Open Access

Clustering subspecies of Aeromonas salmonicida using IS630typing

  • Nicole Studer1,
  • Joachim Frey1Email author and
  • Philippe Vanden Bergh1
Contributed equally
BMC Microbiology201313:36

DOI: 10.1186/1471-2180-13-36

Received: 19 November 2012

Accepted: 5 February 2013

Published: 13 February 2013

Abstract

Background

The insertion element IS630 found in Aeromonas salmonicida belongs to the IS630-Tc1-mariner superfamily of transposons. It is present in multiple copies and represents approximately half of the IS present in the genome of A. salmonicida subsp. salmonicida A449.

Results

By using High Copy Number IS630 Restriction Fragment Length Polymorphism (HCN-IS630-RFLP), strains of various subspecies of Aeromonas salmonicida showed conserved or clustering patterns, thus allowing their differentiation from each other. Fingerprints of A. salmonicida subsp. salmonicida showed the highest homogeneity while ‘atypical’ A. salmonicida strains were more heterogeneous. IS630 typing also differentiated A. salmonicida from other Aeromonas species. The copy number of IS630 in Aeromonas salmonicida ranges from 8 to 35 and is much lower in other Aeromonas species.

Conclusions

HCN-IS630-RFLP is a powerful tool for subtyping of A. salmonicida. The high stability of IS630 insertions in A. salmonicida subsp. salmonicida indicates that it might have played a role in pathoadaptation of A. salmonicida which has reached an optimal configuration in the highly virulent and specific fish pathogen A. salmonicida subsp. salmonicida.

Keywords

Aeromonas salmonicida HCN-IS630-RFLP IS element Subtyping Tc1 Mariner transposon Salmonidae Pathoadaptation

Background

Aeromonas salmonicida is one of the predominant bacterial species found in fish and water samples [1]. While some Aeromonas species are able to cause opportunistic disease in warm- and cold blooded vertebrates, A. salmonicida seems to be specific for fish. Aeromonas salmonicida subsp. salmonicida a specific primary pathogen of Salmonidae (salmon, trout and char) has been known for decades to cause furunculosis. This bacterial septicaemia has a significant economic impact on aquaculture operations as well as on the wild stock of salmonids and some other fish species [2]. Bergey’s Manual of Systematic Bacteriology recognizes five subspecies of A. salmonicida: salmonicida, achromogenes, smithia, pectinolytica and masoucida[3]. Aeromonas salmonicida subsp. salmonicida is referred to as typical Aeromonas salmonicida by reason that these strains are very homogeneous and considered to be clonal [4, 5]. Clinical strains that cannot be assigned to any of the known subspecies are referred to as A. salmonicida ‘atypical’. In recent years, it has been recognized that ‘atypical’ strains cause diseases in salmonidae and other fish species that differ from furunculosis. Therefore their importance is being increasingly recognized. The most common clinical manifestation observed, following infections with such strains, is chronic skin ulceration [6]. Due to a convoluted history of nomenclature and taxonomy of Aeromonas sp., clear assignment of strains using currently available methods remains sometimes confusing and controversial which makes epidemiological studies difficult [7]. Intraspecies phenotypic variability also makes phenotypic identification challenging on the species level [8]. A variety of molecular genetic methods have been employed for genetic classification of Aeromonads including mol% G + C composition, DNA-DNA relatedness studies, restriction fragment length polymorphism, pulsed-field gel electrophoresis, plasmid analysis, ribotyping, multilocus sequence typing, PCR and more [3, 5]. Combination of 16S rDNA-RFLP analysis and sequencing of the gene rpoD was proposed as a suitable approach for the correct assignment of Aeromonas strains [9]. Moreover, analyzing strains by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) with an extraction method revealed 100% genus-level accuracy and 91.4% accuracy at species level [10]. However, this method was not able to discriminate A. salmonicida at the subspecies level.

Currently, no molecular approach gives a clear genotypic distinction of strains among A. salmonicida species. For this reason we elaborated a molecular genetic technique to achieve an adequate subtyping of all Aeromonas salmonicida subspecies. This method, named High Copy Number IS-Element based Restriction Fragment Length Polymorphism (HCN-IS-RFLP), has been successfully applied in numerous epidemiological studies for other pathogenic bacteria [1115].

