doi:10.1111/jfd.12231
Journal of Fish Diseases 2015, 38, 283–293
Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish ska1, E Pazdzior1 and H Głowacka2 A Pez kala1, A Kozin 1 Department of Fish Diseases, National Veterinary Research Institute, Puławy, Poland 2 Department of Veterinary Hygiene, Laboratory of Fish Diseases, and Mycological and Parasitological Examination, Bydgoszcz, Poland
Abstract
Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55– 95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe. Keywords: bacterial fish diseases, fish disorders, freshwater fish, Shewanella putrefaciens. Correspondence A Pez kala, National Veterinary Research Institute, Department of Fish Diseases, Al. Partyzanto w 57, 24-100 Puławy, Poland (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Introduction
Shewanella putrefaciens is a Gram-negative, motile bacterium with rod-shaped cells, which belongs to the family Alteromonadaceae (Bowman 2005). It comprises mainly organisms isolated commonly from marine and brackish waters, and marine fish (Buller 2004; Al-Harbi & Naim Uddin 2005). The bacterium is particularly abundant in the bacterial flora of the intestine of marine fish (Al-Harbi & Naim Uddin 2004), and less frequently, S. putrefaciens has also been isolated from freshwater fish (Esteve, Biosca & Amaro 1993; Kozi nska & Pez kala 2004; Qin, Zhu & Xu 2012). Shewanella putrefaciens is the most important spoilage bacterium of iced fish and is frequently isolated from poultry meat and beef products (Stenstr€om & Molin 1990; Gennari & Campanini 1991; Borch, Kant-Muermans & Blixt 1996; Gram & Huss 1996; Gram & Melchiorsen 1996). Furthermore, the bacterium is an opportunistic human pathogen and has been reported as a causative agent of bacteriaemia and skin and soft tissue infections (Chen et al. 1997; Aubert et al. 2009). However, the role of S. putrefaciens in fish pathology is poorly understood. Until now, there have been only a few publications concerning the fish infections caused by this microorganism: two reports on marine fish species, that is, rabbitfish Siganus rivulatus (Forssk al and Niebuhr) (Saeed, Alamoudi & Al-Harbi 1987) and European sea bass Dicentrarchus labrax L. (Korun, Akgun-Dar
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Journal of Fish Diseases 2015, 38, 283–293
& Yazici 2009), and two of freshwater fish, viz common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum) (Kozi nska & Pez kala 2004), and loach Misgurnus anguillicaudatus (Cantor) (Qin et al. 2012). In recent years, clinical disease involving skin lesions accompanied by increased mortality has been frequently noted in farmed common carp and rainbow trout as well as in wild population of some fish species in Poland. The clinical signs of the disease were observed mainly in spring as water temperature rose to 7–10 °C. The condition was associated with isolation of orange-coloured colonies characteristic for S. putrefaciens from damaged fish tissues and organs. The aim of this study was to characterize the isolated bacteria that were collected from freshwater fish, particularly common carp and rainbow trout from farms where the condition was observed; to determine whether S. putrefaciens is really the causative agent of such health disorders in freshwater fish; and to select the most suitable method for the identification of the bacterium in routine fish examinations.
Materials and methods
Fish Several episodes of S. putrefaciens isolation from freshwater fish with clinical signs of the diseases, and mortality up to 20%, occurred in different farms in Poland during the period 2007–2012. Tested fish originated from twenty-five common carp and eleven rainbow trout farms. The body weight was from 20 to 350 g and 30 to 120 g of carp and rainbow trout, respectively. Ten fish were examined from each farm, and most of the individuals showed clinical signs. Additionally, the wild fish species such as brown trout Salmo trutta m. trutta L., silver carp Hypophthalmichthys molitrix (Valenciennes), European whitefish Coregonus lavaretus L., pike-perch Sander lucioperca L. were tested. In addition, ornamental fish such as koi carp Cyprinus carpio L. and Pelvicachromis taeniatus (Boulenger) imported to Poland showing clinical signs were included in the study (Table 1). Parasitological examination The samples from skin and gills were collected for parasitological examination. The samples were observed under the microscope. Ó 2014 John Wiley & Sons Ltd
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Bacterial isolation For bacterial isolation, samples from skin lesions, gills and internal organs (kidney, spleen) were aseptically collected. Kidney samples only were taken from fish in which no clinical signs of diseases were observed. The tissues taken were diluted in sterile phosphate-buffered saline (PBS) in the ratio of 1:1 (w/v), homogenized and inoculated onto trypticase soy agar (TSA; BioMerieux) and nutrient medium (Biomed) supplemented with 5% horse blood (BM). Fresh blood samples were spread directly onto the culture media. The bacterial growth was evaluated after 48-h incubation at 27 1 °C, and the number and diversity of colonies were determined. The dominant types of colony were inoculated onto TSA medium and incubated for 24 h at 27 1 °C. Pure cultures were kept frozen at 80 °C in tryptic soy broth (TSB; BioMerieux) supplemented with 15% glycerol for further examination. Phenotypical characterization Bacterial isolates were morphologically, physiologically and biochemically characterized by conventional plate and tube tests, mainly following the schemes of Austin & Austin (2007). The morphology of the bacteria was studied by Gram staining. All isolates were identified using the API 20E, API 20NE and API Zym (BioMerieux) systems, according to the manufacturer’s instructions, except the incubation temperature, which was 27 1 °C. The results of API tests were interpreted using the ‘apiweb’ programme (BioMerieux). Moreover, selected properties such as production of oxidase, motility, NaCl tolerance, hydrolysis of Tween 80, casein and starch were tested using the methods previously described (Kozi nska & Pez kala 2004). In addition, the antimicrobial susceptibility of the isolates was determined by the disc diffusion method on Mueller-Hinton agar (Oxoid). The following chemotherapeutic agents (lg per disc, Oxoid) from different group of drugs were used: oxytetracycline (OT) (30), doxycycline (DO) (30), nalidixic acid (NA) (30), flumequine (UB) (30), enrofloxacin (ENR) (5), norfloxacine (NOR) (10), sulphonamides (S3) (300), sulphamethoxazole/trimethoprim (SXT) (25), ampicillin (AMP) (10), amoxicillin (AML) (25), amoxicillin and clavulanic acid
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Journal of Fish Diseases 2015, 38, 283–293
Table 1 Shewanella putrefaciens strains isolated from freshwater fish – origin, biochemical profiles and isolates selected to 16S rRNA sequencing
Group of fish
Fish species
Farmed fish
Common carp
Rainbow trout
Wild fish
Brown trout Silver carp European whitefish Pike-perch
Ornamental fish
Koi carp Pelvicachromis taeniatus
Origin Date collection (No. of farms)
No. of fish tested
Percentage of fish exhibiting disease signs 50–60
5
55–65
3
50–85
22
Poland 2011 (4)
65–80
12
Poland 2012 (7)
65–80
20
Poland 2007 (3) Poland 2009 (2) Poland 2010 (9)
Poland 2007 (1)
Poland 2009 (3) Poland 2010 (3) Poland 2012 (4) Poland 2012 Poland 2011 Poland 2012 Poland 2011 Israel 2011 Camerun 2012
10 fish from each farm
10 fish from each farm
70
5
52–60
8
55–83
5
55–80
9
18
50
2
7
70
4
21
50–80
4
15
50
4
110
15
4
50
25
11
(AMC) (30), chloramphenicol (C) (30), florfenicol (FFC) (30). 16S rRNA gene sequencing Twenty-nine isolates of S. putrefaciens were used for 16S rRNA gene sequencing. These strains were selected on the basis of the clinical signs observed in examined fish and also on the differences in biochemical properties which allowed us to distinguish a few numeric profiles. Representatives of each profile were used to perform the sequencing analysis (Table 1). Ó 2014 John Wiley & Sons Ltd
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No. of isolated strains
Obtained biochemical profiles API 20NE API 20E API 20NE: 1410754 API 20E: 0502004; API 20NE: 1450754; 1450344 API 20E: 0502004; 0702004 API 20NE: 1411344; 1450344, 1450354; 1450344 API 20E: 0402004; 0502004, 0702004 API 20NE: 14117;4; 1450744; 1410354; 1411354 API 20E: 0702004; 0502004; 0502006 API 20NE: 1411754; 1410354; 1451754 API 20E: 0702004; 0402004; 0502004 API 20NE: 1410354; 1411344; 1451344 API 20E: 0702004; 0402004; 0502004 API 20NE: 1411154; 1410754 API 20E: 0502004; 0402004 API 20NE: 1411154; 1411344 API 20E: 0502004; 0402004 API 20NE: 1411344; 1450744 API 20E: 0502004; 040204 API 20NE: 1451344; 1450744 API 20E: 0402004; 0702004 API 20NE: 1410354 API 20E: 0702004 API 20NE: 1450754; 1451344 API 20E: 0502006; 0702004, API 20NE: 1450744 API 20E: 0502006 API 20NE: 1451354; 1410354 API 20E: 0502004; 0400004 API 20NE: 1411344; 1451354; 1410754 API 20E: 0402006; 0500006
Symbols of the isolates selected for sequencing K313, K530 K575, K604 K677, K678, K679, K680, K696 K734, K717
Sh1, Sh2, Sh3, Sh4
–
