UV fluence rate up to 5 microW cm(-2); an increase in the fluence rate above this level did not yield a proportional increase in inactivation rate.

Rapid immunoassays for detection of UV-induced cyclobutane pyrimidine dimers in whole bacterial cells.

Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active Mycobacterium parafortuitum and Serratia marcescens cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled ((125)I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in M. parafortuitum and S. marcescens cells as well as photoenzymatic repair responses in S. marcescens cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.

Photoreactivation in airborne Mycobacterium parafortuitum.

Photoreactivation was observed in airborne Mycobacterium parafortuitum exposed concurrently to UV radiation (254 nm) and visible light. Photoreactivation rates of airborne cells increased with increasing relative humidity (RH) and decreased with increasing UV dose. Under a constant UV dose with visible light absent, the UV inactivation rate of airborne M. parafortuitum cells decreased by a factor of 4 as RH increased from 40 to 95%; however, under identical conditions with visible light present, the UV inactivation rate of airborne cells decreased only by a factor of 2. When irradiated in the absence of visible light, cellular cyclobutane thymine dimer content of UV-irradiated airborne M. parafortuitum and Serratia marcescens increased in response to RH increases. Results suggest that, unlike in waterborne bacteria, cyclobutane thymine dimers are not the most significant form of UV-induced DNA damage incurred by airborne bacteria and that the distribution of DNA photoproducts incorporated into UV-irradiated airborne cells is a function of RH.

A comparative study of the effects of UVC irradiation on select procaryotic and eucaryotic wound pathogens.

Managing wounds infected with a mixture of several types of microorganisms such as bacteria (procaryotes) and fungi (eucaryotes) is a challenging clinical situation. The purpose of this study was to determine the effectiveness of ultraviolet light (UVC) in eradicating select procaryotic and eucaryotic organisms, in both pure culture and mixed cultures in vitro. Five replications of each organism or mixture of organisms (10(6) organisms/mL singly or 10(15) organisms/mL mixed culture) were plated. The cultures were treated with a UVC light 1 inch from the surface. Irradiation times were 0, 2, 3, 4, 5, 15, 30, 45, 60, 90, 120, and 180 seconds. Bacterial cultures were incubated and colony counts performed. Upon exposure to UVC, a 99.9% kill rate was obtained at 3 to 5 seconds for the procaryotic organisms (Pseudomonas aeruginosa and Mycobacterium abscessus) tested. However, 15 to 30 seconds of UVC treatment was required to obtain 99.9% kill of the eucaryotic organisms (Candida albicans, Aspergillus fumigatus) tested. This study demonstrates a decreasing sensitivity of evolutionarily more complex organisms to UVC. This study also provides further evidence that short exposure times to UVC are detrimental to procaryotic and simple unicellular eucaryotic organisms while sparing more complex multicellular organisms.

Construction by homologous recombination and phenotypic characterization of a DNA polymerase domain polA mutant of Mycobacterium smegmatis.

Gene replacement was achieved by homologous recombination in Mycobacterium smegmatis (Ms) using a cloned segment of the polA gene (encoding the DNA polymerase I) disrupted within the region encoding the C-terminal DNA polymerase domain by a kanamycin-resistance marker. The Ms polA755:aph mutant thus generated displayed a phenotype of hypersensitivity to DNA damage induced by UV irradiation and by hydrogen peroxide challenge.

Killing activity of microwaves in milk.

The killing activity of microwaves of 2450 MHz frequency and 600 W power on Pseudomonas aeruginosa, Escherichia coli, Enterobacter sakazakii, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans, Mycobacterium terrae and poliomyelitis vaccine-virus suspended in five infant formula preparations was investigated. The samples were brought to the boil (85-100 s depending on milk type). They had reached average temperatures of 82-93 degrees C at this point. Most of the vegetative organisms were killed. In those samples where growth was still detectable after microwave treatment, a significant reduction in viable micro-organisms (at least 5000-fold) was noted. We conclude that microwave beating to the boil is a convenient and fast method to reduce microbial contamination of infant feeds. However, care should be taken to ensure that milk is adequately cooled to the required temperature before it is fed to an infant.

Devices for the prevention of TB transmission. Considerations for proper application.

Healthcare facilities throughout the country are confronted with the task of preventing the transmission of tuberculosis among their patients and staff. On October 12, 1993, The Centers for Disease Control and Prevention published the Draft Guidelines for Preventing the Transmission of Tuberculosis in Health Care Facilities, and this is scheduled for final publication before year's end. The pending CDC guidelines are comprehensive and include engineering recommendations which are applicable to all settings in which health care is provided. Facilities that do not intend to use conventional construction or renovation to accomplish TB guideline compliance may consider using some type of environmental control device. These devices typically utilize ultraviolet germicidal irradiation (UVGI) and/or high efficiency particulate air filtration (HEPA), each of which is recognized as a viable TB environmental control. TB environmental control measures for specific areas within a healthcare facility should be based on a careful assessment of the actual risk of TB transmission in that setting.

Methods of introduction of foreign DNA into mycobacteria.

Two methods: triparental conjugation and electrotransformation were used for introduction of plasmid DNA into mycobacterial cells. The introduction of shuttle plasmid pMY10 into M. fortuitum mutant caused the activation of its chromosomal cryptic KmR gene. The used of integration vector pUS 903 allowed to obtain a collection of mutants interesting for studies of genetic determination of steroid biotransformation and drug-resistance in mycobacteria.

Light-induced changes in the phospholipid composition of Mycobacterium smegmatis ATCC 607.

The growth of Mycobacterium smegmatis ATCC 607 with intermittent exposure to light for periods of 4 h d(-1) led to changes in the amounts of the lipid components compared with those present in cells grown in the dark. When grown in continuous light the changes were less marked. Pigmentation was absent in cells grown in the dark..

Ionizing radiation in the disinfection of water contaminated with potentially pathogenic mycobacteria.

Sterile drinking water samples were artificially colonized with M. Kansasii, M. gordonae and M. fortuitum suspensions (the numbers of viable unite in 1 ml were 1.2 x 10(3), 48.5 and 3.2 x 10(3), respectively) prepared from mycobacterial strains replicated in Tween 80-free liquid Dubos medium STO. The contaminated water samples were irradiated from a rotary cobalt-60 source (gamma radiation, E = 1.17 and 1.33 MeV, dose intensity 1 kJ/kg.h at room temperature) with doses 0.7, 1.5, 2.2, 3, 9, 16 and 27 kJ/kg. The disinfecting effectiveness was assessed by direct cultivation tests (0.5 ml volumes of water inoculated on egg medium) and by cultivation on membrane filters after filtrating the whole amount of the water examined (about 500 ml). Fully positive disinfecting effects were recorded in M. kansasii and M fortuitum irradiated with 9 kJ/kg and in M. gordonae after irradiation with 1.5 kJ/kg. The calculated value of D10 = 0.4 kJ/kg (i.e. the dose radiation that reduces the number if viable mycobacteria by an order of magnitude) is suggestive of a strong disinfecting effect of ionizing radiation on the tested strains of potentially pathogenic mycobacteria. The results indicate that the use of ionizing radiation to disinfect mycobacteria that are difficult to remove with other methods which, as a rule, cannot ensure the permanent disinfection.