Phenotypic diversity and potential virulence factors of the Shewanella putrefaciens group isolated from freshwater fish

Molecular identification	Symbol of isolate	APINE profile	Fish species	Symptoms of diseases/mortality
S. xiamenensis	K736	1451354	Common carp	Yes/Yes
S. xiamenensis	K733		Common carp	No/Yes
S. xiamenensis	KK3		Koi carp	Yes/Yes
S. xiamenensis	KOI2		Koi carp	Yes/Yes
S. xiamenensis	W24		European eel	Yes/Yes
S. xiamenensis	W28		European eel	Yes/Yes
Shewanella sp.	Jes15		Siberian sturgeon	No/No
S. xiamenensis	St16		Slender krib	Yes/Yes
S. xiamenensis	St8		Slender krib	Yes/Yes
Shewanella sp.	Am6		Grass carp	No/No
S. putrefaciens	K677	1410344	Common carp	No/No
S. putrefaciens	K678		Common carp	No/No
Shewanella sp.	K769		Common carp	Yes/No
S. oneidensis	Pt400		Rainbow trout	nd/Yes
S. putrefaciens	Pt411		Rainbow trout	Yes/Yes
S. putrefaciens	K313	1450754	Common carp	Yes/Yes
S. putrefaciens	K530		Common carp	Yes/Yes
S. putrefaciens	K604		Common carp	Yes/Yes
S. putrefaciens	K679		Common carp	No/No
S. putrefaciens	K680		Common carp	No/No
Shewanella sp.	K575	1450744	Common carp	Yes/Yes
S. putrefaciens	GIII41(3)		Brown trout	Yes/Yes
S. putrefaciens	St4		Rainbow trout	Yes/Yes
S. putrefaciens	Tp5	1410354	Silver carp	Yes/Yes
S. oneidensis	Plo1		Common roach	No/No
S. oneidensis	Jes34		Siberian sturgeon	No/No
S. putrefaciens	Tsk20	1410754	Brown trout	Yes/Yes
S. putrefaciens	St5		European whitefish	Yes/Yes
S. putrefaciens	E6		Rainbow trout	No/No
S. putrefaciens	Sh1	1411754	Common carp	Yes/Yes
S. putrefaciens	Sh2	1411754	Common carp	Yes/Yes
Shewanella sp.	Jes1	1451754	Siberian sturgeon	No/No
S. putrefaciens	Sh4		Common carp	Yes/Yes
S. putrefaciens	Jaz4		Ide	No/No
S. putrefaciens	TIII	1411744	Brown trout	Yes/Yes
S. putrefaciens	K869	1411744	Common carp	Yes/Yes
S. oneidensis	K696	1450344	Common carp	Yes/Yes
S. putrefaciens	E9	1010344	Rainbow trout	No/No
S. putrefaciens	E3	1011754	Rainbow trout	No/No
S. xiamenensis	16/c/29	1051344	Zebrafish	Yes/Yes
S. putrefaciens	W9	1400354	European eel	Yes/Yes
Shewanella sp.	St15	1411344	Slender krib	Yes/Yes
S. putrefaciens	K751	1450354	Common carp	No/No
S. putrefaciens	GIII46(3)	1451344	Brown trout	Yes/Yes

[i] nd – no data

Table 2

Biochemical properties and growth condition of S. putrefaciens group isolates (n = 44) and reference strains

S. putrefaciens isolates
Biochemical properties	S. xiamenensis (n = 9)	S. oneidensis (n = 4)	S. putrefaciens (n = 25)	Shewanella sp. (n = 6)	ATCC BAA-1097	ATCC 8071	ATCC 8073	ATCC 700550	JCM 16212^T
Ornithine decarboxylase	6	1	14	5	-	-	−	+	+
Citrate utilisation	2	0	4	2	+w	+w	−	+	+
Acid from arabinose	9	0	3	1	+	+	+	−	+
Aesculin hydrolysis	9	4	23	6	+w	+w	+	+	+
Assimilation:
Glucose	8	2	12	0	̶	−	+	−	+
Arabinose	9	0	8	1	−	+	+	−	+
Potassium gluconate	0	0	18	1	−	−	−	−	−
Capric acid	7	1	16	0	+	+	−	−	+
Malic acid	9	4	25	5	+	+	−	−	+
Growth condition NaCl range (%)
0.0–2.0	9	4	25	6	+	+	+	+	+
3.0	9	3	25	5	+	+	+	+	+
4.0	5	0	25	1	+	+	+w	+	+
5.0	3	0	24	1	+w	−	+w	+w	+w
6.0	0	0	0	0	−	−	−	−	−