– Pt400 Pt411; Pt420 GIII41, GIII46 Tp5 St4, St5 Sd3 KK3, Koi2 St8, St15, St16
Total bacterial DNA was extracted from pure bacterial cultures using the DNeazy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions. The DNA concentration was spectrophotometrically quantified and adjusted to the concentration of 100 ng lL 1. Universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 142R (5′-TACGGCTACCTTGTTACGACTT-3′), respectively, were used to amplify the 16S rRNA gene (Weisburg et al. 1991). The 50-lL PCR reaction mixture contained PCR buffer (Sigma), 0.05 mM dNTP (Invitrogen), 0.2 lM forward and reverse primers, JumptStart Taq DNA polymerase
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(Sigma) and 100 ng of DNA template. The polymerase chain reaction (PCR) cycles were performed according to Borrell et al. (1997). Amplified products (about 1500 base pairs) were purified by USB ExoSAP-IT PCR Product Cleanup method (Affymetrix) and sequenced using 3730xl DNA Analyzer (Genomed S.A.). 16S rRNA gene sequences of collected isolates were analysed using MEGA 5.05 software. The similarity between the sequences of tested isolates and the sequences available at GenBank was assessed using MEGA 5.05 software. A phylogenetic tree was constructed by neighbour-joining method. Distance matrices were calculated using Komura’s 2-parameter correction, and stability of groupings, and bootstrap analysis (1000 replicates) was conducted using MEGA 5.05 software (Tamura et al. 2011). Shewanella algae ATCC 51192TM (accession no. HM016087) was used as an outgroup. Additionally, two S. putrefaciens sequences previously submitted to GenBank: S. putrefaciens ATCC 8071TM (accession no. X82133) and S. putrefaciens ATCC 8073TM (accession no. AJ000213), which represented Owen’s genomic group I and Owen’s genomic group III, respectively, were included in phylogenetic analysis (Owen, Legros & Lapage 1978). 16S rRNA gene sequences of S. putrefaciens isolates were deposited in the GenBank database under accession numbers: KC607503 to KC60 7531. Challenge tests The following strains of S. putrefaciens were used for challenge tests: Pt411 (KC60709) isolated from rainbow trout and K530 (KC607513) and K717 (KC60726), both isolated from common carp. The selection of those bacteria was based on the clinical signs of the disease observed in fish and also on most frequently obtained biochemical properties. The bacteria were grown in trypticase soya broth for 24 h at 27 1 °C and then centrifuged at 1830 g for 10 min. The cell pellets were suspended in sterile PBS to the final concentration of 107 cells mL 1. The bacterial concentration was determined using spectrophotometer at the wavelength of 540 nm. Healthy common carp and trout weighing 80–100 g were used in experiments. Before infection, the fish were anaesthetized for 2–5 min. in solution of MS-222 (Sigma), at the concentration from 75 to 150 lg L 1 of water Ó 2014 John Wiley & Sons Ltd
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(lower doses for rainbow trout and higher for common carp). For each strain, 20 common carp and 20 rainbow trout were injected intraperitoneally (Ip) with 0.5 mL of the bacterial suspension. The same number of other individuals was injected with sterile PBS, which were treated as the controls. Each group of fish was maintained in separate 300-L glass tanks in flow-through system with dechlorinated and aerated water before and during experiments. Water temperature was maintained at 11 2 °C for the each group, and clinical signs were recorded daily during 21 days. Freshly dead fish were used for bacteriological and post-mortem examinations to determine the cause for the disease. Damaged gills and internal tissues were taken for bacteriological tests. Additionally, blood samples were examined when systemic infection was suspected. Local Ethic Commission Nr II in Lublin (resolution Nr 40/2012) approved the procedure concerning experiments on fish.
Results
Clinical signs Diseased fish exhibited one or more of the following signs: sluggish behaviour (slow movements, weak reaction to stimuli), emaciation, darkening skin, distended anus, pale or focally necrosis of the gills. In common carp and rainbow trout, necrotic skin lesions or ulcers as well as abdomen distension were found (Fig. 1). Internally, haemorrhages or congestion, swollen kidney, as well as abnormal spleen enlargement were mostly observed. The disorders were accompanied by abnormal mortality (2–3% per day for at least 7 days) in farmed fish. Such clinical signs were also observed among population of wild fish species. Among ornamental fish, weak condition, apathy and abnormal mortality were most commonly observed signs of the infection. In all the farms where the fish disorders were noted, the presence of viral infection was monitored twice a year. These demonstrated the farms to be free from koi herpes virus (KHV), spring viraemia virus (SVCV), viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). Moreover, only occasional parasites such as Trichodina sp. or Gyrodactylus sp. were observed in some fish.
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(a)
(b)
Figure 1 Common carp (a) and rainbow trout (b) necrotic lesions and ulcers on the skin.
Phenotypical characterization Characteristic for the genus Shewanella, orangepigmented colonies on TSA grew in pure cultures or constituted 55–90% of total bacterial flora in samples from individual fish showing clinical signs of diseases. Uniform or dominant growth of one colony type was also observed on BM. In samples taken from some of the apparently healthy fish, a small number of diverse bacterial colonies were occasionally observed. Among these, colonies with orange pigmentation constituted 5–20%. In total, 118 characteristic colonies for Shewanella species were isolated. The majority (n = 89) of those bacteria were isolated from fish showing clinical signs of diseases. Sixty-two isolates were obtained from common carp, and 27 from rainbow trout (Table 1). One to five colonies isolated from the skin and internal organ samples of fish were collected from each farm. Each isolate from wild and ornamental fish was obtained from a separate fish sample. The results of phenotypic tests are presented in Table 2. All strains were Gram negative, motile and oxidase positive. Moreover, all isolates were positive for H2S production, gelatin and esculin hydrolysis, NO3 reduction and N-acetyl-glucosamine, maltose and malic acid assimilation. The isolates revealed Ó 2014 John Wiley & Sons Ltd
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variable results for ornithine decarboxylase, citrate utilization and fermentation of arabinose, as well as glucose, arabinose, capric acid and potassium gluconate assimilation. The tested strains did not require NaCl for their growth. In API 20E system, all tested isolates as well as the reference strains of S. putrefaciens indicated the similar numeric profiles (0502004, 0702004, 0402004, 0502006) and were classified as an excellent (99.9%) or very good (99.0–99.3%) identification level. One isolate (Koi2) appeared to be acceptable (89%), rendering profile 0400004. In the API 20NE system, different numeric profiles (1410354, 1410754, 1411344, 1411354, 1451344, 1450354) were observed, but all of them corresponded to an excellent (99.9%), very good (98.9%) or good (93.8–97.0%) identification level, similar to the reference strain (99.9%) (1411154). A few of the isolates were found to be on the acceptable (92.3%) or doubtful (70.5– 65.3%) level of discrimination, following the numerical profiles: 1451754, 1450754, 1450344. Four of the isolates (numeric profile 1450744) were identified as Aeromonas salmonicida subps. salmonicida on the good level of discrimination (97.7%). The ability to hydrolyse Tween 80, casein and starch was varied, depending on the isolates. Almost all tested bacteria (95%) were positive for casein hydrolysis, more than half (65%) for Tween 80 and only 25% for starch (Table 2). Additionally, API Zym system showed that all of the tested isolates were able to produce alkaline phosphatase, leucine and valine arylamidase, trypsin, acid phosphatase and N-acetyl-b-glucosaminidase. The susceptibility to antibacterial agent was varied. There was a significant and consistent difference in sensitivity between sulphonamide and potentiated sulphonamide groups. The bacteria strains appeared to be completely resistant to sulphonamides, but sensitive to potentiated sulphonamides. All tested isolates were also sensitive to quinolones and fluoroquinolones, as well as to systemic antibacterial agents (Table 3). Intermediate reaction was observed to tetracycline and penicillin groups. Sequencing Sequencing of the 16S rRNA gene confirmed the affiliation of twenty-nine Polish isolates with reference strain S. putrefaciens ATCC BAA-1097TM (accession no. CP000681), on the similarity of >97.2%. In the phylogenetic tree based on the neighbour-joining method, collected isolates were
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Table 2 Phenotypical characteristics of the collected isolates of Shewanella putrefaciens, in comparison with the reference strain ATCC BAA-1097TM and strain of Saeed et al. 1987.
