Annals of Occupational Hygiene Advance Access originally published online on July 27, 2004
Annals of Occupational Hygiene 2004 48(6):541-546; doi:10.1093/annhyg/meh043
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© 2004 British Occupational Hygiene Society Published by Oxford University Press;
Six Month Tracking of Microbial Growth in a Metalworking Fluid After System Cleaning and Recharging
1 Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie and Département de Biochimie et de Microbiologie de l'Université Laval, 2725 Chemin Ste-Foy, Québec, Ste-Foy, G1V 4G5, Canada; 2 Environmental Health Sciences Research Center, University of Iowa, Iowa City, IA, USA and 3 NYU Medical Center, Tuxedo, NY, USA
* Author to whom correspondence should be addressed. Fax: +1 418 656 4509; e-mail: caroline.duchaine{at}bcm.ulaval.ca
Received 3 July 2003; in final form 4 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Large volumes of metalworking fluids (MWFs) are used in manufacturing industries for cooling and lubrication of metal pieces and tools during machining. MWFs accumulate microbial growth through continuous recirculation and reuse. We studied the progression of microbial contamination for 6 months after dumping, cleaning and recharging (DCR) of a large semi-synthetic MWF system managed with several biocides. Fresh, uncontaminated fluid was added to the system after extensive cleaning. The following samples were collected and analyzed: pre-DCR fluid (before system cleaning); neat fluid diluted to 6% with water; in use MWF 12 h and 1, 3 and 6 months post-DCR. Samples were analyzed for total microorganism concentrations by direct counting using fluorescence microscopy and by plate counting on various media (R2A, BHI, Middlebrooks and rose bengal under aerobic conditions). In addition, PCR was performed for the detection of mycobacteria. There was a rapid progression in the total bacterial counts as determined by fluorescence microscopy: 5·7 x 107 cells/ml in the pre-DCR used fluid, no measurable bacteria in the neat fluid, 6·9 x 106 cells/ml after 12 h and 2·2 x 106, 3·6 x 108 and 6·1 x 108 cells/ml after 1, 3 and 6 months, respectively. On average, only 0·2% of the direct count organisms were quantified on R2A cultures. PCR showed the presence of mycobacteria in the used MWF at 3 and 6 months. Mycobacteria were also identified from cultures on Middlebrooks and R2A. This study demonstrates that standard methods for cleaning MWF systems are inadequate since residual bacteria in the system can rapidly repopulate the newly charged MWF.
Keywords: biocides • machining • metal working fluids • mycobacteria • PCR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Metal working fluids (MWF) are commonly used in manufacturing and machining industries for boring, hobbing, tapping, grinding and honing metal components to precise dimensions (Thorne and Sprince, 2004
). There are four major classes of MWF: those which contain lubricant base oils without water (straight oils), those composed of an aqueous oil emulsion with oil in high concentration (soluble oils) or lower concentration (semi-synthetic MWF) and those formulated with no petroleum oils (synthetic fluid). The soluble oil MWF are composed of 3–6% oil emulsified in water. These fluids usually contain a variety of additives, including emulsifiers, biocides, antifoaming agents and corrosion inhibitors (Thorne et al., 1996). Even if biocides are added to the fluids, the soluble oils are propitious for microbial growth such as Gram-negative bacteria, e.g. Pseudomonas spp. (Tant and Benett, 1956; Mattsby-Baltzer et al., 1989a,b; Thorne et al., 1996; Thorne and Sprince, 2004). Cross-sectional studies of respiratory disease among workers in contact with soluble MWF (Ameille et al., 1995; Kriebel et al., 1997; Kennedy et al., 1999; Hodgson et al., 2001) suggest that acute lung function decrements are at least weakly associated with the respirable fraction of airborne bacteria, total particulate matter and possibly endotoxins.
