1. Introduction
Opportunistic premise plumbing pathogens (OPPPs) are normal microbial residents of drinking water, distribution systems, and premise plumbing [
1] which are estimated to cause nearly 30,000 cases of human disease and cost
$850 million a year [
2]. OPPPs grow in drinking water distribution systems, unlike contaminants of drinking water such as
Escherichia coli and
Salmonella spp. Examples of OPPPs are:
Legionella pneumonia,
Mycobacterium avium,
Pseudomonas aeruginosa,
Stenotrophomonas maltophilia, and
Acinetobacter baumannii. OPPPs share a number of traits impacting on their ecology, including growth at low oxygen (microaerobic) and organic matter content (oligotrophic), biofilm formation, and resistance to disinfectants [
1]. Premise plumbing has a number of features that select for OPPPs, including low organic matter content, heating, periods of stagnation, and different pipe materials for biofilm formation.
Members of the genus
Methylobacterium are opportunistic pathogens of humans [
3,
4] found in water and soil.
Methylobacterium spp. infections have been linked to patients with indwelling catheters [
5] and pseudo-outbreaks associated with the use of bronchoscopes and endoscopes that were contaminated by tap water containing
Methylobacterium spp. used for washing bronchoscopes [
5,
6]. Bloodstream infections have been reported due to
M. mesophilicum from bone marrow transplant recipients caused by tap water used for oral irrigation [
7]. In another case, a patient undergoing continuous ambulatory peritoneal dialysis developed a peritoneal infection due to
M. mesophilicum, which originated from water in their bathroom [
8]. In general,
Methylobacterium spp. cause mild clinical symptoms such as fever, but are also responsible for bacteremia, peritonitis, and pneumonia [
9,
10].
Methylobacterium spp. produce pink carotenoid pigments as well as phytochromes (cytokinins and auxins), which are known to stimulate plant growth [
11,
12] and are capable of nitrogen fixation [
13] and help plants to fight pathogens [
14].
Methylobacterium spp. are found in a wide variety of natural habitats including soil, dust, air, fresh water, and aquatic sediments [
15] and are normal inhabitants of drinking water distribution systems and premise plumbing. Specifically, high numbers of
Methylobacterium spp. have been identified in shower curtains [
16] and showerhead biofilms [
17]. Interestingly, if
Mycobacterium spp. were detected in showerhead biofilms,
Methylobacterium spp. were absent, and vice-versa [
17]. This observation was confirmed in a study of households containing patients with
Mycobacterium avium pulmonary infections; when
Methylobacterium spp. were present in biofilm samples,
M. avium was absent [
18]. The basis for that mutual exclusion appears to be based upon an inhibition of adherence of
M. avium cells to biofilms of
Methylobacterium spp. [
19].
Methylobacterium spp. share a number of characteristics in common with OPPPs, including: chlorine resistance [
20,
21,
22], biofilm formation [
23,
24], and desiccation tolerance [
23,
25].
Methylobacterium spp. are recognized as resistant to elevated (>50 °C) temperatures [
26] and were isolated from hot tap water in a household [
27], suggesting that the laboratory-measured temperature resistance [
26] is of ecological consequence. Finally, in common with other OPPPs,
Methylobacterium extorquens was isolated from amoebae in drinking water systems, making it an amoeba-resistant microorganism [
28]. It is our objective to describe further characteristics of
Methylobacterium spp. to reinforce the claim that they are opportunistic premise plumbing pathogens.
3. Discussion
The experimental approaches were devised to determine whether representative
Methylobacterium spp. strains had characteristics found in OPPPs. The findings indicate
Methylobacterium spp. are hydrophobic, survive in drinking water (some even growing in drinking water), adhere to many plumbing surfaces, form biofilms, and are relatively thermally tolerant. All these characteristics are shared by opportunistic premise plumbing pathogens [
1].
New methods need to be developed to accurately measure the growth of the aggregating Methylobacterium spp. strains without disturbing their natural growth. M. hispanicum strain JM1-5 was the only strain that did not aggregate under laboratory conditions and thus provides the best measure of the variables reported here without the influence of aggregation.
Evidence that the
Methylobacterium spp. strains survived and even grew in sterile tap water (
Table 1) is in agreement with the discovery that
Methylobacterium spp. were found to survive in autoclaved and filtered river water for up to 260 days [
30]. The
Methylobacterium spp. strain and the coupon type were major factors influencing surface adherence and biofilm formation. Hydrophobicity strongly influences surface colonization [
31]. Therefore, it is not surprising that the number of adherent cells of the more hydrophobic and spontaneously aggregating
M. extorquens strain was greater than those of the non-aggregating, less hydrophobic
M. hispanicum strain JM1-5 (
Table 3 and
Table 4). Both aggregation and surface adherence are influenced by cellular hydrophobicity, as postulated by van Loosdrecht et al. [
31]. The abilities of each
Methylobacterium spp. strain to aggregate have been listed in
Table 7. The materials in each plumbing surface also influenced the number of adherent cells. Galvanized (Zn) steel and PVC materials are generally hydrophobic surfaces, which leads to higher adherence and biofilm formation, while glass and steel are more hydrophilic surfaces, leading to less adherence and biofilm formation. There is speculation that high zinc concentrations in soil and water have also been associated with high mycobacterial numbers [
32], and this could be related to the high number of adherent
Methylobacterium spp. cells.
Methylobacterium spp. exhibited relative thermal tolerance at hot water temperatures (
Table 6). Although killing was not seen until the temperatures were raised to 60 °C (
Table 6), these results suggest that one way to reduce
Methylobacterium spp. exposure is to elevate water heater temperatures to 60 °C. In a study of the numbers of nontuberculous mycobacteria (NTM) in household plumbing samples, it was found that NTM were less frequently recovered from household samples whose water heater temperatures were >130 °F (>55 °C) compared to ≤125 °F (≤50 °C) [
33]. Other ways to reduce
Methylobacterium spp. exposure in water heaters would be to drain and refill the water heater periodically.
In summary,
Methylobacterium spp. share characteristics of other OPPPs, including persistence in tap water, hydrophobicity, an ability to aggregate, adhere, and form biofilms on different plumbing surfaces, and thermal tolerance [
1]. Knowledge of the types of pipe material that support the lowest number of
Methylobacterium spp. cells can be used to reduce
Methylobacterium spp. colonization of plumbing in hospitals and homes.