Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network

Abstract

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.

FIG. 1.

Phylogenetic tree showing representative species of alphaproteobacteria. The tree was constructed by using the neighbor-joining method, based on the nearly complete sequence (1,283 nucleotides) of the 16S rRNA gene coding sequence. Only one species per genus was used. Taxonomic names and GenBank accession numbers are indicated at the ends of the branches. Each order is clearly delimited by dashed lines and gray shading and is branched deeply. Best BLAST with known-species isolates recovered in this study, as well as new species recovered in this study, are indicated by boldface type and underlining.

FIG. 2.

Growth of new alphaproteobacterial species in coculture with A. castellanii ATCC 30010. “Afipia sp. strain laus-1” (⧫), “Bosea sp. strain laus-1” (▪), “Craurococcus-related sp. strain laus-1” (▴), and “Rhodoplanes sp. strain laus-1” (•) were grown for 7 days in PAS with (solid lines) or without (dashed lines) A. castellanii. The data are means of three independent experiments. D, day.

FIG. 3.

Phylogenetic neighbor-joining tree showing the relationships of Afipia and Bosea. The tree was derived from an alignment of partial rpoB sequences. Taxonomic names and GenBank accession numbers are indicated at the ends of the branches. Best BLAST with known-species isolates recovered in this study, as well as new species recovered in this study, are indicated by boldface type and underlining. The support for each branch, as determined with 250 bootstrap samples, is indicated by the value at the node (expressed as a percentage). Sinorhizobium meliloti was used as the outgroup.

FIG. 4.

New alphaproteobacterial isolates “Afipia sp. strain laus-1” (A), “Bosea sp. strain laus-1” (B), “Craurococcus-related sp. strain laus-1” (C), and “Rhodoplanes sp. strain laus-1” (D) in A. castellanii after 5 days of coculture. Numerous “Bosea sp. strain laus-1” cells are present within vacuoles (B). Only a few cells of the other strains are present within amoebae (arrows). Bar = 2 μm.