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Applied and Environmental Microbiology, November 2002, p. 5580-5584, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5580-5584.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Presence of a Single Genotype of the Newly Described Species Mycobacterium immunogenum in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis

Richard J. Wallace, Jr.,^1* Yansheng Zhang,¹ Rebecca W. Wilson,^1,2 Linda Mann,¹ and Harold Rossmoore³

Departments of Microbiology,¹ Pathology, The University of Texas Health Center, Tyler, Texas,² Biosan Laboratories, Warren, Michigan³

Received 13 May 2002/ Accepted 1 August 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The recognition of the presence of mycobacterial contamination in used water-based metalworking fluids (MWF) used in industrial (primarily automotive) metal grinding is quite recent. The timing of this finding coincided with the emergence of occupational asthma and of hypersensitivity pneumonitis (HP) in industrial machinists, suggesting that the two events may be related (4). Other key features which may play roles in both of these developments are the aggressive use of formaldehyde condensate biocides used to control growth of gram-negative bacilli, the dominant population in MWF, and changes in the bacterial flora related to the use of these biocides which facilitated the presence of the mycobacteria. Microbial growth is considered important in the degradation of MWF and water-based hydraulic fluids (12, 13).

This story began in 1993, when Muilenberg et al. (M. L. Muilenberg, H. A. Burge, and T. Sweet, abstract from the 49th Meeting of the American Academy of Allergy and Immunology, 12 March 1993, Abstr. 682, J. Allergy Clin. Immunol. 91:311, 1993) reported an outbreak of HP in 10 patients (machinists) in an industrial plant exposed to aerosolized water-based MWF. Culture of the used MWF revealed 10⁶ to 10⁷ mycobacteria (i.e., acid-fast bacilli [AFB]) per ml of the MWF. Although the mycobacterium was not identified to the species level, this was the first recognition of the potential of AFB in the etiology of HP in industrial machinists. The mycobacterium might have been present in earlier samples and/or HP outbreaks, but since its incubation time was 4 to 6 days, it would not have been detected on routine bacterial culture after a 48-h incubation.

Subsequently, in 1995, Bernstein et al. (1) reported additional cases of HP associated with the use of semisynthetic MWF, a disorder he referred to as "machine operator's lung." Although AFB were not identified in the MWF, AFB were recovered on culture of the sputum from one of the six affected patients. By 1997, eight clusters of HP involving almost 100 workers had been described, prompting investigation of the characteristics of the MWF that might be responsible (8).

A review of the microbiology of these outbreaks revealed that used water-based MWF supported a complex population of microbial flora, with numbers that frequently exceeded 10⁷ organisms per ml. In addition to the usual Pseudomonas species, other aerobic gram-negative bacilli, and fungi that routinely colonize these water-based fluids, rapidly growing mycobacteria were also commonly present, as Muilenberg et al. (Muilenberg et al., J. Allergy Clin. Immunol. 91:311, 1993) had first noted (8, 10).

These rapidly growing mycobacteria had previously been identified phenotypically as Mycobacterium chelonae, M. abscessus, or M. chelonae/abscessus, due to the lack of molecular identification methods at that time. A study of mycobacterial colonization in a single industrial plant with recent cases of HP was published in 2000 (10). According to analysis with molecular techniques, the mycobacteria in the used MWF of that plant consisted of a single strain and had a PCR restriction enzyme analysis (PRA) pattern that was different from those of all previously described rapidly growing species (10). Subsequently, Wilson et al. (22) published a detailed taxonomic study of organisms from the plant as well as from human clinical isolates and named the new taxon Mycobacterium immunogenum for its association with HP.

We collected mycobacterial isolates from industrial plants where cases of HP had been reported. These isolates were identified by PRA to determine how many of the isolates recovered from the sites were M. immunogenum. A comparison of the genomic DNA restriction fragment length polymorphism (RFLP) patterns of the MWF isolates with those of the clinical isolates of M. immunogenum was done to determine whether all the industrial plants had been colonized with a single strain, as had been recently reported in the study of isolates from a single industrial plant (10).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organisms.
A total of 107 isolates of mycobacteria recovered from MWF in industrial plants with known cases of metal-grinder HP were obtained from cultures of the MWF directly or procured from the culture collection of one of the authors of the present work (H.R.). These included isolates from 10 sites in six states (Ohio, Illinois, Wisconsin, Michigan, New York, and Oregon) and Ontario, Canada.