Characteristic Orange pigment Gram staining Motility Oxidase ONPG Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization H2S production Urease hydrolysis Tryptophan deaminase Indole production Acetoin production Gelatin hydrolysis Acid from: Glucose, mannitol Inositol, sorbitol Rhamnose, saccharose Melibiose, amygdalin Arabinose NO3 reduction to NO2 Esculin hydrolysis Assimilation: Glucose Arabinose Mannose Mannitol N-Acetyl-glucosamine Maltose Potassium gluconate Capric acid Adipic acid Malic acid Phenylacetic acid Other characteristics: Growth in the presence of NaCl: 0–5% 6% Hydrolysis: Tween 80 Casein Starch +, 100% positive;
Shewanella putrefaciens ATCC BAA1097TM
Shewanella putrefaciens (Saeed et al. 1987)
+
+
nd
+ +
+ +
+ +
+ (weak) +
+ + +
+
+
Shewanella putrefaciens n = 118
66 12 +
+
28 + +
+ (weak) + +
70 49
+
+ + 35 56
+
+
+
+
+ + (weak) + (weak) +
65 95 26
288
nd nd nd nd nd nd nd nd nd nd nd
+ + nd nd nd
, 100% negative; nd, no data; numbers represent percentage of positive reactions.
divided into two branches, described in this work as genogroups I and II (Fig. 2). The genogroup I consisted of nineteen isolates which originated from farmed fish (common carp and rainbow trout) showing severe external and internal disease signs. The other ten strains were classified in genogroup II. This branch comprised most of the isolates collected from wild and ornamental fish as well as some from farmed fish in which only internal lesions were observed. Values of similarity of the reference strain of S. putrefaciens ATCC Ó 2014 John Wiley & Sons Ltd
+
+ nd
BAA-1097TM to the isolates located in genogroup I were 97.2–99.2%, whereas with genogroup II 97.2–98.3%. Moreover, the results of phylogenetic analysis showed that the strain ATCC 8071TM (accession no. X82133) representing Owen’s genomic group I was localized in the genogroup I, while the strain ATCC 8073TM (accession no. AJ000213) representing the Owen’s genomic group III was classified into genogroup II. Similarity of the reference strain of S. putrefaciens ATCC BAA-1097TM to S. algae ATCC
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Journal of Fish Diseases 2015, 38, 283–293
Table 3 Antimicrobial susceptibility of Shewanella putrefaciens collected isolates
Antibacterial agents
Shewanella putrefaciens n = 118 The average of the diameter of the inhibition zones (mm)
Tetracycline: Oxytetracycline 18 mm Doxycycline 26 mm Quinolones and fluoroquinolones Nalidixic acid 25 mm Flumequine 30 mm Enrofloxacin 32 mm Norfloxacine 26 mm Sulphonamides and potentiated sulphonamides Sulphonamides 0 mm Sulphamethoxazole/ 25 mm trimethoprim Penicillins Ampicillin 17 mm Amoxicillin 10 mm Amoxicillin and 14 mm clavulanic acid Systemic antibacterial agents Chloramphenicol 35 mm Florfenicol 35 m S, susceptible; I, intermediate susceptible; R, resistant.
Figure 2 Neighbour-joining phylogenetic tree of Shewanella putrefaciens strains based on 16S rRNA gene sequence. Bootstrap values (expressed as percentages of 1000 replicates), bar = 0.001 substitution per nucleotide position. Ó 2014 John Wiley & Sons Ltd
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51192TM (accession no. HM016087), as an outgroup, was 96.57%. Challenge tests
Sensitivity
I S S S S S R S
I R I
S S
No mortality or any disease signs in fish injected with sterile PBS were observed. Common carp infected with the K530 and K717 and rainbow trout with Pt411 strains showed one or more of the following external signs: darkening skin and pale gills, necrotic skin lesions and ulcers (Fig. 3), distended anus. Individual common carp or rainbow trout showed exophthalmia and abdominal distension due to ascitic fluid in the peritoneum (Fig. 4). Intensive congestions and watery of internal organs were mostly observed as well as intensive haemorrhages in the peritoneum and internal organs with significant enlargement of the spleen (Figs 5 & 6). Cumulative mortalities of infected fish were from 20% to 75%, depending on fish species and strain used for challenge experiments (Fig. 7). Shewanella putrefaciens was reisolated in pure cultures from damaged tissues of each fish
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Figure 3 Necrotic lesions and ulcers on the skin in carp after experimentally infected with the K530 strain.
Figure 7 Mortality of carp and rainbow trout Ip infected with Shewanella putrefaciens K530, K717 and Pt411 strains.
Discussion Figure 4 Rainbow trout – exophthalmia and abdomen distension in rainbow trout experimentally infected with the Pt411 strain.
Figure 5 Common carp – congestion of internal organs, enlargement of the kidney after experimentally infected with the K530 strain.
Figure 6 Rainbow trout – intensive haemorrhages in the peritoneum and internal organs and enlargement of the spleen (narrow) after experimentally infected with the Pt411 strain.
