Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Abstract

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.

Medium-chain-length polyhydroxyalkanoate production by newly isolated Pseudomonas sp. TN301 from a wide range of polyaromatic and monoaromatic hydrocarbons

“…Although the highest homology of 16S rDNA was with P. plecoglossicida L21, the nonfluorescent pseudomonad isolated from fish (Izumi et al . ), phenotypically TN301 was more similar to P. putida F1 and P. putida GB‐1, and it also shared high 16S rDNA sequence homology with these strains (99% identity with 99% coverage). Pseudomonas putida F1 was isolated by enrichment with ethylbenzene from a polluted creek and has the ability to use broad range of monoaromatic hydrocarbons, but has no PAH dioxygenases (Zylstra and Gibson ), while P. putida GB‐1 was isolated from fresh water and is known as a robust manganese oxidizer (Wu et al .…”

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“…Recently, the gyrB region has been used instead of 16S rDNAfor phylogenetic analysis among closely related bacterial taxa, as the sequence database of gyrBhas become substantial (Yamamoto and Harayama, 1995;Watanabe et al, 2001;Parkinson et al, 2007). In addition,gyrB has been successfully used to detect specific pathogenic bacteria directly from fish samples that were heavily contaminatedwith other environmental bacteria (Venkateswaran et al, 1998;Izumi and Wakabayashi, 2000;Izumi et al, 2007b). Tomake this study practical, unidentified whitepigmented bacterial isolates were collected and used as bacteria that can cause a false positive reaction using the PCR method.…”

Draft Genome Sequence of Pseudomonas plecoglossicida Strain NB2011, the Causative Agent of White Nodules in Large Yellow Croaker (Larimichthys crocea)

“…In large yellow croaker, the infection leads to the development of white nodules in the spleen, kidney, and liver of the diseased fish and results in high mortality (4, 5). The causative bacterium, strain NB2011, was preliminarily identified as P. putida by physical and biochemical profiles and 16S rRNA sequencing (5), but the strain was reclassified as P. plecoglossidica based on a gyrB sequence revealed by genome sequencing (6). …”