Fifteen clinical isolates of M. immunogenum were identified among isolates of rapidly growing mycobacteria which had been submitted to the Mycobacterial/Nocardia Laboratory of the University of Texas Health Center at Tyler for identification and/or susceptibility testing. None of the isolates were epidemiologically linked with each other, and none were recovered from patients with HP. Clinical isolates were from Texas, Florida, Louisiana, Michigan, North Carolina, Massachusetts, Indiana, and Iowa. Only one isolate was from a state (Michigan) that also provided MWF isolates for evaluation. Eleven of the clinical isolates of M. immunogenum had already been identified and characterized in a prior taxonomic study (22).

Clinical isolates of M. immunogenum from six medical pseudo-outbreaks involving contaminated automated commercial endoscopic (bronchoscopic) washing machines were also identified. Details of several of these pseudo-outbreaks have been previously published (7, 9, 21, 24). These studies included isolates from tap water, washing machines, and bronchoscopes, and it was found that each pseudo-outbreak was due to a single pulsed-field gel electrophoresis (PFGE) type (genotype). Pseudo-outbreaks occurred in Missouri, Kentucky, Michigan, North Carolina, Pennsylvania, and Maryland.

PRA.
Isolates were identified to the species level by PRA, using the 439-bp Telenti fragment of the 65-kDa heat shock protein (hsp) gene as previously described (16, 17, 22). The DNA fragment was amplified by PCR and then digested with the restriction enzymes BstEII and HaeIII. The restriction fragments were then separated by agarose gel electrophoresis, and the fragment pattern was compared to those of known mycobacterial species. Eleven of the 15 clinical isolates, as well as 5 of the 6 bronchoscope pseudo-outbreak isolates, had been confirmed by PRA as harboring M. immunogenum in the earlier taxonomic study (22).

PFGE.
Clinical and MWF isolates identified as M. immunogenum were studied for their large RFLP patterns, using PFGE as previously described (21). The chromosomal (genomic) DNA was isolated and then cut with the infrequent cutting restriction enzyme DraI or XbaI (these restriction enzymes have rare restriction sites and are commonly used for DNA fingerprinting of rapidly growing mycobacterial strains) (7, 9, 10, 21). The large restriction fragments (bands) generated by this digestion were then separated by PFGE. Strain relatedness was determined using the criteria of Tenover et al. (18). By these criteria, isolates with no band differences with a given enzyme are considered indistinguishable or identical, isolates that differ by 1 to 3 bands are considered closely related (i.e., clonal), isolates that differ by 4 to 6 bands are considered possibly related, and isolates that differ by seven or more bands are considered unrelated (i.e., unique or different). The PFGE patterns of 12 of 40 M. immunogenum isolates recovered from a single manufacturing plant have been published previously (10).

Top Abstract Introduction Materials and Methods Results Discussion References

 
PRA.
Among the 107 mycobacterial MWF isolates analyzed in the present study, 2 were identified as M. abscessus, 1 was identified as M. chelonae, 2 could not be identified, and the remaining 102 isolates (95%) were identified as M. immunogenum. The 6 bronchoscopic isolates and the 15 clinical isolates were identified as M. immunogenum, with the same PRA pattern as that of the MWF isolates.

PFGE.
Among the 80 industrial MWF isolates of M. immunogenum fingerprinted, all gave satisfactory PFGE patterns (i.e., easily readable restriction fragments). Of the 80 isolates, 2 (3%) gave unique or unrelated DNA digestion patterns with the restriction enzyme DraI. Of the remaining 78 MWF isolates cut with DraI, 76 of 78 (97%) were identical, with no differences in their restriction fragment band patterns. The remaining two isolates differed by only one band and two bands, respectively, from the common DraI pattern (Fig. 1) and are classified as closely related to (or clonal with) the common DraI pattern isolates (18).

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FIG. 1. PFGE patterns of M. immunogenum isolates from MWF from different plants. The chromosomal DNA was digested with DraI. Lanes: 1, yeast DNA standards; 2, isolates from Ohio; 3, Illinois; 4, Indiana; 5, Wisconsin; 6, Illinois; 7, Wisconsin; 8, Indiana; 9, Michigan; 10, New York. All of the DraI PFGE patterns were identical.