[i] w – weak reaction; ATCC BAA-1097, ATCC 8071, ATCC 8073 0150 – S. putrefaciens; ATCC 700550 – S. oneidensis; JCM 16212^T – S. xiamenensis

Table 3

Matrix showing homologous titres for serological variant definition (upper part) and Shewanella distribution in defined variants (lower part)

							Antisera*
Titre		S-K604	S-K575	S-St5	S-St16	S-K530	S-ATCC BAA 1097	S-K696	S-KK3	S-Am6	S-TIII	S-ATCC 8071	S-Sh1	S-K678	S-St8
antigens	A-K604	2,560	0	0	0	0	0	0	0	0	0	0	0	0	0
	A-K575	40	2,560	0	0	0	0	0	0	0	0	0	0	0	0
	A-St5	0	0	2,560	0	0	0	0	0	0	0	0	0	0	0
	A-St16	0	0	0	2,560	0	0	0	0	0	0	0	0	0	0
	A-K530	0	0	0	0	2,560	0	0	0	0	0	0	0	0	0
	A- ATCC BAA 1097	0	0	0	0	0	2,560	0	0	0	0	0	160	0	0
	A-K696	0	0	0	0	0	0	1,280	0	0	0	0	0	0	0
	A-KK3	0	0	0	0	0	160	0	1,280	0	0	0	0	0	0
	A-Am6	0	0	0	0	0	0	0	0	1,280	0	0	0	0	0
	A-TIII	0	0	0	0	0	0	0	0	0	1,280	0	0	0	0
	A-ATCC 8071	0	0	0	0	0	0	0	0	0	0	1,280	0	0	0
	A-Sh1	0	0	0	0	0	0	0	0	0	0	0	640	0	0
	A-K678	0	0	0	0	0	0	0	0	0	0	0	0	320	0
	A-St8	0	0	0	0	0	0	0	0	0	0	0	0	0	160
Variant		I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII	XIV
isolates	ID	K604 ATCC 8073 Tsk20	K575 E9, Tp5 K313 K677	St5 W24 W28	St16	K530	ATCC BAA 1097	K696 Jes1 K733 K736	KK3 St15 Pt400 Plo1	Am6 Jes15	TIII E6 E3 Jaz4	ATCC 8071	Sh1 KOI2 Sh4	K678	St8
	n**	3	5	3	1	1	1	4	4	2	4	1	3	1	1
	%	6%	11%	6%	2%	2%	2%	9%	9%	4%	9%	2%	6%	2%	2%

[i] * only antisera used for serological variant identification are listed; sera showing multiple heterologous reaction were omitted (n = 9; S-GEIII46(3), S-Pt411, S-St4, S-St15, S-K679, S-K751, S-K869, S-ATCC 8073, S-KOI2); ** 13 (28%) isolates remained unrecognised due to reactions of their antigens with multiple sera: autoagglutination/rough antigen structure (n = 8; GEIII46(3), Pt411, St4, K869, K769, 16/c/29, GEIII41(3), W9) or no reaction with relevant sera (n = 5; K679, K751, Jes34, Sh2, K680)

Table 4

Distribution of inhibition zone diameter values of different antimicrobial agents for the Shewanella isolates (n = 44)

