Hypersensitivity pneumonitis in a cystic fibrosis patient.

Hypersensitivity pneumonitis (HP) is a chronic inflammatory lung disease caused by repeated inhalation of antigenic substances. We present a case of metalworking fluids (MWFs)-HP sensitized to Pseudomonas oleovorans in a cystic fibrosis patient. This case illustrates that HP diagnosis remains challenging, especially in patients with another pulmonary disease, and that serodiagnosis contributes to identifying the precise microorganism involved. It also demonstrates that P. oleovorans is an important secondary aetiological agent in MWF-HP, less known than Mycobacterium immunogenum.

Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production.

As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks.

Can oil lubricated dental handpieces be sterilized?: Part 2. Literature review.

UNLABELLED: A literature search revealed six laboratory-based microbial challenge studies which support the opinion that oil contamination does not prevent successful sterilization of the lumen of dental handpieces. However, a detailed appraisal of these papers revealed lack of detail in the cleaning methods used prior to sterilization and none of the papers considered the risks of prion contamination. CLINICAL RELEVANCE: An examination of the evidence base on sterilization of oil-lubricated handpieces is of potential value to dental clinicians and their teams.

Demulsifying properties of extracellular products and cells of Pseudomonas aeruginosa MSJ isolated from petroleum-contaminated soil.

A strain of Pseudomonas aeruginosa isolated from a site contaminated with refined oil products exhibited demulsification capabilities against Tween 80-Span 80 stabilized oil-in-water (O/W), Tween 80-stabilized water-in-oil (W/O) model emulsions (kerosene-water), and an industrial emulsion (Daido Dairoll PA-5A). GC-MS analysis confirmed the presence of fatty acids and carbohydrates in the extracellular biodemulsifier. The demulsifying activity of cells and culture supernatants was favored by growth in media containing 1% diesel oil. There was a correlation between culture age, de-emulsification and cellular hydrophobicity, and highest activities were observed for cells and supernatants from 96-h cultures. Activity increased with addition of up to 60 mg cells or 300 µL supernatant to emulsions. The activity was relatively stable at 20-40 °C and to freezing, but was reduced by 69% by washing the cells with chloroform-methanol-water. This demulsifier has potential for application in biotreatment of emulsified oily wastewaters to promote recovery and/or degradation of oil.

Factors influencing the microbial composition of metalworking fluids and potential implications for machine operator's lung.

Hypersensitivity pneumonitis, also known as "machine operator's lung" (MOL), has been related to microorganisms growing in metalworking fluids (MWFs), especially Mycobacterium immunogenum. We aimed to (i) describe the microbiological contamination of MWFs and (ii) look for chemical, physical, and environmental parameters associated with variations in microbiological profiles. We microbiologically analyzed 180 MWF samples from nonautomotive plants (e.g., screw-machining or metal-cutting plants) in the Franche-Comté region in eastern France and 165 samples from three French automotive plants in which cases of MOL had been proven. Our results revealed two types of microbial biomes: the first was from the nonautomotive industry, showed predominantly Gram-negative rods (GNR), and was associated with a low risk of MOL, and the second came from the automotive industry that was affected by cases of MOL and showed predominantly Gram-positive rods (GPR). Traces of M. immunogenum were sporadically detected in the first type, while it was highly prevalent in the automotive sector, with up to 38% of samples testing positive. The use of chromium, nickel, or iron was associated with growth of Gram-negative rods; conversely, growth of Gram-positive rods was associated with the absence of these metals. Synthetic MWFs were more frequently sterile than emulsions. Vegetable oil-based emulsions were associated with GNR, while mineral ones were associated with GPR. Our results suggest that metal types and the nature of MWF play a part in MWF contamination, and this work shall be followed by further in vitro simulation experiments on the kinetics of microbial populations, focusing on the phenomena of inhibition and synergy.

Implementation of a quantitative real-time PCR assay for the detection of Mycobacterium immunogenum in metalworking fluids.