tested and from blood of common carp or rainbow trout challenged with the K530, K717 or Pt411 strain, respectively. Ó 2014 John Wiley & Sons Ltd
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Physiological and biochemical analyses demonstrated that collected isolates had phenotypic properties consistent with S. putrefaciens, which was confirmed by 16S rRNA sequencing. This is the first description of S. putrefaciens strains as pathogens of freshwater fish. Abundant and uniform or predominant growth of S. putrefaciens was found in all samples originated from fish showing clinical signs of disease. It is noteworthy that uniform growth of S. putrefaciens was found in some gill samples from affected fish. It shows that under certain circumstances, the bacterium significantly suppresses the growth of other bacteria, which are usually present in this tissue in clinically healthy fish. A similar phenomenon has been previously observed after the experimental infection of fish with S. putrefaciens when pure cultures of the bacterium have been even obtained from the intestinal tract (Kozi nska & Pez kala 2004). These findings suggest that S. putrefaciens has an antagonistic effect on other bacteria. The inhibitory effect of this bacterium on Aeromonas sp. has been found (Goldschmidt-Clermont et al. 2008). The biochemical identification of collected strains of S. putrefaciens, performed on API 20E, API 20NE, as well as enzymatic activity tested on API Zym showed a good discrimination level of the bacteria. On the basis of our studies, we were able to compare API 20E and API 20NE tests, for the first time, in order to choose the best diagnostic methods. Excellent or very good identification levels were obtained in the cases of all examined isolates on API 20E test. One of our API 20E profiles (0702004) corresponded to the
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profile of S. putrefaciens isolated from sea bass (Korun et al. 2009). Similar reactions in API 20E test revealed S. putrefaciens strains originated from loach (Qin et al. 2012). In API 20NE, four bacteria strains (GIII41-12-Poland, St4-12-Poland, Sd4-12-Poland and K575-10-Poland) isolated from brown trout, European whitefish, pike-perch and common carp, respectively, were improperly identified as A. salmonicida subsp. salmonicida (numeric profile 1450744). Moreover, five isolates (Sh4-09-Poland, K680-12-Poland, K679-12Poland, K604-11-Poland and K313-05-Poland) all originated from common carps and were classified as S. putrefaciens, but the result of this identification was indicated as a doubtful (numeric profile 1450754). In order to avoid incorrect identification of S. putrefaciens, diagnosis should be performed very carefully, taking into consideration the morphology of the bacterial cells and their colonies. Our results indicate that S. putrefaciens is more correctly identified by API 20E test, which has been confirmed by other laboratories (Korun et al. 2009; Qin et al. 2012). The vast majority of the phenotypical properties of the tested isolates corresponded with the characteristic of the reference strain ATCC BAA1097TM and the strain of S. putrefaciens isolated by Saeed et al. (1987). API Zym tests showed a high enzymatic activity of collected strains of S. putrefaciens, which can play an important role in the pathogenicity of the bacteria. The ability to produce N-acetyl-bglucosaminidase suggests that these isolates should belong to the genomic group II (Zimke et al. 1998). Antimicrobial susceptibility of S. putrefaciens of collected strains was studied in more extended range than in our previous work (Kozi nska & Pez kala 2004). In the present study, we showed that the tested isolates are still sensitive to drugs from quinolone and fluoroquinolone group. The same observations concerning S. putrefaciens isolated from clinical cases of marine and freshwater fish were also previously described (Korun et al. 2009; Qin et al. 2012). Additionally, our studies confirmed a complete resistance of these bacteria to sulphonamides (Korun et al. 2009). For such reason, this group of drugs should be completely withdrawn from the therapy against shewanellosis. However, the question concerning the use of trimethoprim seems to be reasonable. Some strains showed the resistance to this drug (Korun et al. Ó 2014 John Wiley & Sons Ltd
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2009); meanwhile, all bacteria examined in our study were sensitive to trimethoprim correlated with sulphonamides. Resistance to sulphamethoxazole was observed in our previous research (Kozi nska & Pez kala 2004), but the examination was conducted on a smaller number of isolates. A complete resistance of S. putrefaciens to almost all tested antimicrobial agents was already observed in two cases (data not shown). This suggests development of the resistance mechanisms by the bacterium and therefore a need for a well-thought therapy against shewanellosis, supported by laboratory studies on drug susceptibility. It is known that organisms identified phenotypically as S. putrefaciens group comprise several biovars (Bowman 2005) and are divided into four hybridization groups (Owen et al. 1978). The results of sequencing analysis performed in our study showed that collected strains of S. putrefaciens created two genomic groups. Bacterial strains isolated from farmed fish (common carp and rainbow trout) which caused clinical signs of the diseases were included into our genomic group I. Additionally, phylogenetic analysis showed the similarity of this group to the reference strain ATCC 8071TM, which represents genogroup I by Owen’s hybridization groups (S. putrefaciens sensu stricto) (Owen et al. 1978; Bowman 2005). The correlation between our genomic group II and genogroup III with Owen’s, represented by reference strain ATCC 8073TM, was also confirmed. This branch comprised mostly the isolates collected from wild, ornamental as well as farmed fish, in which only few clinical signs were observed. Our results confirm the previous studies, which suggested that S. putrefaciens species is composed of a series of genetically variable strains (Bowman 2005). Phenotypic properties did not correlate well with the hybridization groups of Owen (Owen et al. 1978; Bowman 2005). According to the ability to produce N-acetyl-b-glucosaminidase, all our collected strains should belong to the genomic group II by Owen’s, which, however, were named as S. baltica (Zimke et al. 1998) or S. algae (Bowman 2005). On the other hand, inability to grow in the presence of 6% NaCl is characteristic for isolates belonging to Gillard biovars 1 and 3, which correlate with genogroups I and III by Owen’s, respectively (Zimke et al. 1998). Strains representing I and III hybridization groups of Owen were originated mostly from food and environment (Zimke et al. 1998), and now the isolates from diseased freshwater fish should be included.
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The virulence of S. putrefaciens for common carp and rainbow trout has previously been documented in experimental challenge tests (Kozi nska & Pez kala 2004). Here, it has been confirmed using three strains isolated from natural diseased fish. Experimental infected common carps and rainbow trout showed clinical signs of disease similar to those observed in the naturally infected fish. Some signs of the disease, such as pale gills, ulcers and spleen enlargement, have been described in the cultured marine fish such as European sea bass (Korun et al. 2009), as well as among the freshwater loach (Qin et al. 2012) and common carps (www.environment-agency.gov.uk). It is worth noting that percentages of mortalities which were estimated 40–85% in different species of fish (Kozi nska & Pez kala 2004; Korun et al. 2009) are similar to our present results. Reisolation of S. putrefaciens from freshly dead experimental fish fulfilled Koch’s postulates. Conclusions
Infections associated with S. putrefaciens appear to be becoming more serious problems in freshwater fish farms, causing even an acute form of the diseases with septicaemia and markedly increased mortalities. The clinical signs of shewanellosis may vary, depending on the strain. The differences between the collected strains were visible on the phenotypic properties, as well as on the molecular level. Diagnosis of S. putrefaciens is not complicated, but it could be problematic. Our results showed that the API 20E tests are most suitable for proper identification of this bacteria. The fulfilled Koch’s postulates presented in this study indicate that the S. putrefaciens should be considered as an opportunistic pathogen, like almost all bacteria, which causes fish disorders. Publication History Received: 3 October 2013 Revision received: 3 January 2014 Accepted: 4 January 2014
This paper was edited and accepted under the Editorship of Professor Ron Roberts.
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niloticus 9 Oreochromis aureus) cultured in earthen ponds in Saudi Arabia. Aquaculture 229, 37–44. Al-Harbi A.H. & Naim Uddin M. (2005) Bacterial diversity of tilapia (Oreochromis niloticus) cultured in brackish water in Saudi Arabia. Aquaculture 250, 566–572. Aubert T., Rovery C., Bourhaba K., Singeorzan S., Heim M. & Cretel E. (2009) Dermohypodermie bacterienne non necrosante et bacteriemie a Shewanella putrefaciens. La Revue de me decine interne 30, 800–802. Austin B. & Austin D.A. (2007) Bacterial Fish Pathogens. Disease of Farmed and Wild Fish, 4th edn. Praxis Publishing Ltd., Chichester. Borch E., Kant-Muermans M.L. & Blixt Y. (1996) Bacterial spoilage of meat and cured meat products. International Journal of Food Microbiology 33, 103–120. Borrell N., Acinas S.G., Figueras M.J. & Martinez-Murcia A. (1997) Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. Journal of Clinical Microbiology 35, 1671–1674. Bowman J.P. (2005) Genus XIII. Shewanella. In: Bergey‘s Manual of Systematic Bacteriology (eds. by D.J. Brenner, N.R. Krieg & J.T. Staley), vol. 2, Part B, pp. 480–491. Springer, Michigan State University, MI, USA. Buller N.B. (2004) Bacteria From Fish and Other Aquatic Animals: A Practical Identification Manual, pp. 31, 181, CABI Publishing, London. Chen Y.S., Liu Y.C., Yen M.Y., Wang J.H., Wann S.R. & Cheng D.L. (1997) Skin and soft-tissue manifestations of Shewanella putrefaciens infection. Clinical Infectious Diseases 25, 225–229. Esteve C., Biosca E.G. & Amaro C. (1993) Virulence of Aeromonas hydrophila and some other bacteria isolated from European eels Anguilla anguilla reared in fresh water. Diseases of Aquatic Organisms 16, 15–20. Gennari M. & Campanini R. (1991) Isolation and characterization of Shewanella putrefaciens from fresh and spoiled fish, fresh and spoiled meat, dairy products, water and soil. Industrial Alimentaria 30, 965–976. Goldschmidt-Clermont E., Wahli T., Frey J. & Burr S.E. (2008) Identification of bacteria from the normal flora of perch, Perca fluviatilis L., and evaluation of their inhibitory potential towards Aeromonas species. Journal of Fish Diseases 31, 353–359. Gram L. & Huss H.H. (1996) Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 33, 121–137. Gram L. & Melchiorsen J. (1996) Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue. Journal of Applied Bacteriology 80, 589–595.
