Lab Anim Res.  2016 Dec;32(4):208-216. 10.5625/lar.2016.32.4.208.

Multilocus sequence typing analysis of Pseudomonas aeruginosa isolated from pet Chinese stripe-necked turtles (Ocadia sinensis)

• Wendt M
• Heo GJ

• KMID: 2362912
• DOI: http://doi.org/10.5625/lar.2016.32.4.208

Abstract

Our research sought to characterize the phylogeny of Pseudomonas aeruginosa isolated from pet Chinese stripe-necked turtles (Ocadia sinensis) to better understand its evolutionary relation to other isolates and increase understanding of a potential zoonotic pathogen transmitted through direct contact with pet turtles. Thirty-one Pseudomonas aeruginosa isolates were obtained from both immature and adult turtles sold in pet shops in Korea. To characterize the phylogenic position of Chinese stripe-necked turtle-borne P. aeruginosa relative to other strains, multilocus sequence typing (MLST) analysis was performed due to the accessibility and breadth of MLST databases. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were sequenced and the results were compared with data from the MLST database. The genes were further used for phylogenetic analysis of P. aeruginosa using concatenated gene fragments. Both rooted and unrooted phylogenetic trees were generated. Eleven distinct sequence types were present within the isolates among which seven were new. Expanding an unrooted phylogenetic tree to include P. aeruginosa MLST sequences isolated from various other geographic locations and sources revealed a divergent cluster containing the majority of isolates obtained from turtles. This suggests that P. aeruginosa strains particularly well-adapted for inhabiting turtles occupy a distinct phylogenetic position.

Keyword

MLST; Pseudomonas aeruginosa; Chinese stripe-necked turtles (Ocadia sinensis); pet turtles; phylogeny

MeSH Terms

Adult
Asian Continental Ancestry Group*
Genes, Essential
Geographic Locations
Humans
Korea
Multilocus Sequence Typing*
Phylogeny
Pseudomonas aeruginosa*
Pseudomonas*
Trees
Turtles*

Figure

• 

  Figure 1 Unrooted phylogenetic trees using the concatenated sequences of extended PCR fragments from seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) of P. aeruginosa using A. the ML and B. the NJ method with the Kimura 2-parameter model for distance calculations. The total length of the concatenated sequences was 5,784bp. Bootstrap percentages received from 1,000 replications were used and values over 50% are shown in the trees. The scale bars (0.001) indicate the number of nucleotide substitutions per site.

• 

  Figure 2 Rooted phylogenetic tree using the concatenated sequences of extended PCR fragments from seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) of P. aeruginosa using the NJ method with the Kimura 2-parameter model for distance calculations. The total length of the concatenated sequences was 5,784bp. Bootstrap percentages retrieved from 1,000 replications were used and values over 50% are shown in the tree. The scale bar (0.02) indicates the number of nucleotide substitutions per site. Data for reference and outgroup strains were collected from the NCBI GenBank database.

• 

  Figure 3 Radiation tree using the concatenated sequences of internal MLST fragments from seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) of 57 strains of P. aeruginosa (31 isolates from this study, 25 from the MLST database, and PAO1 from the NCBI GenBank database).The NJ method was used with the Kimura 2-parameter model for distance calculations. The total length of the concatenated sequences was 2,8744bp. Nodes were supported by 1,000 bootstrap replications. The large cluster containing exclusively isolates from this study was demarcated as it contained the majority of isolates from immature turtles. The scale bar (0.001) indicates the number of nucleotide substitutions per site.

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