Results

Optimization of HCN-IS630-RFLP conditions

IS630 was selected because it is the IS element with the highest copy number in the genome of A. salmonicida[16]. Primers internal to the highly conserved IS630 genes [GenBank: ABO88357.1] were designed to generate a probe on an intact IS fragment from the A. salmonicida subsp. salmonicida JF2267 genome. To obtain the most distinct banding pattern, the digestion by several restriction enzymes on a set of sequenced genomes (A. salmonicida subsp. salmonicida A449, A. hydrophila ATCC7966 and A. veronii B565) was predicted by computer analysis. XhoI that does not cut within our probe for IS630 revealed a good resolution with a clear banding pattern and was therefore selected. A size window of 1375 bp to 21226 bp was defined on all southern blots as some hybridizing patterns with very large or small fragments were not sufficiently resolved (Figure 1). The genomic DNA sequence of A. salmonicida strain A449 [GenBank: CP000644.1] predicted that the probe would hybridize with 35 copies of IS630 on XhoI fragments ranging from 1277 bp to 17948 bp (Additional file 1: Table S1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-13-36/MediaObjects/12866_2012_Article_1923_Fig1_HTML.jpg
Figure 1

Southern blot of Xho -I digested DNAs from different A. salmonicida strains hybridized with an IS 630 -specific probe. Lanes 1 and 16, molecular size marker (sizes are indicated on the left in kilobase pairs); lanes 2 to 5 and 11, A. salmonicida subsp. salmonicida (JF2267, JF3224, JF3996, JF3507, JF3121 [formerly identified as atypical]); lanes 6 to 8 and 13, A. salmonicida subsp. achromogenes (JF3115, JF3116, JF2997, JF3123 [formerly identified as atypical]); lane 9, A. salmonicida subsp. pectinolytica (JF3120); lane 10, A. salmonicida subsp. masoucida (JF3118); lanes 12, 14 and 15, A. salmonicida atypical (JF3122, JF3124, JF3125).

We analyzed the IS630 RFLP-fingerprints of 87 Aeromonas sp. strains of various geographical origins, which comprised 31 A. salmonicida subsp. salmonicida, 4 subsp. achromogenes, 4 subsp. smithia, 2 subsp. masoucida, one subsp. pectinolytica, 12 A. salmonicida atypical strains, 8 A. popoffii, 5 A. sobria and A. bestiarum, 2 A. hydrophila, one A. trota, A. enteropelogenes, A. simiae, A. eucrenophila, A. ichthiosmia, A. jandaei, A. molluscorum, A. bivalvium, A. allosaccharophila, A. media, A. veronii, A. caviae and A. culicicola (Table 1). The fingerprints (Figure 1) of the analyzed strains were subjected to similarity analysis and are shown in Figure 2.
Table 1

Aeromonas strains used in this study

JF N°

Synonyme

Species

Subspecies

Origin

Identified virulence characteristics

Pigment production (Day 6)

Ref

JF2996

Austin98

salmonicida

salmonicida

Sediment in Riccarton Loch, Scotland

ascV-, ascU-, aexT+, aopP+, aopO-, aopH+

+++

[17, 18]

JF3507

ATCC 33658 T, NCIMB 1102 T

salmonicida

salmonicida

Salmo salar, Scotland

ascV-, aexT+, aopP+, aopO+, aopH+, acrD-

+++

[1820]

JF3327

F330/04

salmonicida

salmonicida

Arctic char, Switzerland, 2004

ascV+, aexT+, aopP+, aopO+, aopH+

++++

[18]

JF3517

4757

salmonicida

salmonicida

Turbot, Norway

ascV+, aexT+, aopP+, aopO+, aopH+

++++

[18]

JF2267

Fi 94 G

salmonicida

salmonicida

Arctic char, Switzerland, 1999

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+, acrD+

+++

[17, 18, 20]

JF2869

CCUG 47405 (A)

salmonicida

salmonicida

Arctic char (Savelinus alpinus)

aexT+, SacrD 3 +

++++

-

JF3223

Fi 210

salmonicida

salmonicida

White fish, Switzerland, 1997

ascV+, aexT+, aopP+, aopO+, aopH+

++++

[18]

JF3224

R04/170

salmonicida

salmonicida

Brown trout, Switzerland, 2004

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+

++++

[17, 18]

JF3518

4704

salmonicida

salmonicida

Turbot, Norway

ascV+, aexT+, aopP+, aopO+, aopH+

++++

[18]

JF2509

CC72 - D640

salmonicida

salmonicida

Atlantic salmon, Canada, before 1960

ascV+, aexT+, aopP+, aopO+, aopH+, acrD+

++++

[18, 20]

