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 Table of Contents  
Year : 2016  |  Volume : 5  |  Issue : 2  |  Page : 240-243

Methylobacterium spp. as an indicator for the presence or absence of Mycobacterium spp.

1 Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA
2 Division of Pulmonary and Critical Care Medicine, Lankenau Medical Center, Lankenau Institute for Medical Research, Wynnewood, PA, USA

Date of Web Publication9-Feb-2017

Correspondence Address:
Joseph O Falkinham III
Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061-0406
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2016.03.001

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Objective/Background: A published survey of bacteria in showerhead biofilm samples revealed that Methylobacterium spp. and Mycobacterium spp. seldom coexisted in biofilms.
Method: To confirm that information, biofilm samples were collected from household plumbing of Mycobacterium avium patients and Methylobacterium spp. and M. avium numbers were measured by direct colony counts.
Results: The results demonstrated that if Methylobacterium spp. were present, Mycobacterium spp. were absent, and the opposite.
Conclusion: The data demonstrate that microbial populations in biofilms can influence the presence or absence of opportunistic premise plumbing pathogens and, thereby, increase the range of strategies to reduce exposure to waterborne pathogens. Finally, by assessing for the visual presence of methylobacteria as pink pigmentation on showers and shower curtains, homeowners and managers of hospitals and other buildings can quickly determine whether a premise plumbing biofilm sample has mycobacteria with a high degree of assurance.

Keywords: Biofilms, Methylobacterium, Mycobacterium, Mycobacterium avium, Pigmentation, Premise plumbing

How to cite this article:
Falkinham III JO, Williams MD, Kwait R, Lande L. Methylobacterium spp. as an indicator for the presence or absence of Mycobacterium spp. Int J Mycobacteriol 2016;5:240-3

How to cite this URL:
Falkinham III JO, Williams MD, Kwait R, Lande L. Methylobacterium spp. as an indicator for the presence or absence of Mycobacterium spp. Int J Mycobacteriol [serial online] 2016 [cited 2020 Jul 3];5:240-3. Available from: http://www.ijmyco.org/text.asp?2016/5/2/240/199938

  Introduction Top

Nontuberculous mycobacteria (NTM) are opportunistic human pathogens whose source of infection is the environment [1]. Mycobacterium species are found in drinking water distribution systems [2], hospital plumbing [3], and household plumbing [4] and cause chronic and life-threatening pulmonary infections that are difficult to treat [5],[6].

The incidence of NTM disease in the USA and Canada is rising [7],[8]. In Toronto, Canada NTM disease incidence has risen from 1.5 per 100,000 to 9.0 per 100,000 over the period 1997–2003 [7]. Similarly, NTM disease is increasing in the USA based on reports of NTM lung disease in hospitalized persons [8]. A major fraction of these cases are found in older, slender women, lacking any of the classic risk factors for NTM disease, yet have a greater tendency than the general population to develop NTM pulmonary disease [9],[10],[11]. It follows that as the population of the USA continues to age—25% of the US population will be over 60years by 2025 [12]—the incidence of NTM pulmonary disease will continue to increase. Further, as NTM-infected patients are subject to the reemergence of infection or reinfection by other environmental NTM [13], it is of value to identify means to reduce NTM exposure.

A recent study demonstrated the widespread presence and high numbers of Mycobacterium spp. and Mycobacterium avium in showerhead biofilms across the USA [14]. Although not highlighted by the authors, examination of the data indicate a potentially important pattern; namely, the presence of clones of pink-pigmented Methylobacterium spp. were associated with the absence of Mycobacterium spp. and the presence of Mycobacterium spp. with the absence of Methylobacterium spp. [14]. Specifically, of 10 clone libraries with >37.5% Methylobacterium spp. sequences, nine had <6.4% Mycobacterium spp. sequences [14]. Correspondingly, of eight clone libraries with >50% Mycobacterium spp. sequences all had >7.5% Methylobacterium spp. sequences [14]. In only one showerhead sample was there both substantial Mycobacterium spp. (46.7%) and Methylobacterium spp. (38.9%) sequences [14].