References
Korun J., Akgun-Dar K. & Yazici M. (2009) Isolation of Shewanella putrefaciens from cultured European sea bass, (Dicentrarchus labrax) in Turkey. Revue de Medecine Veterinaire-Toulouse 160, 532–536.
Al-Harbi A.H. & Naim Uddin M. (2004) Seasonal variation in the intestinal bacterial flora of hybrid tilapia (Oreochromis
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of the fish. Bulletin of the European Association of Fish Pathologists 24, 199–203. Owen R.J., Legros R.M. & Lapage S.P. (1978) Base composition, size and sequence similarities of genome deoxyribonucleic acid from clinical isolates of Pseudomonas putrefaciens. Journal of General Microbiology 104, 127–138. Qin L., Zhu M. & Xu L. (2012) First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach, Misgurnus anguillicaudatus. Aquaculture Research 2012, 1–7. Saeed M.O., Alamoudi M.M. & Al-Harbi A.H. (1987) A Pseudomonas associated with disease in cultured rabbitfish Siganus rivulatus in the Red Sea. Diseases of Aquatic Organisms 3, 177–180.
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Stenstr€om I. & Molin G. (1990) Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens. Journal of Applied Bacteriology 68, 601–608. Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28, 2731–2739. Weisburg W.G., Barns S.M., Pelletier D.A. & Lane D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173, 697–703. Zimke F., H€ofle M.G., Lalucat J. & Rossello-Mora R. (1998) Reclassification of Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. International Journal of Systematic Bacteriology 48, 179–186.
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Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish ska1, E Pazdzior1 and H Głowacka2 A Pez kala1, A Kozin 1 Department of Fish Diseases, National Veterinary Research Institute, Puławy, Poland 2 Department of Veterinary Hygiene, Laboratory of Fish Diseases, and Mycological and Parasitological Examination, Bydgoszcz, Poland
Abstract
Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55– 95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe. Keywords: bacterial fish diseases, fish disorders, freshwater fish, Shewanella putrefaciens. Correspondence A Pez kala, National Veterinary Research Institute, Department of Fish Diseases, Al. Partyzanto w 57, 24-100 Puławy, Poland (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Introduction
Shewanella putrefaciens is a Gram-negative, motile bacterium with rod-shaped cells, which belongs to the family Alteromonadaceae (Bowman 2005). It comprises mainly organisms isolated commonly from marine and brackish waters, and marine fish (Buller 2004; Al-Harbi & Naim Uddin 2005). The bacterium is particularly abundant in the bacterial flora of the intestine of marine fish (Al-Harbi & Naim Uddin 2004), and less frequently, S. putrefaciens has also been isolated from freshwater fish (Esteve, Biosca & Amaro 1993; Kozi nska & Pez kala 2004; Qin, Zhu & Xu 2012). Shewanella putrefaciens is the most important spoilage bacterium of iced fish and is frequently isolated from poultry meat and beef products (Stenstr€om & Molin 1990; Gennari & Campanini 1991; Borch, Kant-Muermans & Blixt 1996; Gram & Huss 1996; Gram & Melchiorsen 1996). Furthermore, the bacterium is an opportunistic human pathogen and has been reported as a causative agent of bacteriaemia and skin and soft tissue infections (Chen et al. 1997; Aubert et al. 2009). However, the role of S. putrefaciens in fish pathology is poorly understood. Until now, there have been only a few publications concerning the fish infections caused by this microorganism: two reports on marine fish species, that is, rabbitfish Siganus rivulatus (Forssk al and Niebuhr) (Saeed, Alamoudi & Al-Harbi 1987) and European sea bass Dicentrarchus labrax L. (Korun, Akgun-Dar
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& Yazici 2009), and two of freshwater fish, viz common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum) (Kozi nska & Pez kala 2004), and loach Misgurnus anguillicaudatus (Cantor) (Qin et al. 2012). In recent years, clinical disease involving skin lesions accompanied by increased mortality has been frequently noted in farmed common carp and rainbow trout as well as in wild population of some fish species in Poland. The clinical signs of the disease were observed mainly in spring as water temperature rose to 7–10 °C. The condition was associated with isolation of orange-coloured colonies characteristic for S. putrefaciens from damaged fish tissues and organs. The aim of this study was to characterize the isolated bacteria that were collected from freshwater fish, particularly common carp and rainbow trout from farms where the condition was observed; to determine whether S. putrefaciens is really the causative agent of such health disorders in freshwater fish; and to select the most suitable method for the identification of the bacterium in routine fish examinations.
Materials and methods
Fish Several episodes of S. putrefaciens isolation from freshwater fish with clinical signs of the diseases, and mortality up to 20%, occurred in different farms in Poland during the period 2007–2012. Tested fish originated from twenty-five common carp and eleven rainbow trout farms. The body weight was from 20 to 350 g and 30 to 120 g of carp and rainbow trout, respectively. Ten fish were examined from each farm, and most of the individuals showed clinical signs. Additionally, the wild fish species such as brown trout Salmo trutta m. trutta L., silver carp Hypophthalmichthys molitrix (Valenciennes), European whitefish Coregonus lavaretus L., pike-perch Sander lucioperca L. were tested. In addition, ornamental fish such as koi carp Cyprinus carpio L. and Pelvicachromis taeniatus (Boulenger) imported to Poland showing clinical signs were included in the study (Table 1). Parasitological examination The samples from skin and gills were collected for parasitological examination. The samples were observed under the microscope. Ó 2014 John Wiley & Sons Ltd
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Bacterial isolation For bacterial isolation, samples from skin lesions, gills and internal organs (kidney, spleen) were aseptically collected. Kidney samples only were taken from fish in which no clinical signs of diseases were observed. The tissues taken were diluted in sterile phosphate-buffered saline (PBS) in the ratio of 1:1 (w/v), homogenized and inoculated onto trypticase soy agar (TSA; BioMerieux) and nutrient medium (Biomed) supplemented with 5% horse blood (BM). Fresh blood samples were spread directly onto the culture media. The bacterial growth was evaluated after 48-h incubation at 27 1 °C, and the number and diversity of colonies were determined. The dominant types of colony were inoculated onto TSA medium and incubated for 24 h at 27 1 °C. Pure cultures were kept frozen at 80 °C in tryptic soy broth (TSB; BioMerieux) supplemented with 15% glycerol for further examination. Phenotypical characterization Bacterial isolates were morphologically, physiologically and biochemically characterized by conventional plate and tube tests, mainly following the schemes of Austin & Austin (2007). The morphology of the bacteria was studied by Gram staining. All isolates were identified using the API 20E, API 20NE and API Zym (BioMerieux) systems, according to the manufacturer’s instructions, except the incubation temperature, which was 27 1 °C. The results of API tests were interpreted using the ‘apiweb’ programme (BioMerieux). Moreover, selected properties such as production of oxidase, motility, NaCl tolerance, hydrolysis of Tween 80, casein and starch were tested using the methods previously described (Kozi nska & Pez kala 2004). In addition, the antimicrobial susceptibility of the isolates was determined by the disc diffusion method on Mueller-Hinton agar (Oxoid). The following chemotherapeutic agents (lg per disc, Oxoid) from different group of drugs were used: oxytetracycline (OT) (30), doxycycline (DO) (30), nalidixic acid (NA) (30), flumequine (UB) (30), enrofloxacin (ENR) (5), norfloxacine (NOR) (10), sulphonamides (S3) (300), sulphamethoxazole/trimethoprim (SXT) (25), ampicillin (AMP) (10), amoxicillin (AML) (25), amoxicillin and clavulanic acid
