Microbiological Research 169 (2014) 547–552

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Phenotypic and molecular characteristics of an Aeromonas hydrophila

strain isolated from the River Nile
Beata Furmanek-Blaszk ∗
Department of Microbiology, University of Gdansk, 80-308 Gdansk, Wita Stwosza 59, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Aeromonas hydrophila, an inhabitant of aquatic ecosystems found in most parts of the world, has consid-
Received 14 June 2013 erable virulence potential. The polymerase chain reaction technique was used to assay for the presence
Received in revised form 28 October 2013 of five virulence factor genes: haemolytic toxins aerA and ahh1, elastase ahyB, the enterotoxin act, and
Accepted 5 November 2013
the polar flagella flaA/flaB in the A. hydrophila strain isolated from the River Nile. Drug screening showed
Available online 15 November 2013
high levels of resistance to ␤-lactam antibiotics and tetracycline. Slime production was determined by
the Congo red agar plate test. The isolate produced two restriction enzymes named AehI and AehII which
Keywords:
are isoschizomers of XhoI and StuI respectively. The complete nucleotide sequence of the cryptic plasmid
Virulence properties
Antimicrobial resistance
pAhy2.5 (2524 bp) from this strain was determined. Sequence analysis revealed the presence of two open
Plasmid reading frames (ORFs) encoding putative proteins. The protein coded by ORF1 is homologous with Rep
proteins of plasmids belonging to the pC194 family, which are known to replicate by the rolling-circle
mechanism. The putative double-strand origin of replication and a region with palindromic sequences
that could function as a single-strand origin were detected in pAhy2.5.
© 2013 Elsevier GmbH. All rights reserved.

1. Introduction The objectives of this study were to (i) search for the presence
of the virulence gene in the isolate, (ii) determine the antimicrobial
The genus Aeromonas comprises a collection of Gram-negative sensitivity of the isolate, (iii) test the strain for the presence of site-
bacteria that have been implicated in a wide spectrum of diseases specific restriction endonuclease activity and (iv) investigate the
in both humans and fish (Janda and Abbott 2010). Aeromonads can small plasmid pAhy2.5 present in this strain. This plasmid was fully
be divided into two groups; the first includes the psychotrophic sequenced and further characterized at the genomic level.
Aeromonas, represented by Aeromonas salmonicida and the sec-
ond by the mesophilic Aeromonas such as Aeromonas hydrophila
(Seshadri et al. 2006). Among the species belonging to this genus, 2. Materials and methods
A. hydrophila has attracted attention due to its frequent association
with infection in humans, including septicaemia, wound infec- 2.1. Growth conditions
tions and gastroenteritis. It is a major etiologic agent of motile
aeromonads septicaemia in a variety of aquatic animals. A number The studied A. hydrophila strain was isolated from the River Nile,
of virulence factors derived from A. hydrophila have been proposed near the Aswan Dam, Egypt. Bacterial colonies were grown on Luria
in an effort to explain the pathogenesis of infections due to these Bretani (LB) agar plates and subjected to the Gram stain. The strain
organisms, including toxins with haemolysins, enterotoxins, was identified using different biochemical methods: the VITEK2
endotoxins and adhesions which together contribute to overall system (bioMerieux) and an API 20E test kit (bioMerieux). Molecu-
disease progress. lar sequencing of the DNA fragment containing the 16S–23S inter-
Naturally occurring plasmids, encoding different virulence fac- genic spacer corresponding to the conserved region of 16S rDNA
tors, have been identified in the genus Aeromonas (Brown et al. (Martinez-Murcia et al. 1992) allowed for an unambiguous classi-
1997; Majumdar et al. 2007). The presence of plasmids in these fication of the isolate as A. hydrophila. The bacteria was maintained
potentially pathogenic microorganisms may present a potential on LB agar plates at room temperature and for long-term storage
public health hazard, because they may be transferred from animals kept frozen at −70 ◦ C in LB broth supplemented with 20% glycerol.
to man (Bello-Lopez et al. 2010; Janda and Abbott 2010).
2.2. Virulence factors and antimicrobial susceptibility