Like M. avium and other NTM, Methylobacterium spp. are normal inhabitants of drinking water distribution systems and buildings, including hospitals [15],[16],[17],[18],[19],[20],[21]. Further, a substantial proportion of Methylobacterium spp. isolates are chlorine-resistant [22], form biofilms [23],[24], and belong to the group of amoeba-resisting bacteria of drinking water [25]. Household environments influenced by municipal water are evidently also habitats, as Methylobacterium spp. have been shown to be abundant among DNA clones recovered from shower curtains [26].

For this study, it was hypothesized that the presence of the pink-pigmented Methylobacterium spp. would be associated with the absence of Mycobacterium spp. and that the presence of Mycobacterium spp. would be associated with the absence of Methylobacterium spp. to test the hypothesis, household water systems were sampled to directly measure numbers of Methylobacterium spp. and Mycobacterium spp.

  Materials and methods Top


The Falkinham laboratory is currently participating in a study of households of patients from the Philadelphia, Pennsylvania area with M. avium pulmonary disease, with the objective of determining whether their household plumbing (including showerheads) could be the source of their infection. Approval for that study was granted by the Main Line Health Hospitals Institutional Review Board.

Collection of household water and biofilm samples

Surface biofilms of water taps and showerheads from households of patients with NTM pulmonary disease and their neighbors were swabbed (5–10 cm2) by study personnel and placed in 3mL of tap water from the residence. Containers and swabs were sent to the Virginia Tech laboratory for isolation, enumeration, and identification of mycobacteria and methylobacteria. Patients did not collect samples, lest patient M. avium contamination occur.

Isolation of Mycobacterium spp. and Methylobacterium spp. from biofilm samples

Isolation, enumeration, and identification of Mycobacterium spp. was performed as described [27]. In addition, samples were also spread on R2A agar (BD, Sparks, MD, USA) and incubated at 30°C for 3days for isolation and enumeration of the pink-pigmented, presumptive Methylobacterium spp.

Identification of Methylobacterium spp. isolates

Pink-pigmented colonies on R2A agar were picked, purified using single colony isolation, and identified on the basis of cultural, biochemical, and enzyme tests as described [19].

  Results Top

Isolation of Methylobacterium spp. and Mycobacterium spp. in household plumbing biofilm samples

To confirm that the presence of Methylobacterium spp. was associated with an absence of Mycobacterium spp. [14], 153 biofilm samples from the plumbing of 20 Philadelphia NTM patient households and neighboring control households received by the Virginia Tech laboratory were processed to isolate, enumerate, and identify pink-pigmented colonies as well as Mycobacterium spp. Pink-pigmented colonies were picked and purified. Seven percent of pink colonies proved to be yeast by microscopic morphology, 9% were cocci (presumably Deinococcus spp.), and the remaining 84% were gram-negative rods. The rod-shaped isolates were identified as Methylobacterium spp. on the basis of the presence of catalase activity, absence of urease activity, their failure to grow at 37°C or 42°C, absence of growth on MacConkey agar, inability to hydrolyze Tween 80, or change the pH of triple sugar iron agar [19]. A majority grew on glycerol or xylose as sole carbon sources, but failed to grow on glucose, lactose, or mannitol [19]. As noted by others [19], a high percentage (92%) of the Methylobacterium spp. isolates recovered here spontaneously aggregated in Tryptic Soy broth cultures.

All the acid-fast isolates from the biofilm samples collected from the plumbing of the patients' and neighbors' households sampled for this study proved to be M. avium.