The same two isolates that gave unique DraI restriction patterns also gave unique patterns with the second restriction enzyme, XbaI. A total of 68 of the isolates exhibited identical (i.e., indistinguishable) XbaI restriction patterns (designated pattern A1). An additional four isolates differed by one band, and another six isolates differed by two bands (i.e., exhibiting the closely related or clonal pattern). These one- and two-band differences were seen in plants that also contained the common XbaI pattern. This common pattern or genotype was the predominant pattern for all but one plant site.

All six bronchoscopic isolates which were representative of the six pseudo-outbreak genotypes gave satisfactory PFGE patterns. The isolates from the pseudo-outbreaks gave patterns unrelated to the common MWF pattern (Fig. 2).

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FIG. 2. PFGE patterns of DraI digests of M. immunogenum from six contaminated bronchoscope pseudo-outbreaks. Lanes: 1, yeast DNA standards; 8, lambda DNA standards; 2, isolates from Missouri; 3, Maryland; 4, Kentucky; 5, Pennsylvania; 6, Michigan; 7, North Carolina. Note that the patterns for lanes 2 and 7 show only a two-band difference.

All 15 patient isolates of M. immunogenum gave satisfactory PFGE patterns. The genotype of one strain, from a case of disseminated skin infection in a child with a severe immune disorder from Texas (22), exactly matched the common DraI and XbaI genotype of the MWF isolates. A second isolate differed from the common pattern by five bands with DraI and four bands with XbaI (i.e, was considered possibly related). The patterns of the remaining 13 patient isolates were unrelated to the common MWF RFLP pattern or to each other according to analysis by PFGE (Fig. 3). Thus, the industrial MWF DraI restriction pattern was matched by only 1 of 15 (7%) of the human clinical isolates and 0 of 6 of the contaminated bronchoscope pseudo-outbreak isolates.

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FIG. 3. PFGE patterns of XbaI digests of 14 of the 15 epidemiologically unrelated clinical isolates of M. immunogenum from multiple states within the United States. Lane 15, lambda DNA standards.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two major observations resulted from this study. The first is that 95% of mycobacterial isolates recovered from industrial MWF in plants with cases of machinist HP belong to the recently described rapidly growing, nonpigmented species M. immunogenum (22). Rapidly growing mycobacteria are present in 90% of municipal water systems, the most common species being M. mucogenicum (2, 3, 5, 14, 19). M. abscessus, M. chelonae, and M. fortuitum, which are the species of rapidly growing mycobacteria most commonly associated with human disease (6, 19, 20), are also present on occasion in these systems. M. immunogenum has been recovered from hospital water systems (7, 9), but in one study using PRA of U.S. water systems, its presence was not reported (5). Tap water within the plants was the presumed source of the M. immunogenum recovered from the MWF, although it has not been recovered from this source. Slowly growing mycobacteria, especially M. gordonae, are also commonly found in piped water systems (5, 11). The biofilm layer at the water-to-solid surface interface within the system is considered to be the reservoir for these organisms (5).

So why was M. immunogenum found in this setting? A previous study showed that a MWF isolate of M. immunogenum was more resistant to a fresh dilution of the water-soluble MWF than isolates of reference strains of M. chelonae or M. abscessus (10). Those latter two species are the species most closely related taxonomically to M. immunogenum, as their 16S rRNA genes differ by only 8 and 4 nucleotides, respectively, out of almost 1,600 base pairs (22). M. chelonae and M. abscessus were found in some industrial MWF samples in the present study. Formaldehyde-based biocides are commonly added to MWF, and no studies of the resistance of different genotypes of M. immunogenum to these biocides compared to that of other mycobacterial species has been published. Resistance to formaldehyde has been suggested as a key feature for survival in these fluids. Of interest, this species appeared to be the major mycobacterial species that contaminated automated machines that cleaned bronchoscopes (7, 9, 22). Glutaraldehyde (2%) was used in the machines, and relative resistance to this biocide may help explain the presence of this species here as well. Thus, formaldehyde resistance and relative resistance to degraded MWF are potential factors in the almost exclusive presence of this species compared to that of other mycobacterial species.