∗ Tel.: +48 58 523 60 69. To determine the haemolytic activity, the A. hydrophila strain
E-mail address: [email protected] was plated onto blood agar plates. Haemolysin production was

0944-5013/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
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548 B. Furmanek-Blaszk / Microbiological Research 169 (2014) 547–552

recorded as clearance of the medium around bacterial colonies after approximately 2.5 kb could be observed (data not shown). This
24 h at 37 ◦ C. fragment was gel eluted, followed by ligation into HincII digested
The Congo Red Agar plate test was prepared by adding 0.8 g of cloning vector pUC18. The ligation mixture was transformed
Congo red and 50 g of sucrose, both of which had been previously into Escherichia coli DH5␣ cells. The transformant containing the
autoclaved separately into 1L of Brain Heart Infusion agar (Freeman desired recombinant plasmid was designated pAhy5.2. For ini-
et al. 1989). The studied A. hydrophila strain was grown on plates tial sequence information, cycle sequencing reactions were carried
for 24 h in 37 ◦ C and 48 h at room temperature. out using recombinant plasmid and the vector-specific universal
Antibiotic susceptibility was determined by the standard disk- primers M13 forward or M13 reverse. The nucleotide sequence
diffusion method on Mueller-Hinton (MH) agar plates, using OXOID was determined by the cycle sequencing method using the BigDye
antibiotic disks. The A. hydrophila strain was grown overnight in Terminator V3 1 Cycle Sequencing Kit as recommended by the
LB broth, the turbidity of the cell suspensions was adjusted to manufacturer [Applied Biosystems Inc., USA]. The sequence infor-
that equivalent of a 0.5 McFarland standard and used to inoculate mation obtained from the pAhy5.2 recombinant clone was used to
MH agar plates, which were incubated at 30 ◦ C for 24 h. Antibi- design sequence specific primers. Using these primers, the com-
otic susceptibility was determined by measuring the diameter of plete nucleotide sequence of the plasmid pAhy2.5 was determined
the clear zone. Resistance profiles were assigned using Clinical and and has been deposited in the NCBI GenBank database under the
Laboratory Standards Institute (2011) breakpoints. accession number KC525245.