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Table 1 Shewanella putrefaciens strains isolated from freshwater fish – origin, biochemical profiles and isolates selected to 16S rRNA sequencing
Group of fish
Fish species
Farmed fish
Common carp
Rainbow trout
Wild fish
Brown trout Silver carp European whitefish Pike-perch
Ornamental fish
Koi carp Pelvicachromis taeniatus
Origin Date collection (No. of farms)
No. of fish tested
Percentage of fish exhibiting disease signs 50–60
5
55–65
3
50–85
22
Poland 2011 (4)
65–80
12
Poland 2012 (7)
65–80
20
Poland 2007 (3) Poland 2009 (2) Poland 2010 (9)
Poland 2007 (1)
Poland 2009 (3) Poland 2010 (3) Poland 2012 (4) Poland 2012 Poland 2011 Poland 2012 Poland 2011 Israel 2011 Camerun 2012
10 fish from each farm
10 fish from each farm
70
5
52–60
8
55–83
5
55–80
9
18
50
2
7
70
4
21
50–80
4
15
50
4
110
15
4
50
25
11
(AMC) (30), chloramphenicol (C) (30), florfenicol (FFC) (30). 16S rRNA gene sequencing Twenty-nine isolates of S. putrefaciens were used for 16S rRNA gene sequencing. These strains were selected on the basis of the clinical signs observed in examined fish and also on the differences in biochemical properties which allowed us to distinguish a few numeric profiles. Representatives of each profile were used to perform the sequencing analysis (Table 1). Ó 2014 John Wiley & Sons Ltd
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No. of isolated strains
Obtained biochemical profiles API 20NE API 20E API 20NE: 1410754 API 20E: 0502004; API 20NE: 1450754; 1450344 API 20E: 0502004; 0702004 API 20NE: 1411344; 1450344, 1450354; 1450344 API 20E: 0402004; 0502004, 0702004 API 20NE: 14117;4; 1450744; 1410354; 1411354 API 20E: 0702004; 0502004; 0502006 API 20NE: 1411754; 1410354; 1451754 API 20E: 0702004; 0402004; 0502004 API 20NE: 1410354; 1411344; 1451344 API 20E: 0702004; 0402004; 0502004 API 20NE: 1411154; 1410754 API 20E: 0502004; 0402004 API 20NE: 1411154; 1411344 API 20E: 0502004; 0402004 API 20NE: 1411344; 1450744 API 20E: 0502004; 040204 API 20NE: 1451344; 1450744 API 20E: 0402004; 0702004 API 20NE: 1410354 API 20E: 0702004 API 20NE: 1450754; 1451344 API 20E: 0502006; 0702004, API 20NE: 1450744 API 20E: 0502006 API 20NE: 1451354; 1410354 API 20E: 0502004; 0400004 API 20NE: 1411344; 1451354; 1410754 API 20E: 0402006; 0500006
Symbols of the isolates selected for sequencing K313, K530 K575, K604 K677, K678, K679, K680, K696 K734, K717
Sh1, Sh2, Sh3, Sh4
–
– Pt400 Pt411; Pt420 GIII41, GIII46 Tp5 St4, St5 Sd3 KK3, Koi2 St8, St15, St16
Total bacterial DNA was extracted from pure bacterial cultures using the DNeazy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions. The DNA concentration was spectrophotometrically quantified and adjusted to the concentration of 100 ng lL 1. Universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 142R (5′-TACGGCTACCTTGTTACGACTT-3′), respectively, were used to amplify the 16S rRNA gene (Weisburg et al. 1991). The 50-lL PCR reaction mixture contained PCR buffer (Sigma), 0.05 mM dNTP (Invitrogen), 0.2 lM forward and reverse primers, JumptStart Taq DNA polymerase
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(Sigma) and 100 ng of DNA template. The polymerase chain reaction (PCR) cycles were performed according to Borrell et al. (1997). Amplified products (about 1500 base pairs) were purified by USB ExoSAP-IT PCR Product Cleanup method (Affymetrix) and sequenced using 3730xl DNA Analyzer (Genomed S.A.). 16S rRNA gene sequences of collected isolates were analysed using MEGA 5.05 software. The similarity between the sequences of tested isolates and the sequences available at GenBank was assessed using MEGA 5.05 software. A phylogenetic tree was constructed by neighbour-joining method. Distance matrices were calculated using Komura’s 2-parameter correction, and stability of groupings, and bootstrap analysis (1000 replicates) was conducted using MEGA 5.05 software (Tamura et al. 2011). Shewanella algae ATCC 51192TM (accession no. HM016087) was used as an outgroup. Additionally, two S. putrefaciens sequences previously submitted to GenBank: S. putrefaciens ATCC 8071TM (accession no. X82133) and S. putrefaciens ATCC 8073TM (accession no. AJ000213), which represented Owen’s genomic group I and Owen’s genomic group III, respectively, were included in phylogenetic analysis (Owen, Legros & Lapage 1978). 16S rRNA gene sequences of S. putrefaciens isolates were deposited in the GenBank database under accession numbers: KC607503 to KC60 7531. Challenge tests The following strains of S. putrefaciens were used for challenge tests: Pt411 (KC60709) isolated from rainbow trout and K530 (KC607513) and K717 (KC60726), both isolated from common carp. The selection of those bacteria was based on the clinical signs of the disease observed in fish and also on most frequently obtained biochemical properties. The bacteria were grown in trypticase soya broth for 24 h at 27 1 °C and then centrifuged at 1830 g for 10 min. The cell pellets were suspended in sterile PBS to the final concentration of 107 cells mL 1. The bacterial concentration was determined using spectrophotometer at the wavelength of 540 nm. Healthy common carp and trout weighing 80–100 g were used in experiments. Before infection, the fish were anaesthetized for 2–5 min. in solution of MS-222 (Sigma), at the concentration from 75 to 150 lg L 1 of water Ó 2014 John Wiley & Sons Ltd
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(lower doses for rainbow trout and higher for common carp). For each strain, 20 common carp and 20 rainbow trout were injected intraperitoneally (Ip) with 0.5 mL of the bacterial suspension. The same number of other individuals was injected with sterile PBS, which were treated as the controls. Each group of fish was maintained in separate 300-L glass tanks in flow-through system with dechlorinated and aerated water before and during experiments. Water temperature was maintained at 11 2 °C for the each group, and clinical signs were recorded daily during 21 days. Freshly dead fish were used for bacteriological and post-mortem examinations to determine the cause for the disease. Damaged gills and internal tissues were taken for bacteriological tests. Additionally, blood samples were examined when systemic infection was suspected. Local Ethic Commission Nr II in Lublin (resolution Nr 40/2012) approved the procedure concerning experiments on fish.
Results
Clinical signs Diseased fish exhibited one or more of the following signs: sluggish behaviour (slow movements, weak reaction to stimuli), emaciation, darkening skin, distended anus, pale or focally necrosis of the gills. In common carp and rainbow trout, necrotic skin lesions or ulcers as well as abdomen distension were found (Fig. 1). Internally, haemorrhages or congestion, swollen kidney, as well as abnormal spleen enlargement were mostly observed. The disorders were accompanied by abnormal mortality (2–3% per day for at least 7 days) in farmed fish. Such clinical signs were also observed among population of wild fish species. Among ornamental fish, weak condition, apathy and abnormal mortality were most commonly observed signs of the infection. In all the farms where the fish disorders were noted, the presence of viral infection was monitored twice a year. These demonstrated the farms to be free from koi herpes virus (KHV), spring viraemia virus (SVCV), viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). Moreover, only occasional parasites such as Trichodina sp. or Gyrodactylus sp. were observed in some fish.
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(a)
(b)
Figure 1 Common carp (a) and rainbow trout (b) necrotic lesions and ulcers on the skin.