JF3519

3294

salmonicida

salmonicida

Arctic char, Switzerland, 1986

ascV-, aexT+, aopP+, aopO+, aopH-

++++

[18]

JF2506

CC 27–80/9-1

salmonicida

salmonicida

Atlantic salmon Norway

ascV+, aexT+, aopP+, aopO+, aopH+, acrD+

++++

[18, 20]

JF2507

CC 29 - 74/2

salmonicida

salmonicida

Atlantic salmon, Scotland

ascV+, aexT+, aopP+, aopO+, aopH+, acrD+

++++

[18, 20]

JF2508

CC 63- D-615

salmonicida

salmonicida

Atlantic salmon, Canada

ascV+, aexT+, aopP+, aopO+, aopH+, acrD+

++++

[18, 20]

JF2510

CC 23/8019-5

salmonicida

salmonicida

Atlantic salmon Norway

ascV+, aexT+, aopP+, aopO+, aopH+, acrD+

++++

[18, 20]

JF3521

2265

salmonicida

salmonicida

Wild atlantic salmon, Norway 1991

ascV-, aexT+, aopP+, aopO+, aopH-

++++

[18]

JF3496

F05/160

salmonicida

salmonicida

Wild brown trout, Switzerland, 2005

ascV+, aexT+, aopP+, aopO+, aopH+

+++

[18]

JF3844

F06/417

salmonicida

salmonicida

Arctic char, Switzerland, 2006

ascV+, aexT+, aopP+, aopO+, aopH+

+++

[18]

JF2505

MT 44/SS 10

salmonicida

-

non virulent for trout, Canada

A+, LPS+, acrD-

+++

[20]

JF3791

F06/385

salmonicida

-

Arctic char Salvelinus alpinus, Switzerland, 2006

ascV+, aexT+, aopP-aopO+, aopH+

+++

[18]

JF4111

F07/357(NiA)

salmonicida

-

Salvelinus, Switzerland, 2007

ND

+++

-

JF4112

F07/357 (NiB)

salmonicida

-

Salvelinus, Switzerland, 2007

ND

+++

-

JF4113

F07/357 (NiC)

salmonicida

-

Salvelinus, Switzerland, 2007

ND

+++

-

JF3121

As209

salmonicida

salmonicida [formerly atypical]

Wolf fish, UK

ascV-, ascU-

+++

[17]

JF4714

IMD1520

salmonicida

-

Thymallus thymallus (skin), Switzerland, 2009

ND

+++

-

JF4715

IMD 1521

salmonicida

-

Thymallus thymallus (kidney), Switzerland, 2009

ND

+++

-

JF4114

F07/357(LeA)

salmonicida

-

Salvelinus (liver), Switzerland, 2007

ND

+++

-

JF4115

F07/357 (LeB)

salmonicida

-

Salvelinus (liver), Switzerland, 2007

ND

+++

-

JF4116

F07/357 (LeC)

salmonicida

-

Salvelinus (liver), Switzerland, 2007

ND

+++

-

JF4118

F07/(MiB)

salmonicida

-

Salvelinus (kidney), Switzerland, 2007

ND

+++

-

JF4119

F07/357 (MiC)

salmonicida

-

Salvelinus (kidney), Switzerland, 2007

ND

+++

-

JF4117

F07/357 (MiA)

salmonicida

salmonicida

Salvelinus (spleen), Switzerland, 2007

ND

++++

-

JF3122

As204

salmonicida

atypical

Wrasse UK

ascV+, aexT+, aopP + aopO-, aopH+

++

[18]

JF3500

aAs 4143

salmonicida

atypical

Atlantic cod, Norway

ascV+, aexT+, aopP + aopO+, aopH-

++

[18]

JF3666

F06/211

salmonicida

atypical

Bleak (Alburnus alburnus), Switzerland, 2006

ascV+, aexT+, aopP- aopO-, aopH+

-

[18]

JF3124

As93

salmonicida

atypical

Plaice, Denmark

ascV+, aexT+, aopP + aopO+, aopH+

-

[18]

JF3520

4818

salmonicida

atypical

Atlantic Halibut, Norway, 2003

ascV+, aexT-, aopP + aopO-, aopH+

-

[18]

JF3115

ATCC 19261, NCIMB 1109

salmonicida

achromogenes

Salmo trutta

ND

+

-

JF3116

NCIMB 1110 T

salmonicida

achromogenes

Trout, Scotland

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+

++

[1719]

JF2997

F-265/87

salmonicida

achromogenes

Atlantic salmon, Iceland

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+

++

[17, 18]

