A novel selective growth medium-PCR assay to isolate and detect Sphingomonas in environmental samples

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DOIResolve DOI: http://doi.org/10.1016/j.mimet.2010.03.012
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Journal titleJournal of Microbiological Methods
Pages1927; # of pages: 9
SubjectPCR detection of Sphingomonas; Serine palmitoyltransferase gene; Sphingomonas selective growth medium; Streptomycin; Piperacillin; biofilms
AbstractSphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin–piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 µg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt–PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt–PCR test. None of the unpigmented isolates was positive to the spt–PCR assay. The 16S rDNA of 17% of the spt + ve and − ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin–piperacillin selective growth medium in combination with the colour pigmentation and the positive spt–PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.
Publication date
AffiliationNational Research Council Canada; NRC Plant Biotechnology Institute
Peer reviewedYes
NRC number54653
NPARC number21268523
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Record identifier03afa974-5383-4b7a-b682-b7c32238119a
Record created2013-09-09
Record modified2016-05-09
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