Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance

Abstract

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease, which causes significant problems to aquaculture worldwide. A recent study (Soule M, Cain K, LaFrentz S, Call DR [2005] Infect Immun 73:3799-3802) identified two 16S rRNA gene sequence variants (6 base differences) within the variable stem-loop region 3 for F. psychrophilum strains ATCC 49418 and CSF 259-93. That study also hypothesized that F. psychrophilum is composed of at least 2 distinct genetic lineages (I and II) described by a microarray-based comparative genomics study. In the present study, we augmented an existing 16S rDNA microarray to detect both 16S rRNA sequence variants from F. psychrophilum. Subsequent microarray experiments showed that CSF 259-93 hybridized as expected, but ATCC 49418 was positive for both sequence variants. We then developed a PCR-restriction fragment length polymorphism (RFLP) assay (MnlI and MaeIII) to distinguish between the 2 sequences. Gel isolation of PCR-RFLP products, cloning, and sequencing confirmed that ATCC 49418 harbors both 16S rRNA sequences. Microarray experiments showed that 11 of 14 strains from genetic Lineage I harbor both the CSF 259-93 and ATCC 49418 16S rRNA sequence variants, whereas all 15 Lineage II strains were only positive for the CSF 259-93 sequence (p < 0.0001). Elastin hydrolysis and tetracycline resistance were most closely associated with the latter strains (p < 0.0001 and p = 0.024, respectively). These data support the hypothesis that F. psychrophilum is composed of at least 2 distinct genetic lineages that are closely associated with host origin. KEY WORDS: 16S rRNA · Flavobacterium psychrophilum · Bacterial coldwater disease · Elastin hydrolysis · Microarray hybridization · Tetracycline resistance Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [209][210][211][212][213][214][215][216] 2005 In general, Flavobacterium psychrophilum strains are biochemically homogeneous, with the exception of some variation in the presence or absence of caseinases, gelatinases, and elastases (Bertolini et al. 1994, Lorenzen et al. 1997, Madetoja et al. 2001. Not all strains of F. psychrophilum are capable of causing the same degree of disease (Holt 1987, Madetoja et al. 2002, LaFrentz et al. 2003 and they can be antigenically diverse (Evensen & Lorenzen 1996, Crump et al. 2001, Madetoja et al. 2002. Immunization with killed cells of one strain does not necessarily convey protective immunity from infection by other strains (Holt et al. 1989, Obach & Laurencin 1991.Genetic variation has been assessed between strains using ribotyping, plasmid profiles, randomly amplified polymorphic DNA (RAPD), and PCR-RFLP of specific gene fragments (Chakroun et al. 1997, Chakroun et al. 1998, Madetoja et al. 2001, Izumi et al. 2003. Studies of outbreak strains from Danish trout farms suggest dominance by a single ribotype , Madetoja et al. 2002, but other studies show greater genetic variation across geographic scales (Chakroun et a...

Flavobacterium psychrophilum associated with septicaemia and necrotic myositis in Atlantic salmon Salmo salar: a case report

“…Although the degradation of elastin is considered to be a virulence factor in F. psychrophilum (Madsen & Dalsgaard 1998, and has been correlated to strains in genetic lineage II (Soule et al 2005b), this trait was not reported in extracellular preparations of F. psychrophilum recorded from a case with necrotic myositis (Ostland et al 2000).…”

Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

“…The disease mainly affects fingerlings, and is currently considered one of the most significant bacterial infections in the salmonid industry, causing high mortality and serious economic losses worldwide. Although F. psychrophilum is considered a rather fastidious bacterium because it is difficult to isolate and handle (Michel et al, 1999), significant advances have been made over the last few years in culture (Michel et al, 1999;Cepeda et al, 2004;Á lvarez & Guijarro, 2007), typing (Izumi et al, 2003;Soule et al, 2005;Arai et al, 2007;Nicolas et al, 2008), molecular diagnosis Cepeda & Santos, 2000;del Cerro et al, 2002;Fujiwara-Nagata & Eguchi, 2009) and genomics (Duchaud et al, 2007). Moreover, a genetic manipulation system based on conjugation, transformation and transposon mutagenesis has been developed (Á lvarez et al, 2004).…”

A Review of Flavobacterium Psychrophilum Biology, Clinical Signs, and Bacterial Cold Water Disease Prevention and Treat

“…Ramsrud et al [145] differentiated strains using a simple PCR assay. Tiirola et al [146], Izumi et al [147], and Soule et al [79], used PCR-RFLP (restriction fragment length polymorphism), whereas del Cerro et al [148] developed a multiplex PCR method combining the use of 16S rDNA with gyrB based primers to improve reliability and accuracy.…”

“…psychrophilum has at least three main serotypes [18,24,43]. There are also a number of distinct genetic lineages, with the number appearing to increase with the refinement of molecular DNA techniques [19,20,40,79] with considerable genetic variation and variation in virulence among strains [19,25,45,80,81]. Strain virulence may also be fish species specific [42].…”