JF3123

As183

salmonicida

achromogenes [formerly atypical]

Arctic char, Iceland

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+

++

[17, 18]

JF3499

aAs4101

salmonicida

achromogenes

Atlantic Cod, Iceland

ascV+, aexT+, aopP + aopO+, aopH-

-

[18]

JF3125

As 51

salmonicida

atypical

Rainbow trout, Norway

ascV+, aexT+, aopP- aopO+, aopH+

-

[18]

JF4097

-

salmonicida

smithia

Salvelinus alpinus lepeschini, Austria

ascV+, aexT+, aopP+, aopO-, aopH+

-

[21]

JF4460

-

salmonicida

smithia

Salvelinus alpinus lepeschini, Austria

ascV-, aexT+, aopP+, aopO-, aopH+

-

[21]

JF4439

-

salmonicida

smithia

Salvelinus alpinus lepeschini, Austria

ascV+, aexT+, aopP+, aopO-, aopH+

-

[21]

JF3117

NCMB13210, ATCC 49393

salmonicida

smithia

Roach, England

ascV+, ascU+, aexT+, aopP-, aopO+, aopH+

-

[1719]

JF3126

As 54

salmonicida

atypical

Rainbow trout, Norway

ascV-, aexT+, aopP-, aopO-, aopH-

++

[18]

JF3502

aAs 4067

salmonicida

atypical

Spotted wolffish, Norway

ascV+, aexT+, aopP+, aopO+, aopH+

+

[18]

JF3118

ATCC 27013 T

salmonicida

masoucida

Salmon, Japan

ascV+, ascU+, aexT+, aopP-, aopO-, aopH+

-

[1719]

JF3119

NCMB 2020

salmonicida

masoucida

same as ATCC 27013 (salmon, Japan)

ND

-

-

JF2512

CC 30/8038

salmonicida

atypical

Atlantic salmon, Canada, before 1960

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+, acrD+

-

[17, 18, 20]

JF2513

CC 34/8030

salmonicida

atypical

Atlantic salmon, Canada, before 1960

ascV+, ascU+, aexT+, aopP+, aopO+, aopH+, acrD+

-

[17, 18, 20]

JF3328

848 T

molluscorum

-

Type strain

ND

-

[22]

JF3071

ATCC 51106, bg sobria HG8

veronii

-

?

ND

-

[19]

JF2635

429/01 # 1c; official JF2635

sobria

-

Perca fluviatilis, Switzerland, 2001

ascV+, ascU+, acrD+

-

[17]

JF3326

-

popoffii

-

Urinary tract infection, France

ND

-

[23]

JF3120

DSM 12609 T

salmonicida

pectinolytica

River water

ascV-, aexT-, aopP-, aopO-, aopH-

++++

[17, 19]

JF3240

LMG 17542, IK-B-r-15-1

popoffii

-

Drinking water production plant, Belgium

ND

-

[24]

JF2796

CECT 4199

allosaccharophila

-

Type strain

ND

-

[19]

JF3242

LMG 17547, AG-9

popoffii

-

Drinking water treatment plant, Scotland

ND

-

[24]

JF2797

LMG 17541T, IK-0-a-10-3

popoffii

-

Drinking water production plant, Belgium

ND

-

[19, 24]

JF3241

LMG 17544, IK-E-a- 14- 1

popoffii

-

Drinking water production plant, Belgium

ND

-

[24]

JF2905

Fi 125

sobria

-

Perch

ascV+

-

[25]

JF2791

ATCC 33907

media

-

Type strain NENT Nr. 2346-98

ascV+, ascU+

-

[17, 19]

JF2899

F86/03-2

sobria

-

Perch

ascV+

-

[25]

JF2806

F533E

popoffii

-

Tap water, Switzerland, 2003

ND

-

[19]

JF2808

F600C

popoffii

-

Tap water, Switzerland, 2003

ND

-

[19]

JF2807

F548B

popoffii

-

Tap water, Switzerland, 2003

ND

-

[19]

JF 3954

868ET

bivalvium

-

Bivalve molluscs; Type strain

ND

-

[26]

JF2637

Fi 303

hydrophila

-

Ornamental fish

ND

-

-

JF2794

ATCC 49657, NENT Nr.2360-98

trota (enteropelogenes)

-

Human feces, India

ND

-

[19]

JF2785

CDC 9533-76

bestiarum

-

Type strain NENT Nr: N2341-98

ND

-

[19]

