Quantitative Real-Time PCR with Metalworking Fluids
Metalworking Fluid users are well aware of the problems caused by unwanted bacterial and fungal colonization of their fluids. Numerous species of microorganisms can cause problems in functional fluids and, barring use of clean-room techniques, all industrial fluids are plagued by constant inoculation with microorganisms.
Numerous species of microorganisms create unwanted odors, and almost all bacteria and fungi selectively consume specific fluid components leading to the loss of fluid function. There can also be adverse health effects caused by microbial contamination, and a number of industrial health syndromes have been linked to systemic infections by and adverse allergic reactions to microbes present in contaminated industrial fluids.
For a number of years, the metalworking fluid industry has suspected that an occupational lung disease known as Hypersensitivity Pneumonitus may be caused by exposure to metalworking fluid mists.
In November 2001, the Center for Disease Control reviewed a situation that involved the hospitalization for respiratory illness of three machinists from an automobile brake manufacturing facility in Ohio. A review of the facility personnel records found that 107 (27 percent) of 400 workers had been placed on work restriction by their physicians during the preceding 11 months because of respiratory conditions; 37 (35 percent) of these 107 workers remained on medical leave and 70 (65 percent) had returned to work. Thirty-two workers had either full or part-time work duties in the machining portion of the plant; the median length of time working in the plant was 18 years (range 3 to 32 years). Initial symptom onset for this cluster occurred during October 2000 with continued cases being reported through April 2001. The peak reporting frequency occurred in December 2000. From February through July 2001, multiple samples of buld metalworking fluid from all central plant systems were analyzed. Thistesting revealed the predominant growth of a newly proposed species of the Mycobacterium abscessus / Mycobacterium chelonae group, with the name of Mycobacterium immunogenum. This bacterium was found at population leves of up to 1 million bacteria per milliliter.
Based on this an related incidents, a limited number of scientific and industrial health-care professionals have proposed a link between M. immunogenum and metalworking fluid related Hypersensitivity Pneumonitis (HP). However, the link between Mycobacterium and HP remains a hypothesis at this point with a number of research groups around the world starting to work on definitively proving or disproving the connection.
What are Mycobacteria?
The species of the genus Mycobacterium are broadly grouped into two major categories on the basis of pathogenicity for animals and humans. The extremely pathogenic organisims M. tuberculosis and M. leprae are obligate parisites (the must transfer directly from host-to-host without residence in the environment), but the majority of the species, such as M. smegmatis and M. terrae, are ubiquitous in the environment. However, as mentioned above, the species M. immunogenum has been implicated as a cause of the infrequent but serious lung condition Hypersensitivity Pneumonitis. The occurence of HP has been stronly correlated with chronic exposure to metalworking fluid mists, and the bulk of the metalworking industry has started to examine methods for the control of mycobacteria with an aim toward elimination of HP.
Giving the ubiquitous nature of the genus Mycobacterium, on might ask: Just how dangerous are environmental mycobacteria? How does one detect and quantify mycobacteria? Why would mycobacteria proliferate in preference to other environmental bacteria?
The mycobacteria are unicellular, aerobic, weakly Gram-positive but still acid-fast bacteria (i.e., once stained with one of the basic dyes such as fuchsin, they resist decolorization with mineral acids or with acidified organic solvents).
The early subdivision of the "atypical" mycobacteria into "groups," on the basis of pigment production and speed of growth, was proposed to provide a more systematic basis for study and discusion of these organisms.
How to rapidly quantify the microbial population is a fundamental problem in the investigation of the occurrence of Mycobacterium in complex environments like metalworking fluids. Mycobacterium are slow-growing organisms that are difficult to quantify with traditional culture techniques. The problem of accurate measurement is so fundamental to this field that an entire symposium was held Dec. 5, 2004 in Tampa, Fla. The symbosium, "Recovery and Enumeration of Mycobacteria from the Metalworking Fluid Environment," was jointly sponsored by ASTM Committee E34.50 on Health and Safety of Metalworking Fluids and Committee D02.L.1 on Metalworking Fluids (see www.astm.org).
Tools and Tests
More recently, DNA technology has initiated a new era in environmental microbiology. Nucleic acid based methods provide specific sensitive detection or microorganisms from a variety of environments.
Quantitative PCR (qPCR) is possible through the combined use of specialized PCR reagents (e.g., TaqMan) and refined instrumentation. This advance is partiularly useful in environmental microbiology because the population size, the amount of a particular organism, can be determined, and thus population changed can be tracked over time or in response to a change in the environment. The authors of this article have been applying these methods to Mycobacterium detection, identification, and population size determination in metalworking fluids.
The undisputed success of detection assays based on the polymerase chain reacation has been largely due to PCR's speed in comparison with many conventional diagnostic methods, such as cultures.
PCR has the ability to amplify specific DNA sequences in an exponential fashion by in vitro DNA synthesis. The PCR technique can be used to detect, identify, and differentiate microbial agents present in either clinical or environmental samples.
With PCR based techniques, a thorough and complete analysis of suspect metalworking fluids has become possible. Through statistical analysis of a large number of metalworking fluids, along with clinical analysis of any assoicated lung disease, and accurate assessment of the connection between mycobacterium and HP will ultimately by made.
ARKEMA is currently conducting several startisical studies of the influence of our high-performing SYNERGEXTM amine additives as inhibitors of mycobacteria.
Larger and more thorough studies aimed at a comprehensive understanding of the real danger of Mycobacterium contamination in metalworking fluids are being carried out by a number of other groups too, such as NIOSH.
With a combination of commercial and public efforts, the role of Mycobacterium species in metalworking fluid related respiratory health issues will ultimately be understood and controlled.
So how does the formulator deal with the contradictory goals of absolute biological control and minimization of biocide concentrations? One possible approach, in many cases just a start, is the use of biocide synergy.
Formulating a Metalworking Fluid
The modern formulator still uses empirical methods, but empiricism is supplemented with conceptual knowledge. For examples, the moder formulator understands the function of all the components in a formulation. We could call this the function concept. The function concept allows for the rapid selection of candidate materials to test in a given formula. Even with the function concept, the formulation process can be time consuming. Without it, the process is impossible.
What additional concepts can formulators use to accelerate discovery? One important idea could be called the "range of phsyical properties" concept.
After an assessment of function and the range of physical properties needed, the formulator considers a variety of other issues like availability, cost, biodegradability, toxicity, and general customer acceptance. After all of this, a much smaller group of possible candidate components will emerge, and these materials can then be obtained in sample quantities for laboratory testing. After the final formula components are chosen and the appropriate level of each component is defined, a functional fluid will hopefully result.
Dr. Bruce C. Hemming, president, CEO, and founder of St. Louis, MO based Microbe Inotech Laboratories, Inc. has conducted R&D in the microbiological arena for over 30 years. He has joined Dr. Michael D. Gernon, a senior research scientist with ARKEMA in King of Prussia, PA, who has worked on metal finishing and metal forming problems for over 50 years and has written several articles concerning unwanted bacterial and fungal colonization occuring in metalworking fluids.