Hypersensitivity pneumonitis (HP) is a granulomatous allergic lung desease (Kreiss and Cox-Ganser, 1997), most common among farmers, that has been reported in workers exposed to MWF (Muilenberg and Burge, 1993; Bernstein et al., 1995; Embil et al., 1997; Freeman et al., 1998; Fox et al., 1999; Shelton et al., 1999; Hodgson et al., 2001). This disease was associated with antibody responses to environmental microorganisms (Pseudomonas fluorescens) that were isolated from the used MWF. In that study, the presence of other HP-causing microorganisms was not evaluated. Mycobacterium chelonae has frequently been recovered from MWF associated with HP (Kreiss and Cox-Ganser, 1997; Moore et al., 2000). The presence of mycobacteria in MWF can be explained by the fact that (i) M.chelonae is more resistant to chlorine than coliform bacteria and is found in 83–90% of samples of domestic water purification systems (Carson et al., 1988a,b) and (ii) M.chelonae is resistant to up to 8% formaldehyde (Carson et al., 1978). More recently, HP cases related to MWF have been associated with a new mycobacteria species, Mycobacterium immunogenum (closely related to M.chelonae/abscessus) (Rose, 1996; Shelton et al., 1999; Weiss, 2002). It was shown that several MWF systems that tested positive for M.immunogenum became negative and then changed back to positive (Moore et al., 2000). The culturability state of the cells may explain this shift. The main distinguishing differences between M.chelonae and M.immunogenum is the number of copies of the rRNA operon and the ability of M.chelonae to use citrate as carbon source (Wilson et al., 2001). Culture counts grossly underestimate the actual concentration of bacteria in MWF (Mattsby-Baltzer et al., 1989b; Thorne et al., 1996; Lange et al., 1997). In fact, culture-based enumeration represents only a small proportion of the viable and culturable (VC) microorganisms and doesn't recover the viable but non-culturable (VNC) cells. Obviously, culturing does not account for dead or non-viable (NV) cells that are present. However, most dead cells retain their immunogenicity and can cause sensitization and possibly HP. Because classical culture methods underestimate the total microbial load of MWF, tools like the direct count method (Thorne et al., 1996; Hobbie et al., 1977; Lange et al., 1997) using epifluorescence microscopy are needed to estimate the concentration of microorganisms of all viability statuses (VC, VNC and NV) present in MWF samples. In addition to the direct count method, PCR can be used to give a better idea of the presence of specific organisms in MWF, including mycobacteria.
In this study, a MWF system where HP cases had been described was emptied, cleaned and recharged. The aims of the present study were to:
- quantify the microbial load of MWF before and after dumping, cleaning and recharging (DCR);
- compare culturing using a broad range of growth conditions with direct counting using epifluorescence microscopy for the quantification of microorganisms in MWF;
- optimize and apply a PCR protocol for the detection of mycobacteria and compare PCR with culture-based detection.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Samples
A MWF sump system that had been associated with prior cases of HP was selected for study. Samples of the original MWF (called pre-DCR), the new replacement fluid and the newly charged in-use MWF 12 h and 1, 3 and 6 months after the system was cleaned and recharged were taken. The MWF system in this facility was maintained by the supplier and fluid maintenance procedures were typical of the industry. No changes in fluid maintenance were made after DCR. The storage tank was emptied as completely as possible and then washed using high pressure hot water. The washing included all regions that were accessible. Interiors of pipes were not accessible and, thus, were not cleaned. The system was next charged with a cleaning cocktail, which was circulated for 5 h. The cleaning cocktail comprised: a solvent-based cleaner with surfactants; an industrial detergent containing potassium hydroxide, trisodium orthophosphate, amide soap, sodium tetraborate and sodium metasilicate; 0·1% Kathon biocide. This solution was then dumped and accessible areas of the system were further washed and scraped. The system was then charged with new coolant. During operation, the system was treated continuously with a formaldehyde-releasing compound and monitored to keep levels between 0·08 and 0·12% with Grotan and at pH 9·6· Bacteria and fungus levels were monitored 3 times/week with dipsticks. Kathon was added if fungal colonies exceeded 10 c.f.u./ml.
Plate count
Samples were collected and then shipped express mail under refrigeration. Upon receipt, 100 µl of three serial dilutions from 100 to 10–7 were plated on different media: BHI agar incubated at 35°C for 48 h; R2A incubated at 25°C for 48 h and 2 weeks; Middlebrooks 7H10 with OADC enrichment were incubated at 35°C for 2 weeks. Culturing of molds was done on Rose bengal agar at 25°C for 7 days.
Direct count method
The direct count method (Thorne et al., 1996; Hobbie et al., 1977; Lange et al., 1997) using fluorescence microscopy was performed using a Nikon ECLIPSE 660 equipped with a SPOT RT slider digital camera and the SPOT advanced image capture software. Two staining kits were used: a DAPI staining kit (Molecular Probes, Eugene, OR) and the Live/Dead Baclight (Molecular Probes), using a concentration ratio of 3 [Syto9:propidium iodide (PI)]. The stained samples were filtered through a 25 mm black stained polycarbonate nucleopore membrane (0·22 µm) (Millipore, Cambridge, ON). A minimum of 10 different fields were randomly pictured from the same filter using a standardized pattern that eliminates double counting of the same field. Knowing the ratio between the picture area and the filtration area (20:500), the volume and the dilution filtered, the counts in cells/ml (Eduard et al., 2001) can be evaluated.