JF 4032

A28)A28B/1-1

bestiarum

-

Wild perch (Perca fluviatilis), Switzerland, 2007

ND

-

-

JF 4608

A28) 28B/1-1

bestiarum

-

Wild perch, Switzerland, 2009

ascV+

-

[22]

JF2804

F 530 D

bestiarum

-

Tap water

ND

-

-

JF3018

68

bestiarum

-

River water

ND

-

-

JF3070

S 6874 T

simiae

-

Type strain

ND

-

[19]

JF2786

ATCC 15468

caviae

-

Type strain NENT Nr. N2344-98

ND

-

[19]

JF2789

ATCC 7966

hydrophila

-

Type strain NENT Nr. : N2339-98

ND

-

[19]

JF2793

CIP 7433; ATCC 43979

sobria

-

Type strain NENT Nr.2352

ND

-

[19]

JF2929

Fi 179a

sobria

-

Perch, Switzerland

ascV + SacrD+

-

[22]

JF2788

NCMB 74; ATCC 23309

eucrenophila

-

Type strain NENT Nr. N2348-98

ND

-

[19]

JF3069

ATCC 49904 T

ichthiosmia

-

Type strain Antonella Demarta

ND

-

-

JF2790

ATCC 49568

jandaei

-

Type strain NENT Nr. 2355-98

ND

-

[19]

JF3067

CIP 107763 T

culicicola

-

Type strain

ND

-

[19]

JF3068

ATCC 49803 T

enteropelogenes

-

Type strain

ND

-

-

ND: not determined.

HCN-IS630-RFLP profiles and stability of IS630insertions

A high degree of IS630 polymorphism, both in a numerical and positional sense, was observed between the various A. salmonicida subspecies (Figure 1). However, the patterns revealed that IS630 copy numbers and positions are well conserved within the given subspecies (Figure 1). The dendogram in Figure 2 is a RFLP tree that reveals the evolutionary relationship between strains analyzed. Strains of the subspecies salmonicida, smithia, achromogenes and masoucida each grouped together showing a similar banding pattern. The number of IS630-positive bands varied from 27 to 35 in A. salmonicida subsp. salmonicida, 23 to 33 in achromogenes and 19 to 21 in smithia. Within a subspecies, several bands were conserved: 21 in salmonicida, 20 in achromogenes and 13 in smithia subspecies. About 15 distinct patterns were observed in A. salmonicida subsp. salmonicida without showing geographical association. The IS630 pattern of A. salmonicida subsp. salmonicida strain A449 as calculated from the genome sequence data closely clusters with these 15 patterns. In contrast, each pattern in the achromogenes cluster was different. In A. salmonicida subsp. masoucida 15 to 21 positive bands were detected and only 8 in the subspecies pectinolytica. Even though the copy numbers vary within the subspecies, the patterns form clusters for each subspecies. The most remarkable tight clustering was found for A. salmonicida subsp. salmonicida. This latter presents IS630 patterns that only show minute differences among strains that were isolated from various continents and over a period of half a century. No pattern was specific of a given geographical region. The results showed also that strains JF3121 and JF3123, formerly classified as A. salmonicida atypical, clustered with A. salmonicida subsp. salmonicida (JF3121) and subsp. achromogenes (JF3123) (Figures 1 and 2) showing that they were misclassified previously.

The IS630 pattern of A. salmonicida subsp. salmonicida strain JF 2267 that was subcultured for 4 days at 18°C and 25°C (in stressing conditions) to reach approximately 20 generations remained unchanged (results not shown) indicating a good stability of IS630 under experimental growth conditions.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-13-36/MediaObjects/12866_2012_Article_1923_Fig2_HTML.jpg
Figure 2

Dendogram generated from the IS 630 -RFLP patterns of the 87 Aeromonas strains used in this study. The tree was established by using the UPGMA clustering analysis with the BioNumerics software. In red (), the A. salmonicida subsp. salmonicida cluster; in green (), the A. salmonicida subsp. achromogenes cluster; in blue (), the A. salmonicida subsp. smithia cluster; in pink (➜), the A. salmonicida subsp. masoucida cluster; and in brown (), A. popoffii strains clustering together.

Copy number of the IS630 element and RFLP among other Aeromonasspecies

Other Aeromonas species revealed lower copy numbers of IS630: 5 in A. molluscorum, 5 to 8 in clinical A. sobria strains, 9 in A. veronii, 5 in A. allosaccharophila and A. media. Only one copy was found in A. bivalvium and a clinical strain of A. hydrophila. No signal for IS630 was obtained in A. caviae, A. trota, A. simiae, A. eucrenophila, A. ichthiosmia, A. jandaei, A. culicicola, A. enteropelogenes, A. bestiarum and the type strains of A. hydrophila and A. sobria. Among the 8 strains of A. popoffii we found 6 very distinct patterns.