The bacterium Mycobacterium immunogenum has been implicated in causing the lung condition hypersensitivity pneumonitis (HP) in factory workers exposed to colonized metalworking fluids (MWFs). M. immunogenum-specific, real-time quantitative PCR detection technique (MiRT-qPCR) was implemented on a large scale to 363 MWFs of varying types, originating from the United States and Europe, that had been in use for between 30 days and 1 year. In MWFs that contained between 10(3) and 10(9) culturable general heterotrophs mL(-1) the technique detected between 5 and 2 × 10(6) mL(-1) M. immunogenum cell equivalents (CE) in 12.2% (23 of 189) of U.S. samples and between 8 and 6 × 10(5) mL(-1) CE in 39.1% (68 of 174) of samples from Europe. In contrast, only three cultured presumptive mycobacterial isolates across all samples were confirmed as M. immunogenum. Implementation of the assay demonstrated its practicality and further emphasized the limitations of using cultivation alone. Interestingly, no M. immunogenum were detected in mineral oil-based Bio-Concept MWFs from the United States, while it was more commonly detected in used MWFs based on formaldehyde-releasing biocides than in MWFs free of formaldehyde-depot biocides.

Hypersensitivity pneumonitis and metalworking fluids contaminated by mycobacteria.

Metalworking fluids (MWF) are responsible for hypersensitivity pneumonitis (HP). The aim of the present study was to identify the antigen (Ag) responsible for MWF-associated HP, and to optimise serological diagnosis by definition of a threshold allowing discrimination between HP patients and asymptomatic exposed workers. 13 patients, who were workers at a car engine manufacturing plant, were suspected of MWF-associated HP. Microbial analysis of 83 used MWFs was carried out. Sera from 13 MWF-associated HP patients, 12 asymptomatic exposed workers and 18 healthy unexposed controls were tested to determine their immunological responses to three Ags, including Mycobacterium immunogenum. M. immunogenum was identified in 40% of used fluids by culture and confirmed by DNA sequencing. The threshold for differentiating MWF-associated HP patients from asymptomatic exposed workers was five arcs of precipitation (sensitivity 77% and specificity 92%), as determined by electrosyneresis (ES). Using ELISA methods with protein extract from M. immunogenum, a threshold leading to 92% sensitivity and 100% specificity was established. The detection of specific antibodies against M. immunogenum Ag at high levels in case sera suggests that M. immunogenum-contaminated MWF is responsible for MWF-associated HP. To discriminate MWF-associated HP patients from asymptomatic exposed workers, we suggest a five-arc threshold for ES and a 1.6-AU threshold for ELISA methods.

Development of a species-specific colorimetric-PCR assay for detection and species differentiation of Mycobacterium immunogenum and Mycobacterium chelonae and its comparison with quantitative real-time PCR for field metalworking fluids.

Mycobacterium immunogenum and Mycobacterium chelonae are closely related species associated with occupational hypersensitivity pneumonitis (HP) and nosocomial infections. There is a need to develop specific and readily adaptable methods for detection and speciation of these agents. Here we report development of a probe-based colorimetric-PCR assay involving heat shock protein (hsp) gene amplification (228 bp) and its detection in an ELISA-like reaction. A quantitative format of this assay was developed and validated on metalworking fluids (MWF). The assay showed a minimum detection limit of 10 fg genomic DNA or 1 mycobacterial cell, albeit with variations depending on type and composition of the MWF matrix. When applied to the field MWF samples, the developed assay was found to be comparable to the real-time PCR assay, and allowed direct speciation of MWF mycobacteria without sequencing and/or restriction pattern analysis. In conclusion, the developed colorimetric PCR allows detection and quantification of MWF mycobacteria without culturing and is the first probe-based assay for unambiguous differentiation between the two phylogenetically closely related species, M. immunogenum and M. chelonae. Considering that the assay offers high throughput format involving relatively simpler instrument infrastructure, it has a potential for applications in routine assessment of MWF mycobacteria in diagnostic and industrial laboratories.

Production of hydroxy-fatty acid derivatives from waste oil by Escherichia coli cells producing fungal cytochrome P450foxy.

Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7-16 carbon atoms to their omega-1 to omega-3 hydroxy derivatives. Most products were recovered from the culture supernatant. Decanoic acid was most efficiently converted to omega-1 to omega-3 hydroxy decanoic acids in the order of omega-1>omega-2>omega-3, with a total product yield of 47%. We also found that P450foxy was more active against physiological fatty acyl esters such as monopalmitoyl glycerol, monopalmitoyl phospholipid, and palmitoyl CoA than free palmitic acid. The bacteria producing P450foxy were applicable as biocatalysts in the production of omega-1 hydroxy palmitic acid from lard, vegetable, and soy sauce oil wastes from the food industry.

Microbial and endotoxin contamination of water-soluble metalworking fluids.