Phenotypical characterization Characteristic for the genus Shewanella, orangepigmented colonies on TSA grew in pure cultures or constituted 55–90% of total bacterial flora in samples from individual fish showing clinical signs of diseases. Uniform or dominant growth of one colony type was also observed on BM. In samples taken from some of the apparently healthy fish, a small number of diverse bacterial colonies were occasionally observed. Among these, colonies with orange pigmentation constituted 5–20%. In total, 118 characteristic colonies for Shewanella species were isolated. The majority (n = 89) of those bacteria were isolated from fish showing clinical signs of diseases. Sixty-two isolates were obtained from common carp, and 27 from rainbow trout (Table 1). One to five colonies isolated from the skin and internal organ samples of fish were collected from each farm. Each isolate from wild and ornamental fish was obtained from a separate fish sample. The results of phenotypic tests are presented in Table 2. All strains were Gram negative, motile and oxidase positive. Moreover, all isolates were positive for H2S production, gelatin and esculin hydrolysis, NO3 reduction and N-acetyl-glucosamine, maltose and malic acid assimilation. The isolates revealed Ó 2014 John Wiley & Sons Ltd
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variable results for ornithine decarboxylase, citrate utilization and fermentation of arabinose, as well as glucose, arabinose, capric acid and potassium gluconate assimilation. The tested strains did not require NaCl for their growth. In API 20E system, all tested isolates as well as the reference strains of S. putrefaciens indicated the similar numeric profiles (0502004, 0702004, 0402004, 0502006) and were classified as an excellent (99.9%) or very good (99.0–99.3%) identification level. One isolate (Koi2) appeared to be acceptable (89%), rendering profile 0400004. In the API 20NE system, different numeric profiles (1410354, 1410754, 1411344, 1411354, 1451344, 1450354) were observed, but all of them corresponded to an excellent (99.9%), very good (98.9%) or good (93.8–97.0%) identification level, similar to the reference strain (99.9%) (1411154). A few of the isolates were found to be on the acceptable (92.3%) or doubtful (70.5– 65.3%) level of discrimination, following the numerical profiles: 1451754, 1450754, 1450344. Four of the isolates (numeric profile 1450744) were identified as Aeromonas salmonicida subps. salmonicida on the good level of discrimination (97.7%). The ability to hydrolyse Tween 80, casein and starch was varied, depending on the isolates. Almost all tested bacteria (95%) were positive for casein hydrolysis, more than half (65%) for Tween 80 and only 25% for starch (Table 2). Additionally, API Zym system showed that all of the tested isolates were able to produce alkaline phosphatase, leucine and valine arylamidase, trypsin, acid phosphatase and N-acetyl-b-glucosaminidase. The susceptibility to antibacterial agent was varied. There was a significant and consistent difference in sensitivity between sulphonamide and potentiated sulphonamide groups. The bacteria strains appeared to be completely resistant to sulphonamides, but sensitive to potentiated sulphonamides. All tested isolates were also sensitive to quinolones and fluoroquinolones, as well as to systemic antibacterial agents (Table 3). Intermediate reaction was observed to tetracycline and penicillin groups. Sequencing Sequencing of the 16S rRNA gene confirmed the affiliation of twenty-nine Polish isolates with reference strain S. putrefaciens ATCC BAA-1097TM (accession no. CP000681), on the similarity of >97.2%. In the phylogenetic tree based on the neighbour-joining method, collected isolates were
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Table 2 Phenotypical characteristics of the collected isolates of Shewanella putrefaciens, in comparison with the reference strain ATCC BAA-1097TM and strain of Saeed et al. 1987.
Characteristic Orange pigment Gram staining Motility Oxidase ONPG Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization H2S production Urease hydrolysis Tryptophan deaminase Indole production Acetoin production Gelatin hydrolysis Acid from: Glucose, mannitol Inositol, sorbitol Rhamnose, saccharose Melibiose, amygdalin Arabinose NO3 reduction to NO2 Esculin hydrolysis Assimilation: Glucose Arabinose Mannose Mannitol N-Acetyl-glucosamine Maltose Potassium gluconate Capric acid Adipic acid Malic acid Phenylacetic acid Other characteristics: Growth in the presence of NaCl: 0–5% 6% Hydrolysis: Tween 80 Casein Starch +, 100% positive;
Shewanella putrefaciens ATCC BAA1097TM
Shewanella putrefaciens (Saeed et al. 1987)
+
+
nd
+ +
+ +
+ +
+ (weak) +
+ + +
+
+
Shewanella putrefaciens n = 118
66 12 +
+
28 + +
+ (weak) + +
70 49
+
+ + 35 56
+
+
+
+
+ + (weak) + (weak) +
65 95 26
288
nd nd nd nd nd nd nd nd nd nd nd
+ + nd nd nd
, 100% negative; nd, no data; numbers represent percentage of positive reactions.
divided into two branches, described in this work as genogroups I and II (Fig. 2). The genogroup I consisted of nineteen isolates which originated from farmed fish (common carp and rainbow trout) showing severe external and internal disease signs. The other ten strains were classified in genogroup II. This branch comprised most of the isolates collected from wild and ornamental fish as well as some from farmed fish in which only internal lesions were observed. Values of similarity of the reference strain of S. putrefaciens ATCC Ó 2014 John Wiley & Sons Ltd
+
+ nd
BAA-1097TM to the isolates located in genogroup I were 97.2–99.2%, whereas with genogroup II 97.2–98.3%. Moreover, the results of phylogenetic analysis showed that the strain ATCC 8071TM (accession no. X82133) representing Owen’s genomic group I was localized in the genogroup I, while the strain ATCC 8073TM (accession no. AJ000213) representing the Owen’s genomic group III was classified into genogroup II. Similarity of the reference strain of S. putrefaciens ATCC BAA-1097TM to S. algae ATCC
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Table 3 Antimicrobial susceptibility of Shewanella putrefaciens collected isolates
Antibacterial agents
Shewanella putrefaciens n = 118 The average of the diameter of the inhibition zones (mm)
Tetracycline: Oxytetracycline 18 mm Doxycycline 26 mm Quinolones and fluoroquinolones Nalidixic acid 25 mm Flumequine 30 mm Enrofloxacin 32 mm Norfloxacine 26 mm Sulphonamides and potentiated sulphonamides Sulphonamides 0 mm Sulphamethoxazole/ 25 mm trimethoprim Penicillins Ampicillin 17 mm Amoxicillin 10 mm Amoxicillin and 14 mm clavulanic acid Systemic antibacterial agents Chloramphenicol 35 mm Florfenicol 35 m S, susceptible; I, intermediate susceptible; R, resistant.
Figure 2 Neighbour-joining phylogenetic tree of Shewanella putrefaciens strains based on 16S rRNA gene sequence. Bootstrap values (expressed as percentages of 1000 replicates), bar = 0.001 substitution per nucleotide position. Ó 2014 John Wiley & Sons Ltd
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51192TM (accession no. HM016087), as an outgroup, was 96.57%. Challenge tests
Sensitivity
I S S S S S R S
I R I
S S
No mortality or any disease signs in fish injected with sterile PBS were observed. Common carp infected with the K530 and K717 and rainbow trout with Pt411 strains showed one or more of the following external signs: darkening skin and pale gills, necrotic skin lesions and ulcers (Fig. 3), distended anus. Individual common carp or rainbow trout showed exophthalmia and abdominal distension due to ascitic fluid in the peritoneum (Fig. 4). Intensive congestions and watery of internal organs were mostly observed as well as intensive haemorrhages in the peritoneum and internal organs with significant enlargement of the spleen (Figs 5 & 6). Cumulative mortalities of infected fish were from 20% to 75%, depending on fish species and strain used for challenge experiments (Fig. 7). Shewanella putrefaciens was reisolated in pure cultures from damaged tissues of each fish
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Figure 3 Necrotic lesions and ulcers on the skin in carp after experimentally infected with the K530 strain.
Figure 7 Mortality of carp and rainbow trout Ip infected with Shewanella putrefaciens K530, K717 and Pt411 strains.
