Fatty Acid Composition in Yersinia ruckeri Strains Isolated from Rainbow Trout Farms

In this study, a total of 33 Yersinia ruckeri isolates were obtained from rainbow trout farms in the five different region of Turkey. We determined to changes in fatty acids group of the strains. All the isolates were identified as Y. ruckeri based on colonical, cellular morphology and biochemical characters. All Y. ruckeri 8 major fatty acids, including 12:0 (Lauric acid), 14:0 (Myristic acid), 16:0 (Palmitic acid), 16:1n-7 (Palmitoleic acid), 17:1 (Heptadecanoic acid), 18:1n-9c (Oleic acid), 18:2n-6c (Linoleic acid), 18:3n-3c (Alfa-linoleic acid). Compared to isolates biochemical property, there was no difference between fatty acids and biochemical characteristics. The results of this study showed that the fatty acids composition of Y. ruckeri isolated strains from rainbow trout farms in Turkey is on a large scale homogenous.


INTRODUCTION
Enteric red mouth disease, known as ERM or Yersiniosis, was first reported in 1950's in the rainbow trout farms in the USA as a septicemic disease with high mortality rates in sub-acute and acute forms (Bullock and Anderson, 1984;Busch, 1982;Frerichs and Roberts, 1989;Lucangeli et al., 2000;Ross et al., 1966). ERM disease is widespread in many parts of the world (Bullock et al., 1977;Roberts, 1983;Lesel et al., 1983;Stevenson and Daly, 1982).
In Turkey, Yersiniosis caused by Yersinia ruckeri was identified in rainbow trouts in 1991. As the trout farms increased in number, the disease became more widespread, starting to cause economic losses. This disease is currently frequently found in trout farms and has become the most important bacterial disease in Turkey (Çağırgan and Yüreklitürk, 1991;Kubilay, 1997;Kubilay and Diler, 1999;Timur and Timur, 1991).
A standartdized and rapid identification system is offered by the API 20E system. Miniaturized multitest system was originally developed for the identification of Gram-negative enteric bacteria in clinical laboratories. During the last decade, this system has been increasingly used for the identification of marine and fresh water fish pathogens (Tanrıkul et al., 2004).
Lipids are one of the most basic organic compounds in bacteria, as in other living things in the world. Lipids, which play an important role in the functions of cytoplasmic membranes, are affected by different environmental factors such as temperature, surface tension, osmotic pressure and pH. Fatty acids that make up lipids in defining and characterizing bacteria are accepted as one of the basic taxonomic criteria. It is considered that free fatty acid complexes, which are used in bacterial systematics, will be a good chemotaxonomic property in different growth environments in terms of microbial ecology (Kariptas, 1999).
The detection of the fatty acids of the bacteria using gas chromatography dates back to the 1960s. It has been suggested that mobile Aeromonas strains of clinical and non-clinical origin can be classified by liquid-gas chromatography according to their fatty acid metal ester profiles (Canonica and Pisono, 1985).
In this study, the analysis of the heterogeneity of Y. ruckeri isolated from fish in the trout farming plants in different regions of Turkey was carried out in order to reveal their fatty acid profiles. For this purpose, the biochemical properties of the strains used in the study, in addition to the traditional biochemical tests, the relationship of epidemiological characters with the fatty acid composition were revealed.

Bacteria
Y. ruckeri (33) isolates used in the study were isolated from classical fish farms in different regions of Turkey between 2005 and 2014 by classical methods. All strains were cultured at 25 0 C using Tryptic Soy Agar (TSA). Isolates that were revealed to be Y. ruckeri by biochemical tests were confirmed using the Polymerase Chain Reaction (PCR) method reported by Şeker et al. (2012). Sowing was performed in Shotts-Waltman Medium (SWM) and Ribose Ornitine Deoxycholate (ROD) selective fattening sites.
Isolates, which were previously isolated and added to glycerin in Triptic Strain Broth (TSB), kept at -20 0 C, were freshly cultured in Triptic Strain Agar (TSA) for sterility control before the study started, and it was checked whether the strains were pure. DSM18506 was used as the reference strain. Table 1 shows the geographical distribution of the isolates used in the study. Fatty acid extraction and analysis Bacteria were incubated in 100ml TSB placed in 500ml flasks at 25 0 C for 48 hours and collected by centrifugation at 2500 rpm for 10 minutes. The bacteria were washed twice with 0.85% NaCl and kept at room temperature for 2 hours. For hydrolysis of approximately 55 mg bacteria biomass, NaOH and 1 ml of methanol mixture were added and incubated for 30 minutes in a water bath at 95 0 C. Then, 2 ml of 6 N HCl prepared in methanol was added to methylation of free fatty acids and kept for 10 minutes in a water bath at 80 0 C. The aqueous phase for fatty acid extraction was collected and 1.15 ml of the hexane/methyl tertbutyl ether (1:1) mixture was added to each tube and inverted for 10 minutes. After the supernatant was removed, 3ml of 1.2% NaOH was added and shaken for 5 minutes. The supernatant organic phase was taken to the chromatography bottles (viallere) for fatty acid determination.
Fatty acid analysis was carried out by using Ultra 2-HP (25m x 0,22 mm x 0,33mm) column and hydrogen gas in gas chromatography-mass spectrophotometer (GC-MS).

RESULTS and DISCUSSION
It was determined that 33 Y. ruckeri isolates used in the study showed a homogeneous structure in terms of biochemical tests.
Results revealed that Y. ruckeri forms a ground glasslike zone due to the precipitation of calcium salts and hydrolysis of Tween 80 by forming green colored colonies in Shotts-Waltman medium (SWM) selective medium. Y. ruckeri was found to give yellow colonies in Ribose Ornitine Deoxycholate (ROD) medium as a result of fermentation of ribose and precipitation of sodium desoxycholate salts. The positive result of KSU J. Agric Nat 24 (6): 1208-1211, 2021 Araştırma Makalesi Research Article isolate 7 in terms of Sorbitol test is that this strain has serotype 2 characteristics. This strain was immobile and found that it did not hydrolyze the tween 80.

CONCLUSION
Results of this study cocluded that the fatty acid composition of Y. ruckeri strains isolated from rainbow trout farms in different geographic regions of Turkey revealed that Y. ruckeri strains sustain a very similar character in terms of fatty acid composition. It has been determined that serotype 1 strain is common in Turkey as in European countries. Since this research has been studied with Y. ruckeri strains isolated from businesses covering different geographic regions of Turkey, the results of the research obtained will contribute to future studies with this bacterium.