Analysis of IS630 abundance, localization and impact on the genome of Aeromonasspecies

In order to study the origin of IS630 in A. salmonicida, we performed a profound analysis and comparison of published Aeromonas genomes (Additional file 2: Table S2). The genetic environment of IS630 copies in the A. salmonicida subsp. salmonicida A449 genome is shown in detail in Additional file 1: Table S1. About 148 loci or DNA sequences forming 108 complete or partial IS units were found in the chromosome of A. salmonicida subsp. salmonicida A449 and on the plasmids pASA4/pASA5 [GenBank: CP000644.1, CP000645.1 and CP000646.1]. IS630 (referred to as ISAs4 in the Genbank genome annotation of A. salmonicida A449 and as ISAs7 in the corresponding manuscript [16]) was found to be present in 38 copies and was the most abundant family representing 35% of transposons in A. salmonicida A449 (Figure 3, Additional file 3: Table S3). The different copies are well-conserved and show 98% nucleotide sequences identity. The other 70 IS elements are ISAs7 (13%), ISAs5 (11%), ISAs6 (6%), ISAs11 (6%), ISAs2 (5%), ISAs9 (4%), ISAs8 (4%), and unclassified ISAs (16%) (Figure 3). 90% of the IS630 copies reside in chromosomal regions that are specific to A. salmonicida subsp. salmonicida and were not found in other Aeromonas. Interestingly most of these loci correspond to known genes in bacterial genera other than Aeromonas. This is the case for instance for the hypothetical gene ASA_1385 (homology to VOA_002034 of Vibrio sp. RC586) that is directly linked to IS630 in A. salmonicida subsp. salmonicida and is not found in other Aeromonads (Additional file 2: Table S2). In ISAs families other than IS630, 34 (31%) are directly adjacent to IS630 showing that 66% of A. salmonicida A449 transposons are associated to genomic domains of variability. In comparison to other Aeromonas sp., A. salmonicida A449 contains 4 to 54 fold more transposases (Figure 3) which are not responsible for a genome-reductive evolution [27] because the total number of ORFs is stable in comparison to other Aeromonads (Figure 4). However they explain the high abundance of pseudogenes (170) in A. salmonicida subsp. salmonicida[16] in contrast to A. hydrophila ATCC 7966 which only contains 7 pseudogenes and 2 transposases.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-13-36/MediaObjects/12866_2012_Article_1923_Fig3_HTML.jpg
Figure 3

Number of transposases and IS family affiliation in Aeromonas sp. A. salmonicida A449 [GenBank: CP000644.1, CP000645.1 and CP000646.1], A. hydrophila ATCC 7966 and SSU [GenBank: CP000462.1 and AGWR00000000.1], A. caviae Ae398 [GenBank: CACP00000000.1], A. veronii B565, AMC34, AMC35, AER39 and AER397 [GenBank: CP002607.1, AGWU00000000.1, AGWW00000000.1, AGWT00000000.1 and AGWV00000000.1], and A. aquarorium AAK1 [GenBank: AP012343.1].

https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-13-36/MediaObjects/12866_2012_Article_1923_Fig4_HTML.jpg
Figure 4

Numerical comparison of common, shared and specific ORFs between several Aeromonas species. The number of ORFs was calculated from Additional file 2: Table S2 without taking into account IS elements, tRNA and rRNA. In dark grey, the number of ORFs that are common among Aeromonas sp. In white, ORFs that are shared with at least one other Aeromonas species. In light grey, ORFs that are unique to the species. A. salmonicida subsp. salmonicida A449 and 01-B526, A. hydrophila ATCC 7966 and SSU, A. caviae Ae398, A. veronii B565, AMC34, AMC35, AER39 and AER397, and A. aquarorium AAK are illustrated in the graph.

Discussion

HCN-IS6110-RFLP has been applied as a standard method to subtype Mycobacterium tuberculosis strains for years [28]. Moreover, RFLP based on IS elements has been employed to type numerous other pathogenic bacteria [14, 15, 2931]. The published genome of A. salmonicida subsp. salmonicida A449 shows numerous IS elements among which 38 belong to the IS630 family [GenBank: CP000644.1]. We therefore used HCN-IS630-RFLP as a new typing methodology for Aeromonas species.