Coexistence of Methylobacterium spp. and Mycobacterium spp. in biofilms

Based only on the number of Methylobacterium spp. isolates, the results ([Table 1]) show that biofilm samples with Methylobacterium spp. seldom yielded Mycobacterium spp. and that samples lacking Methylobacterium spp. were more likely to yield Mycobacterium spp. The criterion for the presence of Methylobacterium spp. colonies in samples required ≥10 or more colonies (i.e., 300CFU/cm2) on RA2 agar. Any sample yielding an M. avium isolate was considered positive. Based on the assumption that the distribution of Mycobacterium spp. and Methylobacterium spp. should be equal and random, the variation in the four groups was significantly different than expected (p = .0015, Fisher's exact test). Thus, the results of the survey of the homes of NTM patients were in agreement with the previously published molecular survey of shower heads [14].
Table 1: Mycobacterium spp. and Methylobacterium spp. in biofilm samples of household plumbing.a

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  Discussion Top

We conclude that the presence of a Methylobacterium spp. biofilm is associated with the absence of M avium. These observations contribute to the emerging study of drinking water biofilm microbiomes [23],[24], here describing a unique interaction between M. avium and Methylobacterium spp. Further, the results are of significance to the estimated 30,000 individuals in the USA with pulmonary mycobacterial disease [7],[8], as they are innately susceptible to continued mycobacterial infections [13]. Specifically, the presence or absence of the visibly apparent pink-pigmented Methylobacterium spp. could be used to rapidly determine whether a water tap or showerhead had Mycobacterium spp. by inspecting shower surfaces or a shower curtain for presence of a pink stain or deposit [14]. As Mycobacterium spp. are only isolated from 30% of household taps or showerheads in a home [4], at-risk individuals or Mycobacterium spp.-infected patients could select taps and showers most likely to lack Mycobacterium spp. Furthermore, the advantage of this discovery is that laboratory culture detection of Methylobacterium spp. can be rapidly performed (2–3days at 30°C). The study of showerhead biofilms employing 16Sr ribosomal RNA sequence for identification of Mycobacterium clones [14], showed that a great variety of Mycobacterium spp. clones were absent when Methylobacterium spp. were detected. Thus, we suggest that the presence of Methylobacterium spp. is not only associated with the absence of M. avium, but is also associated with the absence of other Mycobacterium species.

The fact that Mycobacterium spp. are seldom recovered from taps or showerheads that have Methylobacterium spp., could potentially be exploited as a strategy to limit adherence and biofilm formation by M. avium and likely other Mycobacterium species to reduce exposure of individuals to these opportunistic premise plumbing pathogens in household and hospital plumbing and in instruments with water reservoirs. Rather than suggest the “inoculation” of household plumbing with Methylobacterium spp. cells, further investigation of the possible inhibition of M. avium adherence by cellular fractions of Methylobacterium spp. has been initiated as well as studies to elucidate the mechanism whereby Methylobacterium spp. presence signals the absence of Mycobacterium spp.

  Conflicts of interest Top

None declared.

  References Top

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J.O. Falkinham III, C.D. Norton, M.W. LeChevallier, Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other mycobacteria in drinking water distribution systems, Appl. Environ. Microbiol. 67 (2001) 1225– 1231.  Back to cited text no. 2
G.C. duMoulin, K.D. Stottmeier, P.A. Pelletier, et al, Concentration of Mycobacterium avium by hospital water systems, J. Am. Med. Assoc. 260 (1988) 1599–1601.  Back to cited text no. 3
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R.J. Wallace Jr., Y. Zhang, B.A. Brown-Elliott, et al, Repeat positive cultures in Mycobacterium intracellulare lung disease after macrolide therapy represent new infections in patients with bronchiectasis, J. Infect. Dis. 186 (2002) 266–273.  Back to cited text no. 13
L.M. Feazel, L.K. Baumgartner, K.L. Peterson, et al, Opportunistic pathogens enriched in showerhead biofilms, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 16393–16399.  Back to cited text no. 14
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A. Hirashi, K. Furuhata, A. Matsumoto, et al, Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments, Appl. Environ. Microbiol. 61 (1995) 2099–2107.  Back to cited text no. 22
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