Discussion Figure 4 Rainbow trout – exophthalmia and abdomen distension in rainbow trout experimentally infected with the Pt411 strain.
Figure 5 Common carp – congestion of internal organs, enlargement of the kidney after experimentally infected with the K530 strain.
Figure 6 Rainbow trout – intensive haemorrhages in the peritoneum and internal organs and enlargement of the spleen (narrow) after experimentally infected with the Pt411 strain.
tested and from blood of common carp or rainbow trout challenged with the K530, K717 or Pt411 strain, respectively. Ó 2014 John Wiley & Sons Ltd
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Physiological and biochemical analyses demonstrated that collected isolates had phenotypic properties consistent with S. putrefaciens, which was confirmed by 16S rRNA sequencing. This is the first description of S. putrefaciens strains as pathogens of freshwater fish. Abundant and uniform or predominant growth of S. putrefaciens was found in all samples originated from fish showing clinical signs of disease. It is noteworthy that uniform growth of S. putrefaciens was found in some gill samples from affected fish. It shows that under certain circumstances, the bacterium significantly suppresses the growth of other bacteria, which are usually present in this tissue in clinically healthy fish. A similar phenomenon has been previously observed after the experimental infection of fish with S. putrefaciens when pure cultures of the bacterium have been even obtained from the intestinal tract (Kozi nska & Pez kala 2004). These findings suggest that S. putrefaciens has an antagonistic effect on other bacteria. The inhibitory effect of this bacterium on Aeromonas sp. has been found (Goldschmidt-Clermont et al. 2008). The biochemical identification of collected strains of S. putrefaciens, performed on API 20E, API 20NE, as well as enzymatic activity tested on API Zym showed a good discrimination level of the bacteria. On the basis of our studies, we were able to compare API 20E and API 20NE tests, for the first time, in order to choose the best diagnostic methods. Excellent or very good identification levels were obtained in the cases of all examined isolates on API 20E test. One of our API 20E profiles (0702004) corresponded to the
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profile of S. putrefaciens isolated from sea bass (Korun et al. 2009). Similar reactions in API 20E test revealed S. putrefaciens strains originated from loach (Qin et al. 2012). In API 20NE, four bacteria strains (GIII41-12-Poland, St4-12-Poland, Sd4-12-Poland and K575-10-Poland) isolated from brown trout, European whitefish, pike-perch and common carp, respectively, were improperly identified as A. salmonicida subsp. salmonicida (numeric profile 1450744). Moreover, five isolates (Sh4-09-Poland, K680-12-Poland, K679-12Poland, K604-11-Poland and K313-05-Poland) all originated from common carps and were classified as S. putrefaciens, but the result of this identification was indicated as a doubtful (numeric profile 1450754). In order to avoid incorrect identification of S. putrefaciens, diagnosis should be performed very carefully, taking into consideration the morphology of the bacterial cells and their colonies. Our results indicate that S. putrefaciens is more correctly identified by API 20E test, which has been confirmed by other laboratories (Korun et al. 2009; Qin et al. 2012). The vast majority of the phenotypical properties of the tested isolates corresponded with the characteristic of the reference strain ATCC BAA1097TM and the strain of S. putrefaciens isolated by Saeed et al. (1987). API Zym tests showed a high enzymatic activity of collected strains of S. putrefaciens, which can play an important role in the pathogenicity of the bacteria. The ability to produce N-acetyl-bglucosaminidase suggests that these isolates should belong to the genomic group II (Zimke et al. 1998). Antimicrobial susceptibility of S. putrefaciens of collected strains was studied in more extended range than in our previous work (Kozi nska & Pez kala 2004). In the present study, we showed that the tested isolates are still sensitive to drugs from quinolone and fluoroquinolone group. The same observations concerning S. putrefaciens isolated from clinical cases of marine and freshwater fish were also previously described (Korun et al. 2009; Qin et al. 2012). Additionally, our studies confirmed a complete resistance of these bacteria to sulphonamides (Korun et al. 2009). For such reason, this group of drugs should be completely withdrawn from the therapy against shewanellosis. However, the question concerning the use of trimethoprim seems to be reasonable. Some strains showed the resistance to this drug (Korun et al. Ó 2014 John Wiley & Sons Ltd
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2009); meanwhile, all bacteria examined in our study were sensitive to trimethoprim correlated with sulphonamides. Resistance to sulphamethoxazole was observed in our previous research (Kozi nska & Pez kala 2004), but the examination was conducted on a smaller number of isolates. A complete resistance of S. putrefaciens to almost all tested antimicrobial agents was already observed in two cases (data not shown). This suggests development of the resistance mechanisms by the bacterium and therefore a need for a well-thought therapy against shewanellosis, supported by laboratory studies on drug susceptibility. It is known that organisms identified phenotypically as S. putrefaciens group comprise several biovars (Bowman 2005) and are divided into four hybridization groups (Owen et al. 1978). The results of sequencing analysis performed in our study showed that collected strains of S. putrefaciens created two genomic groups. Bacterial strains isolated from farmed fish (common carp and rainbow trout) which caused clinical signs of the diseases were included into our genomic group I. Additionally, phylogenetic analysis showed the similarity of this group to the reference strain ATCC 8071TM, which represents genogroup I by Owen’s hybridization groups (S. putrefaciens sensu stricto) (Owen et al. 1978; Bowman 2005). The correlation between our genomic group II and genogroup III with Owen’s, represented by reference strain ATCC 8073TM, was also confirmed. This branch comprised mostly the isolates collected from wild, ornamental as well as farmed fish, in which only few clinical signs were observed. Our results confirm the previous studies, which suggested that S. putrefaciens species is composed of a series of genetically variable strains (Bowman 2005). Phenotypic properties did not correlate well with the hybridization groups of Owen (Owen et al. 1978; Bowman 2005). According to the ability to produce N-acetyl-b-glucosaminidase, all our collected strains should belong to the genomic group II by Owen’s, which, however, were named as S. baltica (Zimke et al. 1998) or S. algae (Bowman 2005). On the other hand, inability to grow in the presence of 6% NaCl is characteristic for isolates belonging to Gillard biovars 1 and 3, which correlate with genogroups I and III by Owen’s, respectively (Zimke et al. 1998). Strains representing I and III hybridization groups of Owen were originated mostly from food and environment (Zimke et al. 1998), and now the isolates from diseased freshwater fish should be included.
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The virulence of S. putrefaciens for common carp and rainbow trout has previously been documented in experimental challenge tests (Kozi nska & Pez kala 2004). Here, it has been confirmed using three strains isolated from natural diseased fish. Experimental infected common carps and rainbow trout showed clinical signs of disease similar to those observed in the naturally infected fish. Some signs of the disease, such as pale gills, ulcers and spleen enlargement, have been described in the cultured marine fish such as European sea bass (Korun et al. 2009), as well as among the freshwater loach (Qin et al. 2012) and common carps (www.environment-agency.gov.uk). It is worth noting that percentages of mortalities which were estimated 40–85% in different species of fish (Kozi nska & Pez kala 2004; Korun et al. 2009) are similar to our present results. Reisolation of S. putrefaciens from freshly dead experimental fish fulfilled Koch’s postulates. Conclusions
Infections associated with S. putrefaciens appear to be becoming more serious problems in freshwater fish farms, causing even an acute form of the diseases with septicaemia and markedly increased mortalities. The clinical signs of shewanellosis may vary, depending on the strain. The differences between the collected strains were visible on the phenotypic properties, as well as on the molecular level. Diagnosis of S. putrefaciens is not complicated, but it could be problematic. Our results showed that the API 20E tests are most suitable for proper identification of this bacteria. The fulfilled Koch’s postulates presented in this study indicate that the S. putrefaciens should be considered as an opportunistic pathogen, like almost all bacteria, which causes fish disorders. Publication History Received: 3 October 2013 Revision received: 3 January 2014 Accepted: 4 January 2014
This paper was edited and accepted under the Editorship of Professor Ron Roberts.
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