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1Department of Internal Medicine, Division of Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, Texas 79912
1Department of Internal Medicine, Division of Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, Texas 79912
1Department of Internal Medicine, Division of Infectious Disease, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, Texas 79912
We read with interest the article by Beumer et al. (1) on the detection of Mycobacterium avium subsp. paratuberculosis in 81% of drinking water and biofilm samples by PCR. Although these findings could have a significant influence on our understanding and the potential public impact of M. avium subsp. paratuberculosis as an agent of disease, much of the information on M. avium subsp. paratuberculosis based on genomic sequence detection and the environment tends to raise more questions than answers and fuels an ever-increasing mass of conflicting data.
It must first be questioned whether the detection of a specific sequence in bulk-extracted DNA actually represents a microbe, an in situ microbe, and a member of the microbial community without establishing a direct link between the molecular sequence and an actual in situ active microbe. It may be a leap of faith to assume that it does. Perhaps it would be more accurate to express such studies as detection of a genomic sequence rather than detection of an actual microbial species?
Although IS900 has proved to be species specific and reliable for the detection and confirmation of M. avium subsp. paratuberculosis infection in animals with Johne's disease (ruminant paratuberculosis) (2, 4), it may again be a leap of faith to extrapolate that specificity to other biological systems such as the environment and human tissues. In today's era of gene sequencing and genomic databases, we tend to assign sequence specificity based on information contained within those databases without appreciating that they reflect only a small fraction of known bacteria and even a smaller percentage of the genomic diversity of wild-type strains. Within the human gut microbiome, for example, only 20% of ribosomal gene sequences align with known cultivable species of bacteria, suggesting that 80% of all bacteria within the human gut are unknown and/or uncultivable (7). We should expect similar findings in other ecosystems and microbial communities.
Beumer et al. (1) have recognized and shown that other mycobacterial species that contain IS900 and/or IS900-like sequences have been identified (although not in genomic databases) and that some strains of M. avium subsp. paratuberculosis may not contain IS900. We agree with the authors' premise that bona fide strains of M. avium subsp. paratuberculosis are IS900 and 251F positive, as documented in their specificity studies (1), those of others (8), and our own. But what do we make of those strains that are IS900 negative but 251F positive and those that are IS900 positive but 251F negative? Are they or are they not M. avium subsp. paratuberculosis?
Historically, the identification of M. avium subsp. paratuberculosis has not been stringent and has been based solely on acid fastness, slow growth, and mycobactin dependency (4). With the advent of liquid culture systems and molecular biological methods, identification has been diminished further to simply acid fastness and IS900 positivity (2). Combined with the knowledge that “species-specific” insertion sequences have been shown to cross species barriers (5), the sole reliance on IS900 as a means of identifying M. avium subsp. paratuberculosis would also seem to be a misguided leap of faith when applied to remote ecosystems.
Accepting that the detection of IS900 is prima facie evidence of M. avium subsp. paratuberculosis and that M. avium subsp. paratuberculosis is widely distributed in the environment, the data still fail to hold water. If M. avium subsp. paratuberculosis was as widespread as suggested by Beumer et al. and others (6) and the primary biomass of M. avium subsp. paratuberculosis was the environment, why are not all domestic and wild ruminants infected? Was it not these precise inconsistencies that led to the realization that M. avium subsp. avium (the causative agent of avian tuberculosis) was not an environmental organism and to the designation of M. avium subsp. hominissuis as the only bona fide environmental M. avium strain (9)? Is the overreliance on IS900 as the sole basis for the identification of M. avium subsp. paratuberculosis going to result in a state of confusion similar to that created by strain 18 (3)?
REFERENCES
1.Beumer, A., et al. 2010. Detection of Mycobacterium avium subsp. paratuberculosis in drinking water and biofilms by quantitative PCR. Appl. Environ. Microbiol. 76:7367-7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
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We appreciate Chiodini and Chamberlin's comments on and interest in our work. The Safe Drinking Water Act of 1974 requires the U.S. Environmental Protection Agency to prioritize research on emerging chemical and microbial contaminants in drinking water and specifically contaminants thought to occur in drinking water but for which data are lacking to conduct a comprehensive human-health based risk assessment. Mycobacterium avium is such a contaminant. The goal of our study (1) was to gather occurrence data on M. avium subsp. paratuberculosis, a subspecies of M. avium associated with Crohn's disease, in drinking water. At the time, no occurrence data existed for M. avium subsp. paratuberculosis in drinking water in the United States.
