Halomonas: Three New Species, a Newly Recognized Human Pathogen Causing Infections and Contamination in a Dialysis Center: Three New Species

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Abstract

Our Renal Care Center (RCC) is a separate building, performing almost 2500 outpatient dialysis runs per month. In May 2007, 2 patients developed, days apart, bacteremia with an apparently identical nonfermentative Gram-negative rod. Because of difficulty identifying the organism, testing in the Biolog system identified them as a Halomonas species. Sequencing of ∼1500 bases of the 16S rRNA gene in both organisms in 3 reference laboratories confirmed, searching against 3 databases, that the organisms were identical and were Halomonas species. There were 54 recognized species of this genus, associated with marine or saline sites. Initial attempts at environmental isolation as primary cultures, including a 4% salt agar plate, or initial incubation in 6.5% salt broth enrichment culture with subculture to agar, to exploit the halophilicity of Halomonas, were successful in demonstrating the colonies seen in the blood cultures, only from sites not contaminated with other organisms, because of competing growth. A more selective method was developed for use on samples suspected to be heavily contaminated with other organisms, using the strategy of increased salt concentration in a broth enrichment culture to further exploit Halomonas halotolerance, and thereby inhibit other organisms. A 16.5% salt concentration in brain-heart infusion broth, incubated at 35°C for 48-72 hours, then subcultured to agar plates incubated in room air at 35°C, proved optimal for selection and secondary isolation. With a combination of these techniques, 14/15 cultures of dialysates and 10/38 from the outflow pathways of the machines were Halomonas positive, compared to 0/31 cultures from the inflow side of the machines (including water supplies and storing, mixing, and preparation tanks). The exception was sites associated with or downstream of bicarbonate influx, 12/54 of which were positive. Two other local hospitals' dialysis centers, and our own inpatient dialysis facility, were cultured at sites that yielded Halomonas from our RCC, and Halomonas was not isolated. Further study by 16S rRNA gene sequencing and DNA-DNA hybridization revealed the cultures represented 3 novel species: 1 (H. stevensii sp. nov.) in the patients and environment and 2 (H. hamiltonii sp. nov., H. johnsoniae sp. nov.) in the environment, most closely related to H. magadiensis. Of 35 speciated isolates, 22 were H. stevensii, 10 H. johnsoniae, and 3 H. hamiltonii. We hypothesize that the RCC became contaminated with these halophilic organisms from bicarbonate used to prepare dialysis fluid, and they persist despite cleaning and flushing procedures because of biofilm in machines and bicarbonate fluid inflow sites. Our experience, together with the review of the literature presented here, indicates the genus Halomonas has pathogenic potential.

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