UNLABELLED: The present study is a part of a larger project on assessment of exposure to biological agents and respiratory health effects among metal workers. OBJECTIVES: The aim of this study was to evaluate the level of microbial contamination of metalworking coolants as a potential source of harmful biological agents in three different metal industry plants in Poland. MATERIAL AND METHODS: Ten samples of coolants with a different state of wear, including four fresh fluids, were analyzed. Qualitative and quantitative analysis of bacteria and mould content was performed using standard analytical methods. Bacterial endotoxin concentration was determined by the kinetic, chromogenic version of the LAL test. RESULTS: The analysis showed the total bacterial counts ranging from 1.0x10(1) CFU/ml to 3.2x10(7) CFU/ml, 60% to 100% of which were Gram-negative bacteria capable of producing endotoxins. Among the highly contaminated coolants, one unused concentrate was also found. The predominant species of bacteria was Shewanella putrefaciens present in 60% of the samples. As for moulds, the predominant species was Acremonium butyri. The average concentration of bacterial endotoxins was 773 EU/ml in the old coolants, and much lower in the fresh fluids 285 EU/ml. A correlation (r=0.66) was found between endotoxin concentration and the number of Gram-negative bacteria detected in the coolants. CONCLUSION: Procedures should be developed for cleaning tanks during fluid replacement to minimize the risk of bacterial growth in the coolants and limit bioaerosol emission in workplace. Due to the affinity of microflora for the water phase, it is advisable to use effective biocides with appropriate partition coefficient between the water phase and oil phase.

[Harmful impact of biological agents released at metalworking]. / Szkodliwe dzialanie czynników biologicznych uwalnianych podczas procesów obróbki metali z uzyciem chlodziw.

In metalworking, the temperature of the cutting edge can reach 700 degrees C, which can adversely affect either the surface of the metal processed or the cutting edge itself. To reduce the heat and friction, the metalworking fluids and coolants are used. The most common are the oil-in-water emulsions, which are a mixture of concentrated mineral oils, different additives, and water. The presence of water and organic substrates generates conditions for microbial growth. Bacterial endotoxins can also be frequently detected in metalworking fluids. Rapid rotation of the metalworking tools makes various biological agents to be released as droplet bioaerosols that can be inhaled by the workers, thus producing adverse health effects, mainly on the respiratory system. More than 130 species of bacteria (including pathogens), moulds and yeasts have so far been found in the metalworking fluids and coolants. This paper summarizes the outcomes of projects on the impact of biological agents present in metalworking fluids. Exposure to oil mist, bacteria and endotoxins as well as the variety of health effects resulting from that exposure have been discussed. The findings indicate that the most frequent symptoms reported by the workers include cough with phlegm, wheezing breath, chest tightness and throat irritation. The most common among the diagnosed diseases are chronic allergic bronchitis, asthma, hypersensitivity pneumonitis and acute respiratory dysfunction indicated by decreased FEV1 values. The main skin diseases include oil acne, bacterial infections, dermatomycosis and allergic reactions. The review made the authors conclude that the problem of occupational exposure to biological agents released with coolants during metalworking has not been well recognized in Poland.

Hypersensitivity pneumonitis associated with environmental mycobacteria.

A previously healthy man working as a machine operator in an automotive factory developed respiratory symptoms. Medical evaluation showed abnormal pulmonary function tests, a lung biopsy showed hypersensitivity pneumonitis, and his illness was traced to his work environment. His physician asked the employer to remove him from exposure to metalworking fluids. Symptoms reoccurred when he was later reexposed to metalworking fluids, and further permanent decrement in his lung function occurred. Investigation of his workplace showed that five of six large reservoirs of metalworking fluids (cutting oils) grew Mycobacterium chelonae (or Mycobacterium immunogenum), an organism previously associated with outbreaks of hypersensitivity pneumonitis in automaking factories. His lung function remained stable after complete removal from exposure. The employer, metalworking fluid supplier, union, and the National Institute for Occupational Safety and Health were notified of this sentinel health event. No further cases have been documented in this workplace.

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids.

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

Respiratory symptoms, immunology and organism identification in contaminated metalworking fluid workers. What you see is not what you get.