Another possibility is that M. immunogenum is able to metabolize one or more of the degraded hydrocarbons present in the used fluid, while other mycobacteria may lack such a capability. It appears that there is an association between the presence of M. immunogenum and development of machinist HP. Other sites without cases of machinist HP may be colonized with other mycobacterial species or no mycobacteria. Shelton et al. (15) reported that they recovered mycobacteria from six of seven facilities from which samples were taken for testing as part of the evaluation of cases of HP among metal grinders, while mycobacteria were recovered from only one of eight facilities without cases of machinist HP. M. immunogenum remains the leading candidate for the cause of HP in machinists using MWFs of any type. Whatever the reason, used industrial MWF appears to be a special ecologic niche for M. immunogenum. It clearly can appear in other environmental settings, however, as its association with contaminated bronchoscopic pseudo-outbreaks demonstrates.

The second major observation is that all of the industrial sites are colonized with the same PFGE type (genotype) of M. immunogenum, a genotype that is relatively infrequent (1 of 15 [7%]) among clinical isolates of the species. This is an astonishing finding for an environmental organism that is presumed to be ubiquitous. One would have expected geographically distant industrial sites, as with infected patients, to have unrelated genotypes. Such is clearly not the case. With a few exceptions, all the MWF isolates are the same. The clinical isolates of the species from geographically distant sites were clearly heterogeneous, although one isolate (from a disseminated skin infection in a child from Texas) had the same genotype as the MWF isolates. This would seem to discount some unusual geographic localization of the MWF genotype as an explanation for the high degree of identity of the isolates. Could these isolates have spread from one plant to another? This would be possible if the freshly prepared MWF were contaminated and if all of the plants used the same fluid. This seems very unlikely, as fresh undiluted MWF appears toxic to this strain (10) and all plants do not use the same brand of fluid. Cases of HP have also been reported in association with all three types of MWF: synthetic, semisynthetic, and natural soluble oils (8). Spread or exchange of fluids or contaminated equipment between different industrial plants also seems plausible although highly unlikely. No special measures were taken to prevent the possibility of laboratory cross-contamination to explain the presence of the same genotype among all the isolates. However, these isolates were all recovered at different time periods, and the large number of different sources utilized (10 different plants) also makes this unlikely.

Other studies of genotyping have generally not resulted in findings that show that a specific genomic DNA pattern exhibits differences in virulence, although some genotypes of M. tuberculosis appear to be more contagious than others. Most virulence factors or unique genes among environmental bacteria or mycobacteria are carried on extrachromosomal DNAs (plasmids), which are often readily transmissible from one bacterial strain (genotype) to another. Interestingly, M. tuberculosis is one of the few species to lack plasmids. The presence of plasmids in most species of rapidly growing mycobacteria has been demonstrated (23), although not specifically in isolates of M. immunogenum. Their function is generally unknown. Some drug-resistant bacteria that contain nontransferable resistance genes may show limited clonal diversity when studied by PFGE. Good examples are methicillin-resistant Staphylococcus aureus and multidrug-resistant Streptococcus pneumoniae. Thus, a nontransferable chromosomal biocide resistance gene common for this genotype might help explain its lack of RFLP diversity.

So the explanation for the presence of identical genotypes or clones of M. immunogenum (which means that all the isolates were genetically derived from each other) in cases of machinist HP in these industrial plants remains unknown. This is also an astonishing finding and suggests that more studies of this particular genotype are needed to explain its widespread but exclusive presence in MWF where cases of machinist HP have occurred. These data provide more support for the causative association of M. immunogenum with cases of HP and for the view that this species, and perhaps this genotype, has a special virulence mechanism for producing this disease.

 
We acknowledge Donald Nash and Steven Moore, whose interest and support prompted these studies, and Joanne Woodring for her help in preparing the manuscript.

 
* Corresponding author. Mailing address: The University of Texas Health Center, Department of Microbiology, 11937 U.S. Hwy. 271, Tyler, TX 75708. Phone: (903) 877-7680. Fax: (903) 877-7652. E-mail: Richard.Wallace{at}uthct.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, November 2002, p. 5580-5584, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5580-5584.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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