2.3. DNA isolation and PCR amplification 3. Results and discussion

The genomic DNA and plasmid DNA used in this study 3.1. Virulence factors and antimicrobial susceptibility
were isolated using Genomic Mini and Plasmid Mini Kits (A&A
Biotechnology, Poland). The presence of the following genes encod- Strains of Aeromonas release a variety of virulence factors which
ing virulence factors was determined in the isolate: cytolytic are important for their pathogenicity (Janda and Abbott 2010).
heat-labile enterotoxin act, cytotonic heat-labile enterotoxin alt, The majority of A. hydrophila virulent strains secrete at least
cytotonic heat-stable enterotoxin ast, aerolysin aerA, haemolysin two types of haemolysins (Wang et al. 2003). One of them is
ahh1, ahyB for elastase, flaA/flaB for polar flagella and lafA for lat- channel-forming aerolysin (AerA) and the second a non-channel-
eral flagella. Primers based on the A. hydrophila ATCC 7966 16S rRNA forming haemolysin AHH1 showing 50% identity to a Vibrio cholerae
sequence were used to amplify a portion of the 16S rRNA gene as HlyA haemolytic toxin. Both toxins contribute to virulence in A.
an internal control. The details of primers used and their corre- hydrophila and are widespread within virulent strains of motile
sponding amplicons are shown in Table 1. The identity of each PCR aeromonads (Wong et al. 1998).
product was confirmed by nucleotide sequencing. The A. hydrophila isolate exhibited ␤-haemolytic activity on
blood agar plates (data not shown). To confirm further the presence
2.4. Purification of restriction endonucleases of haemolysins in this strain the PCR method targeting the virulence
genes was used. The sizes of the amplification products were iden-
The occurrence of restriction endonucleases in the A. hydrophila tical to those predicted from the designed primers (Table 1). The
strain was tested by the modified lysozyme and Triton X-100 PCR revealed the presence of an amplification product at 356 bp
method (Belavin et al. 1988). Bacterial cells were collected from characteristic for the genus Aeromonas and allowed the detection
Petri dishes and transferred into 20 ␮l of incubation mixture A of haemolytic gene fragments 130 bp and 309 bp for aerA and ahh1,
containing 20 mM Tris–HCl, pH 8.0, 1000 mM NaCl, 12.5 mM EDTA, respectively (Fig. 1). This is in agreement with earlier studies that
10 mM 2-mercaptoethanol (ME), and lysozyme at a concentration the occurrence of haemolytic factors in aeromonads is widespread
of 10 g/L. The sample was incubated for 30 min at room temperature and their presence can be used as indicators of virulence (Zhang
and then 20 ␮l of incubation mixture B containing 20 mM Tris–HCl, et al. 2000).
pH 8.0, 2% Triton X-100 and 10 mM ME was added for 60 min at
6 ◦ C. The restriction endonuclease activity was assayed in 20 ␮l of
reaction mixture containing 0.1 ␮g ␭DNA, 2 ␮l restriction buffer
Tango and 2 ␮l of bacterial lysate cleared by centrifugation for 1 min
(10,000 × g). For the purpose of further characterization, restriction
endonucleases were purified using ion-exchange chromatography.
From a 1000 ml of overnight culture cells were harvested, resus-
pended in 20 ml of buffer S (10 mM potassium phosphate pH 6.5,
20 mM KCl, 1 mM EDTA, 10 mM ME, 5% glycerol) supplemented
with a protease inhibitor 0.1 mM phenyl-methyl-sulfonyl-fluoride
and disrupted by sonication at 4 ◦ C in 60 × 10-s bursts. After cen-
trifugation, the supernatant was applied to a phosphocellulose
P11 column equilibrated with buffer S. The proteins were eluted
with a 150 ml KCl gradient (20–1000 mM) in the same buffer. The
active fractions were dialysed against buffer S, loaded onto a CM-
Sephadex C-50 column, and eluted with 150 ml of a linear gradient
of KCl from 25 mM to 1000 mM.

2.5. Sequencing strategy

Fig. 1. Detection and identification of Aeromonas hydrophila virulence genes by
The pAhy2.5 DNA was initially separately digested with dif- amplification of fragments in the PCR assay. Lane 1, DNA size marker 50-bp Gene
Ruler (Fermentas); lane 2, the ahh1 gene (130 bp fragment); lane 3, the act gene
ferent restriction endonucleases, to identify suitable fragments
(232 bp fragment); 4, the aerA gene (309 bp fragment); lane 5, A. hydrophila 16S
that could be generated for cloning purposes. When pAhy2.5 rRNA internal control showing a 356 bp fragment; lane 6, the ahyB gene (513 bp
was digested with the enzyme MlsI, one restriction fragment fragment); 7, flaA/flaB genes (608 bp fragment).
 B. Furmanek-Blaszk / Microbiological Research 169 (2014) 547–552 549

Table 1
Primers used for amplification.