DNA extraction
DNA extraction was performed on the pelleted cells from 1·5 ml of MWF sample. The pellet was washed twice in phosphate-buffered saline and digested with proteinase K (Sigma-Aldrich Corp., St Louis, MO) for 2 h at 50°C, in a maximum volume of 100 µl. The digested cells were heat shocked by three cycles of 100°C followed by freezing on dry ice in 500 µl of DNAZOL (Invitrogen, Burlington, ON) containing 15% Chelex 100 (Bio-Rad, Mississauga, ON). After freezing/boiling, 500 µl of DNAZOL without Chelex was added to a final volume of 1·1 ml. DNA precipitation was performed with 99% ethanol as specified by the DNAZOL manufacturer's procedure. The precipitated DNA was then resuspended in 50 µl of ultrapure water (Sigma).
Polymerase chain reaction
PCR was performed using a MJ Research Pelletier Thermocycler (MJ Research, Waltham, MA) under previously published conditions (Kox et al., 1995). The amplified region of 16S rDNA contains several polymorphisms. The composition of the PCR mixture was 10 mM Tris–HCl (pH 8·3), 50 mM NaCl, 3·0 mM MgCl2, 0·2 mM each deoxynucleotides triphosphate (dATP, dCTP, dGTP and dTTP), 0·2 µM each primer (pMyc14 and pMyc7) (Kox et al., 1995) and 1 U Taq polymerase (Promega, Mississauga, ON) per 50 µl reaction mixture volume. To run samples, 5 µl of DNA extract was added to 45 µl of the PCR mixture. A PCR cycle included 60 s of DNA denaturation at 94°C, 90 s of annealing at 64°C, and 90 s extension at 72°C. A total of 70 PCR cycles were performed. After the last cycle, samples were maintained at 4°C until they were stored at –20°C. The control strains used for the PCR are presented in Table 1.
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Identification of isolates
The different isolated strains were classified on the basis of acid-fast properties. Non-acid-fast strains were identified by cellular fatty acid analysis by gas chromatography according to the procedure described by MIDI Inc. (MIDI Inc., Newark, NJ). The analysis was performed with the Sherlock Microbial Identification System using the databases CLIN 40 and TSBA (MIDI Inc.). The acid-fast isolates were identified by sequencing the first 500 base pairs of the 16s rDNA sequence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Quantification of microorganisms
Figure 1 shows the microbial contamination of MWF samples assessed by plate counting on R2A (since this medium gave the highest counts) and direct counting. A rapid progression of the microbial mass in the MWF system was observed. The pre-DCR sample shows a concentration of 4·5 x 105 c.f.u./ml culturable microorganisms and 5·7 x 107 total cells/ml. The neat metalworking fluid used for recharge was essentially sterile. Upon recharging the system, the bacterial concentration increased to 1·6 x 103 c.f.u./ml and 6·9 x 106 cells/ml in just 12 h. After 1 month, the concentration of culturable microorganisms reached 3·1 x 103 c.f.u./ml and the total organisms reached 2·3 x 106 cells/ml. After 3 months of use the concentrations reached 2·7 x 105 c.f.u./ml culturable microorganisms and 3·6 x 108 cells/ml. The last sample (6 months post-DCR) showed a concentration of culturable microorganisms of 3·1 x 105 c.f.u./ml and 6·1 x 108 cells/ml. Non-significant mold growth was observed.
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Table 2 shows the comparison of plate counts and direct counts. The plate count method dramatically underestimated the total bacterial population provided by the direct count method. The results demonstrate that the culture method retrieved <1% of the microbial mass present in MWF samples (0·02–0·78%).