	Inhibition zone diameter (mm)
Antimicrobial (concentration)	Bacteria species		7	11	14														30
		6	-	-	-	17	18	19	20	21	22	23	24	25	26	27	28	29	-	>35	Criteria*	%NWT
	Shewanella		10	13	16														35
sulphonamides (300μg)	putrefaciens (n = 25)	24		1																	n.a.	n.d.
	xiamenensis (n = 9)	9																				n.d.
	oneidensis (n = 4)	4																				n.d.
	sp.** (n = 6)	6																				n.d.
sulphamethoxazole/ trimethoprim (23.75/1.25μg)	putrefaciens (n = 25)	1							2		3		1	5	3	2	4		4		NWT≤19 mm	4
	xiamenensis (n = 9)	3									1			2	1	1			1			33
	oneidensis (n = 4)										1			1		1			1			0
	sp.** (n = 6)											1	1	1				1	2			0
oxolic acid (2μg)	putrefaciens (n = 25)	2								3	5	1	1	7	3	1	2				NWT ≤29 mm	100
	xiamenensis (n = 9)	6									1				1	1						100
	oneidensis (n = 4)								1					1			1		1			75
	sp.** (n = 6)										1						1		4			33
flumequine (30μg)	putrefaciens (n = 25)				1										1	1	5		16	1	n.a.	n.d.
	xiamenensis (n = 9)	5										1				1			1	1		n.d.
	oneidensis (n = 4)													1					3			n.d.
	sp.** (n = 6)														1				4	1		n.d.
enrofloxacin (5μg)	putrefaciens (n = 25)			1			1		2		1	1	1	2	3				12	1	n.a.	n.d.
	xiamenensis (n = 9)	3							2								1		3			n.d.
	oneidensis (n = 4)														1				3			n.d.
	sp.** (n = 6)													1					5			n.d.
norfloxacine (10μg)	putrefaciens (n = 25)	2							3		3	1		3	2	1	3		7		n.a.	n.d.
	xiamenensis (n = 9)	4		1										1		1			2			n.d.
	oneidensis (n = 4)								1					1		1			1			n.d.
	sp.** (n = 6)													1		1				4		n.d.
oxytetracycline (30μg)	putrefaciens (n = 25)	1			1		4	1	7	4	4		1	2							NWT ≤27 mm	100
	xiamenensis (n = 9)	6		2	1																	100
	oneidensis (n = 4)	2					1		1													100
	sp.** (n = 6)	1		1			2		1					1								100
doxycycline (30μg)	putrefaciens (n = 25)				2		2		6		3		2	2	1		1		5	1	n.a.	n.d.
	xiamenensis (n = 9)	2		1	5	1																n.d.
	oneidensis (n = 4)			1	1				1				1									n.d.
	sp.** (n = 6)	1			1				1		1				1	1						n.d.
florphenicol (30μg)	putrefaciens (n = 25)																5		16	4	NWT ≤26 mm	0
	xiamenensis (n = 9)																		8	1		0
	oneidensis (n = 4)																		4			0
	sp.** (n = 6)																2		1	3		0

[i] *NWT criteria for Aeromonas salmonicida according to CLSI, ** isolates not classified to species level, NWT – non-wild-type,

%NWT – the number (%) of isolates potentially resistant to antimicrobial agents, n.a. – not available, n.d. – no data

Adhesion ability of *S. putrefaciens* group to different fish cell lines
The average number of adherent bacteria (CFU) from two repetitions is shown. The standard errors of means were less than 10% of mean values in each case. Cell lines used: EPC – epithelioma papulosum cyprini, BF-2 – bluegill fry, CHSE – chinook salmon embryo, RTG-2 – rainbow trout gonad

Congestion of internal organs in common carp

Exophthalmia, lesions on gill cover (A) and pale liver (B) in rainbow trout

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DOI: https://doi.org/10.2478/jvetres-2019-0046 | Journal eISSN: 2450-8608 (formerly 2300-3235)

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Language: English

Page range: 321 - 332

Submitted on: Mar 7, 2019

Accepted on: Jul 30, 2019

Published on: Sep 13, 2019

Published by: National Veterinary Research Institute in Pulawy

In partnership with: Paradigm Publishing Services

Keywords:

bacterial fish diseases,

Related subjects:

Microbiology and virology,

Life sciences, other,

Medicine,

Veterinary medicine

© 2019 Ewa Paździor, Agnieszka Pękala-Safińska, Dariusz Wasyl, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Volume 63 (2019): Issue 3 (September 2019)

Phenotypic diversity and potential virulence factors of the Shewanella putrefaciens group isolated from freshwater fish

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Table 4

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