Primer pair Sequence (5 –3 ) Size of amplified product (bp) References

AHH1F/AHH1R GCCGAGCGCCCAGAAGGTGAGTT/GAGCGGCTGGATGCGGTTGT 130 Wang et al. (2003)

AH-aerAF/AH-aerAR CAAGAACAAGTTCAAGTGGCCA/ACGAAGGTGTGGTTCCAGT 309 Wang et al. (2003)
A16SF/A16SR GGGAGTGCCTTCGGGAATCAGA/TCACCGCAACATTCTGATTTG 356 Wang et al. (2003)
ElasF/ElasR ACACGGTCAAGGAGATCAAC/CGCTGGTGTTGGCCAGCAGG 513 Sen and Rodgers (2004)
FlaF/FlaR TCCAACCGTYTGACCTC/GMYTGGTTGCGRATGGT 608 Sen and Rodgers (2004)
LafA1/LafA2 GGTCTGCGCATCCAACTC/GCTCCAGACGGTTGATG 550 Merino et al. (2003)
AHCF1/AHCF2 GAGAAGGTGACCACCAAGAACA/AACTGACATCGGCCTTGAACTC 232 Kingombe et al. (1999)
AstF/AstR TCTCCATGCTTCCCTTCCACT/GTGTAGGGATTGAAGAAGCCG 331 Sen and Rodgers (2004)
AltF/AltR TGACCCAGTCCTGGCACGGC/GGTGATCGATCCACCAGC 442 Sen and Rodgers (2004)