BACKGROUND: Metal working fluids (MWF) constitute a significant respiratory hazard, although symptoms experienced by workers are often poorly investigated and attributed. AIMS: A single possible case of extrinsic allergic alveolitis (EAA) led to a formal workplace investigation. It was clear that other exposed workers were affected. The aim of this study was to accurately quantify the clinical, immunological and microbiological findings in MWF workers following presentation of a sentinel case. METHODS: Eleven of 21 individuals participated; eight were assessed by symptom questionnaire, spirometry and serology and three workers provided blood samples only. The microbes cultured from MWF and air samples were used to determine the presence of precipitating antibodies. RESULTS: Work-related respiratory symptoms were reported in six of eight individuals questioned, two of these complaining of 'flu-like' symptoms. Personal breathing zone measures identified 2.1 x 10(3) to 1.1 x 10(5) colony-forming units/m3 air (CFU/m3). Pseudomonas fluorescens was isolated from air samples. Despite visible 'fungal' contamination of MWF, airborne fungi were detectable in only one sample, at 486 CFU/m3 air. MWF cultured Eurotium sp., Fusarium sp. and Pseudomonas sp. Precipitating IgG antibodies to Pseudomonas sp. were identified in 4/11 and to an extract of the MWF in 3/11. IgG to Pseudomonas was elevated in the two individuals who had the strongest precipitating bands to Pseudomonas sp. CONCLUSIONS: Workplaces with possible EAA must be investigated promptly, thus allowing clinical assessment to be contemporary to exposures and accurate microbiological profiling included to identify the likely cause.

Enhanced biodegradation of transformer oil in soils with cyclodextrin--from the laboratory to the field.

The use cyclodextrins for the intensification of bioremediation by improving the mobility and bioavailability of contaminants has recently been studied. In this work, the role of randomly methylated beta-cyclodextrin in the bioremediation of soils contaminated with transformer oil was studied both in bench scale bioreactors and through field experiments. The aims of this research were to (a) establish the scientific background of a cyclodextrin-based soil bioremediation technology, (b) demonstrate its feasibility and effectiveness in the field, and (c) develop an integrated methodology, consisting of a combination of physical, chemical, biological and ecotoxicological analytical methods, for efficiently monitoring the technology performances. The stepwise increasing scale of the experiments and the application of the integrated analytical methodology supported the development of a scientifically established new technology and the identification of the advantages and the limitations of its application in the field. At each phase of the study, randomly methylated beta-cyclodextrin was found to significantly enhance the bioremediation and detoxification of the transformer oil-contaminated soils employed by increasing the bioavailability of the pollutants and the activity of indigenous microorganisms.

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline.

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.

Metalworking fluid--the toxicity of a complex mixture.

Various chemicals are used in the manufacture of cooling and lubricating fluids and fall into the classes of straight, soluble, semisynthetic, and synthetic metalworking fluids. The diversity of chemicals and in-use contaminants makes the risk assessment of metalworking fluids quite difficult. Toxicologists have used a number of methods to evaluate the component(s) responsible for the adverse pulmonary effects of metal working fluid aerosols encountered in the workplace. Although investigators have studied the adverse effects of metalworking fluid chemicals alone and in combination, the majority of evidence strongly suggests that the microbial changes that occur in fluid composition, during use and storage in the workplace, are responsible for the pulmonary effects reported for workers exposed to metalworking fluid aerosols. This review discusses the methodologies used to examine the toxicity of the complex nature of modern metalworking fluids and the findings that point toward bacterial endotoxin as a major contributor to their adverse effects.

Observations on the relationship between triazines and mycobacteria in metal removal fluids.

The purpose of the study was to determine whether hexahydrotriazine ("triazine") bactericides increase the likelihood of elevated mycobacteria growth in metal removal fluids (MRFs). In reaction to an outbreak of hypersensitivity pneumonitis (HP) at a transmission manufacturing facility, machining plants of a large automobile manufacturer were directed to increase the testing of MRF, especially testing for mycobacteria. A working hypothesis was developed that triazine bactericides cause an increase in mycobacteria contamination, which in turn may lead to an increased risk of HP for workers exposed to MRF aerosols. Test results for 277 central MRF systems from nine machining facilities were analyzed for various MRF properties including the presence and type of both bactericide in the MRF formula and bacterial contamination of the MRF. The study included data reflecting usual operating conditions of all of the 277 large MRF systems in the company. The study does not evaluate the effect of tank-side biocide addition. The results show that soluble oils, semi-synthetic, and synthetic MRFs are all capable of supporting mycobacterial growth. The results also demonstrate a highly significant association between routine use of triazine bactericides and mycobacterial contamination of MRF. Based on the hypothesis that mycobacteria are a likely causative agent of hypersensitivity pneumonitis from MRF exposure, it is concluded that elimination of triazines from MRF formulae may be protective against HP in the machining environment.