IS630 was present in different copy numbers and integrated at various sites between the different A. salmonicida subspecies. On the other hand banding patterns were conserved within subspecies (Figure 1). HCN-IS630-RFLP revealed that IS630 is abundant in all subspecies of A. salmonicida allowing a good accuracy for genomic fingerprinting. Our results showed that RFLP profiles can be used to distinguish subspecies of A. salmonicida and to differentiate A. salmonicida from other Aeromonas species. They also indicate a high variability among strains of ‘atypical’ A. salmonicida. All strains of yet unclassified ‘atypical’ A. salmonicida consisted of a high number of IS630 copies and were effectively related to the A. salmonicida cluster. Our method demonstrates that such ‘atypical’ strains represent a heterogeneous group that does not fit into the classification of the five described A. salmonicida subspecies. These strains might represent various subtypes of A. salmonicida subsp. salmonicida or novel subspecies of A. salmonicida that have adapted to particular ecological niches or respective hosts. On the other hand, all A. salmonicida subsp. salmonicida isolated since the 1950s and originating from all over the world have very similar patterns, indicating that they form a single clone showing pathoadaptational stability. Altogether, our results confirm those of a previous study comparing genomic profiles of clinical isolates of Aeromonas salmonicida using DNA microarrays [32]. With the origin and intensification of fish farming, genetic rearrangements occurring through IS transposition events could have been responsible for the selection and the emergence of this pathogenic fish specific clone. Such an adaptation process of a pathogenic bacterium towards its host was recently indicated in the Mycoplasma mycoides cluster for Mycoplasma mycoides subsp. mycoides[33]. Moreover, no unique pattern was associated to a specific geographical region of the world and we assume that this could be explained by the dissemination of A. salmonicida subsp. salmonicida strains between aquaculture countries via the intensification of the international trade in farmed salmon or by the natural migration of wild salmons.

Besides the epidemiologic and phylogenetic interests of IS630 fingerprinting to subtype A. salmonicida, we studied the characteristics of this predominant IS element to reveal information concerning the pathoadaptation towards its specific host. Mobile genetic elements can exert different effects on bacterial genomes [11, 3436]. Through such genomic effects, IS630 family has had an impact on the modulation of virulence genes in other bacteria [3743]. In A. salmonicida 90% of the IS630 copies reside in genomic regions that are variable between Aeromonas sp. (Additional file 1: Table S1) and 80% of these sites contain genes that are specific to A. salmonicida and are not encountered in other Aeromonas sp. suggesting that they constitute genomic islands. A part of these coding sequences are phages or hypothetical genes with homologues of characterized sequences in other environmental bacteria: i.e. the ‘Vibrio Seventh Pandemic cluster I’ (VSP-I), genes for the synthesis of polysaccharide capsule, lipopolysaccharide, S-layer, chitinase, cytolytic insecticidal delta-endotoxin, and some effectors (AopO and ApoH) of the type-three secretion system, the major virulence system of the bacterium. Based on these findings we assume that IS630 elements could be used by environmental bacteria to exchange DNA fragments between each other by horizontal transfer. In the genomic islands where IS630 is present, supplementary IS elements can be found, which might serve as hot spots for further insertions. This would allow the transposon and the genomic island to evolve with acquisition of new genes without disruption of existing loci. These observations could explain why the IS630 elements remained stable within the A. salmonicida subsp. salmonicida genome.

Other interesting characteristics of IS elements homologous to IS630 in A. salmonicida suggest that they could play a role in the co-adaptation of the bacterium with its host by trans-kingdom horizontal gene transfers through the bacterial T3SS: (i) such IS630 elements are mostly present in Gram-negative bacteria that use a T3SS, (ii) their expression can be specifically induced or increased when bacteria are in direct contact with host cells [44] and (iii) several IS630 are predicted to be T3SS effectors [45]. The Modlab® T3SS effector prediction software gives for A. salmonicida IS630 a positive output at 0.69 which means, that the IS630 itself is a potential T3SS effector. Hence, when the bacteria colonize the host, the IS630 expression could be induced and they could begin to exert their transposase activity by excising the transposon (composite if associated to adjacent additional DNA fragments) from the bacterial genome. Subsequently, the transposase linked to its transposon could be translocated into the host cell by the T3SS, reach the host genome in the nucleus, and finally perform its transposition.