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Identification of isolated strains
As showed in Table 3, acid-fast bacilli were the predominant species recovered in all contaminated samples of MWF. Mycobacterium immunogenum was discriminated from M.chelonae by the inability to use citrate as a sole carbon source and the identification was confirmed by sequencing the PCR product. The other isolates were not sequenced but had the same morphology. Gram-positive bacteria were also identified: Staphylococcus, Bacillus and Micrococcus were the predominant non-acid-fast microorganisms isolated. These species most likely came from the work environment (Bacillus and Exiguobacterium) and the workers' microflora (Staphyloccus and Micrococcus). It was observed that the bacterial population was relatively stable over time and the first observed strain (M.immunogenum) was the most important representative of the culturable portion of microorganisms after 6 months.
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PCR detection
Detecting mycobacteria by PCR in MWF samples gave a qualitative estimate of the presence of mycobacteria in metalworking fluids. All the samples that were positive for Mycobacterium sp. by culture were also positive for the genus Mycobacterium as detected by PCR (Table 4). PCR positive samples contained as few as 1·6 x 103 c.f.u./ml culturable mycobacteria.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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When respiratory problems occur, emptying, cleaning and recharging the problematic MWF system is often the recommended solution. In the system studied, an important microbial load remained even after extensive cleaning of the MWF sump system and was able to seed the system within 12 h, clearly showing that the approach was inadequate to ameliorate the risk of HP. Defective cleaning methods and the presence of biofilms in the system could be at the origin of this rapid post-cleaning contamination. It is very difficult to estimate the fraction of MWF that was not removed from the system but the only places that were not thoroughly cleaned were in inaccessible regions of the system. A constant increase in the microbial mass in the 1, 3 and 6 month samples was observed. This reinforces the notion that neat MWF can support microbial growth (Mattsby-Baltzer et al., 1989b
; Chazal, 1995; Lonon et al., 1999) and provide nutritional elements for microorganisms (Lonon et al., 1999). In the setting we have studied, the use of biocides may not significantly limit microbial growth (Izzat and Bennett, 1979; Taylor and Falkinham, 2000). Other biocides used in a different system may have produced a different result.
The recovery rates of plate culture methods compared with direct count methods demonstrated an underestimation of nearly 3 log. It is known that sensitization to antigenic bacteria such as mycobacteria is independent of their culturability (Costabel, 1989). Direct bacterial counting is largely for risk evaluation associated with exposures to immunogenic organisms. Since direct counting using fluorescence microscopy does not allow one to isolate and identify the bacteria present, culture recovery of microorganisms present in MWF samples remains important for identification. The isolation of major strains is essential to aid in the evaluation of the sensitization of workers to MWF airborne bacteria. If culturing were not performed, a complete molecular microbial ecology study would have to be performed for each MWF sample to identify the bacteria present.
The relationship between HP outbreaks and the presence of mycobacteria in MWF systems have not been clearly proved. However, some scientific evidence supports the hypothesis that M.chelonae and M.immunogenum are HP-causing agents (Greaves et al., 1997; Abrams et al., 2000; Wilson et al., 2001). In this study M.immunogenum was the most important culturable microorganism found in the MWF samples studied. The system studied did not show co-colonization of the system by mycobacteria and other microorganisms able to use complex hydrocarbon sources such as Pseudomonas sp.
The detection of mycobacteria in MWF samples by PCR was a rapid and reliable tool. Optimization of the DNA extraction protocol was performed to improve the sensitivity of the mycobacteria PCR protocol from that published by Kox et al. (1995). Other purification steps were added to enhance removal of the PCR inhibitors presents in MWF samples. Under our conditions PCR detection rapidly gave qualitative information about the presence of mycobacteria in MWF samples without culturing. This is especially important for MWF samples containing mycobacteria because of the slow growth rate of these organisms. Work is in progress to extend this method to quantitative real-time PCR.
This study has shown that emptying, cleaning and recharging a problematic MWF system without removing all subsequent contamination sources may not be the best solution to the problem of HP outbreaks. Different cleaning processes or biocides may have led to a different result. Mycobacteria can rapidly repopulate the system and the risk related to exposure may remain the same. Solutions may include the use of biocides that differ from that in the system studied here and recharging with MWF pre-contaminated with a relatively harmless bacterial consortium. A better understanding of MWF microbial ecology is essential to apply workable solutions to microbial contamination of MWF systems.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The authors are thankful to Mrs Carole Pépin (IRSST) for bacterial identification support. This project was supported by grants from the Metal Working Fluid Product Stewardship Group of the Independent Lubricant Manufacturers Association, CDC/NIOSH OH03044 (T.G.) and the UAW-DaimlerChrysler National Training Center. C.D. acknowledges an IRSST/CIHR scholarship.
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