Both culture and molecular-based methods introduce their own biases and limitations. Due to the extreme difficulty of culturing M. avium subsp. paratuberculosis from environmental samples, we chose the rapid and sensitive quantitative PCR technique and required detection of two M. avium subsp. paratuberculosis-specific targets (IS900 and target 251) to reduce the probability of nonspecific detection. We did not report results as the number of M. avium subsp. paratuberculosis cells but rather as the estimated number of target gene copies per volume of water. We agree with Chiodini and Chamberlin that our method is not perfect. The ideal method for detecting these organisms in environmental samples and drinking water specifically has yet to be developed. Should we wait until the ideal method exists to begin collecting occurrence data on M. avium subsp. paratuberculosis? Rather, this first occurrence study justifies the allocation of resources to develop better methods for definitive detection and quantification of M. avium subsp. paratuberculosis in drinking water and to conduct additional surveys.
Chiodini and Chamberlin state that our results demonstrate that M. avium subsp. paratuberculosis is widespread in the environment. Actually, our results suggest the converse. We detected two M. avium subsp. paratuberculosis-specific DNA sequences in approximately 18% of the total drinking water samples (56 of 304 samples). Furthermore, all positive samples were obtained during a 7-month period from a small geographic area. Two years later, all positive taps were negative for M. avium subsp. paratuberculosis-specific targets. In addition, more than 200 water samples from diverse geographical areas in the United States were negative for M. avium subsp. paratuberculosis-specific targets. Our results suggest that the occurrence of M. avium subsp. paratuberculosis may be more episodic than what has been observed for other M. avium strains.
Chiodini and Chamberlin discuss the hypothesis that M. avium subsp. hominissuis is the only subspecies of M. avium that predominates in the environment (8). The hypothesis arises in part from culture-based data that revealed a lower isolation prevalence of M. avium subsp. avium and a higher isolation prevalence of M. avium subsp. hominissuis from humans, suggesting either host specificity or the possibility that humans are not frequently exposed to environments containing M. avium subsp. avium. We are not aware of any study that has identified a large number of environmental M. avium isolates to subspecies level in order to determine if they are mostly subspecies hominissuis. We are aware that most culture-based methods for the isolation of environmental mycobacteria have not been evaluated for the ability to recover all subspecies of M. avium, which could alter one's conclusions about the prevalence of any M. avium subspecies in the environment. How many laboratories that isolate environmental mycobacteria routinely incorporate mycobactin in the culture medium and incubate for 16 to 23 weeks, which is necessary to recover M. avium subsp. paratuberculosis? There are reports of isolation of viable M. avium subsp. paratuberculosis from environmental samples, including surface water and soils (4, 5, 6, 9, 10). Furthermore, it has been demonstrated that M. avium subsp. avium is not transmitted from bird to bird and is thought to be acquired from the environment (2, 7).
Finally, we recognize that any human health-based microbial risk assessment of drinking water would be based in the knowledge that exposure to opportunistic pathogens is different from infection and that infection is different from disease. Crohn's disease is considered multifactorial, involving many unknown host factors, and is not caused by mere exposure to a microorganism (3). This is why humans drinking from taps where M. avium subsp. paratuberculosis genetic targets were detected would not necessarily be expected to develop Crohn's disease. Humans routinely encounter microorganisms and chemicals through daily environmental exposures but do not develop illness or disease. We did not suggest illness in our conclusions. We acknowledge that our study poses more questions than it answers and extend an invitation to all interested laboratories to join us in the quest to develop a better understanding of the occurrence of environmental mycobacteria and their impact on human health.
REFERENCES
1.Beumer, A., et al. 2010. Detection of Mycobacterium avium subsp. paratuberculosis in drinking water and biofilms by quantitative PCR. Appl. Environ. Microbiol. 76:7367-7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
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