Bacterial IS630 elements constitute with the Tc1/mariner eukaryotic DNA transposon family, a superfamily [46]. It was demonstrated in vitro that eukaryotic members of this family are able to transpose into prokaryotic genomes [46]. We suppose that the opposite could also be possible as IS630 itself could be translocated via type three secretion system from the pathogen to its host. In this perspective, our assumption could explain how the adaptive horizontal transfer of a bacterial mannanase gene (HhMAN1) into the genome of an invasive insect pest of coffee (Hypothenemus hampei) occurred in the immediate genetic vicinity of a Tc1/mariner transposon [47].

Conclusions

In this study we describe HCN-IS630-RFLP as an adequate method for subtyping A. salmonicida strains and to differentiate A. salmonicida from other Aeromonas species. The high degree of conservation of HCN-IS630-RFLP profiles among strains of A. salmonicida subsp. salmonicida isolated from geographically most distant areas and over the period of half a century shows that practically all copies of IS630 are stably integrated in this pathogen that has a well-defined host range. We therefore conclude that IS630 might have contributed to the pathoadaptation of A. salmonicida to salmonidae and to the emergence of the subtype A. salmonicida subsp. salmonicida.

Methods

Bacterial strains and growth conditions

Aeromonas strains used in this study are listed in Table 1. Bacteria were grown on trypticase soy agar plates at 18°C for 3 to 6 days until sufficient bacteria were available for DNA extraction.

Southern blot analysis with A. salmonicida subsp. salmonicida IS630probe

Total DNA extraction from each strain was performed with the Peqgold Bacterial DNA extraction Kit (Peqlab Biotechnologie, Erlangen, Germany). One microgram of DNA from each sample was digested overnight with XhoI restriction enzyme (Roche Diagnostics, Mannheim, Germany), loaded on a 0.7% agarose gel and subjected to electrophoresis for 4 to 5 hours. On each gel a DIG-labeled DNA Marker (Roche Diagnostics, Mannheim, Germany) and XhoI digested DNA from A. salmonicida subsp. salmonicida JF2267 were loaded for normalization. DNA bands were stained with ethidium bromide for control and transferred onto a nylon membrane (Roche Diagnostics, Mannheim, Germany) with a VacuGene apparatus (GE Healthcare Bio-Sciences). The IS630 probe was prepared by PCR using primers Clust_asa1052_S6 (5- AGGCAGAACTTGGGGTTCTT-3) and Clust_asa1052_R4 (5- ACAAAAGCGGGTTGTCACTC-3) and DNA of A. salmonicida subsp. salmonicida JF2267 as a template. PCR was performed in 30 μL which contained 0.5 μL of Taq DNA polymerase (5 units/μL) (Roche Diagnostics, Mannheim, Germany), 300 nM of each primer, 1.75 mM MgCl2, 200 μM concentrations of each dNTP and 1 μl of the Digoxigenin-11-dUTP (1 nmol/μL) (Roche Diagnostics, Mannheim, Germany). Each reaction involved a denaturing step at 94°C for 5 min followed by 30 cycles of 10 sec at 94°C, 30 sec at 54°C and 60 sec at 72°C and a final extension step of 7 min at 72°C.

Bioinformatic analysis

The hybridization patterns were scanned and the data were analyzed using the BioNumerics software version 6.6 (Applied Maths, Kortrijk, Belgium). Bands automatically assigned by the computer were checked visually and corrected manually. Cluster analysis of the IS-RFLP patterns was done by the unweighted pair group method with average linkages (UPGMA) using the Dice coefficient and the following parameters: 0.5% Optimization, 0% Band filtering, 0.5% Tolerance and ignore uncertain bands.

Prediction of T3SS effectors was performed with the Modlab® online software (http://gecco.org.chemie.uni-frankfurt.de/T3SS_prediction/T3SS_prediction.html) [45].

Stability of IS630 in cultured A. salmonicida subsp. salmonicida

The stability of IS630 under growth conditions in TSB medium was assessed by daily 100x dilution of a culture of strain JF2267 at 18°C and at 25°C during 4 days to reach 20 generations. Every day DNA was extracted from 109 bacteria, digested with XhoI and submitted to southern blot hybridization.

Notes

Abbreviations

HCN-IS-RFLP: 

High copy number insertion element restriction fragment length polymorphism

T3SS: 

Type-three secretion system

UPGMA: 

Unweighted pair group method with arithmetic mean.

Declarations

Acknowledgements

This research was funded by the Swiss National Science Foundation grant no. 31003A-135808.

Authors’ Affiliations

(1)
Institute of Veterinary Bacteriology, University of Bern

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