Since virulence in Aeromonas is considered to be multifactoral, resistance for A. hydrophila have varied. Differences in antimicrobial
the PCR approach to detect more virulence genes was used. The susceptibility between Aeromonas may be related to the source of
three enterotoxins Act, Alt, and Ast have been implicated as major the isolates and the frequency of use of antimicrobial agents pre-
virulence factors in diarrhoeal disease, however the presence of scribed for treating Aeromonas infection in a specific geographic
these toxins may not be sufficient for virulence (Sha et al. 2002). The area (Son et al. 1997; Aravena-Roman et al. 2012).
temperature-stable metalloprotease AhyB with elastolytic activity
plays an important role in the invasiveness and establishment of 3.2. Complete nucleotide sequence of pAhy2.5
infection (Janda and Abbott 2010). Both lateral (Laf) and polar (Fla)
flagella are involved in the initial attachment of bacteria to the gas- When the total cellular DNA of A. hydrophila was separated by
trointestinal epithelium and the subsequent adherence process and electrophoresis through a 1% agarose gel, a single extra chromo-
biofilm formation (Cascon et al. 2000). somal DNA band was observed (data not shown). To investigate
The A. hydrophila isolate was found to posses genes act, ahyB this extrachromosomal element in greater detail, plasmid DNA was
and flaA/flaB, while genes for enterotoxins Alt and Ast, and the purified. Restriction analysis identified a single plasmid of about
lateral flagella LafA were not identified. The cytolytic enterotoxin 2.5 kb designated pAhy2.5. The occurrence of pAhy2.5 was not
Act, protease AhyB and polar flagella Fla are important virulence affected by the bacterial growth stage or repeated subculturing.
factors in aeromonads (Kingombe et al. 1999; Cascon et al. 2000; To shed further light on the possible significance of pAhy2.5 in
Sen and Rodgers 2004), however the exact combinations of spe- A. hydrophila antibiotic resistance and/or virulence, the nature of
cific microbial determinants involved in the pathogenesis process the plasmid was examined. The nucleotide sequence analysis of
remains unknown. These results show that A. hydrophila isolate pAhy2.5 revealed a circular molecule of 2524 bp with a mean GC
found in environmental water possess a wide variety of virulence- content of 56.5% which is lower than that of A. hydrophila chromo-
associated genes and suggest the importance of examining as many somal DNA (61.5%) (Seshadri et al. 2006). Computer analysis of the
isolates as possible in order to better understand the health risk sequence of pAhy2.5 revealed the presence of seven putative ORFs
these bacteria may present. with at least 95 amino acid codons in length. However, only two
The application of the PCR assay to detect virulence genes has ORFs which have the same transcription direction on the plasmid
become a specific and easy method for identifying potentially were predicted to encode putative proteins (Fig. 2). To attribute
pathogenic Aeromonas spp. The presence of virulence determinants functions to the deduced products of the ORFs, these were com-
as genetic markers has been widely used in many epidemiologi- pared to the gene products available in the databases. Thus ORF1,
cal studies to differentiate pathogenic from non-pathogenic strains which starts with an ATG codon, contains 1284 bp and is found
of Aeromonas (Kingombe et al. 1999; Wang et al. 2003; Sen and between coordinates (140–1423) encodes a protein showing a rel-
Rodgers 2004). atively high amino acid sequence similarity with several plasmid
The production of slime by the A. hydrophila strain under study replication proteins. The Rep protein shares 28% and 27% identity
was assessed by culturing the strain on Congo red agar plates. over its whole length with the putative replication proteins of the
The isolate was found to be slime positive, producing black shiny pC194 plasmid family encoded in plasmids pJS46 (GenBank acces-
colonies with a metallic tinge whereas the non-slime producer, sion no. AAV68396) from Comamonas sp and pBL63 (NCBI reference
Staphylococcus epidermidis reference strain test ATCC 12228, looked sequence YP 227352) from Bacillus licheniformis, respectively. The
red (data not shown). Slime production is thought to be one of highest similarities (39%) between the ORF1 product and these pro-
the virulence factors responsible for biofilm formation. This pro- teins were observed mainly in the central and C-terminal parts of
tective layer is highly significant to the pathogenesis and appears these proteins. Amino acid sequence analysis revealed the presence
to contribute to infections in fish and humans (Janda and Abbott of all five highly conserved characteristic motifs of the replication
2010). initiator proteins of RCR replicons belonging to the pC194 plasmid
The resistance of the A. hydrophila pattern isolate toward 22 family (del Solar et al. 1998). The cysteine-rich motif I is a potential
antimicrobial agents tested is shown in Table 2. As expected this
strain displayed resistance toward most of ␤-lactam antibiotics
but was susceptible to the 1st and 3rd generation cephalosporins. Table 2
The ␤-lactam resistance phenotype may be explained in part The antibiotic sensitivity and resistance pattern of an Aeromonas hydrophila isolate.
by Aeromonas spp.’s naturally occurring, chromosomally encoded Antimicrobial agent Susceptible/resistant
␤-lactamases (Jacobs and Chenia 2007). The A. hydrophila iso-
Amoxicillin, Ampicillin, Clarithromycin, Fusidic R
late showed susceptibility to chloramphenicol, co-trimoxazole, acid, Oxacillin, Penicillin, Tetracycline
aminoglycosides and quinolones. While this strain displayed sus- Amikacin, Azithromycin, Cefazedone, S
ceptibility to doxycycline, resistance to tetracycline was observed. Cefoperazone, Ceftriaxone, Ciprofloxacin,
The isolate in the present study appeared to have a suscepti- Chloramphenicol, Doxycycline, Gentamicin,
Nalidixic acid, Netilmicin, Mupirocin,
bility profile similar to environmental, fish and clinical isolates
Piperacillin, Rifampicin, Co-trimoxazole
(Jacobs and Chenia 2007). However, data regarding antimicrobial (Sulfamethoxazole-Trimethoprim)
550 B. Furmanek-Blaszk / Microbiological Research 169 (2014) 547–552

et al. (1997) to be involved in the termination of primer RNA synthe-

sis. In pAhy2.5, a 59 bp long inverted-repeat sequence, able to form
orf2 BamHI (382) a potential stem-and-loop structure, was located from nt 2275 to
2333. This putative secondary structure shows extensive sequence
homology (84–89%) with the sso of various Gram-negative bacterial
plasmids (GenBank accession number in brackets): p40-95A from
pAhy2.5 Salmonella sp. (JQ418539), pKYM from Shigella sonnei (S77326),
2524 bp pO145-NM from E. coli (HM38194). Within the loop region there is
BamHI (1786) HindIII (696) a sequence 5 -TTGTGG-3 which shares three out of six nucleotides
with the CS-6 motif.
Aeromonads are known to carry plasmids encoding antibiotic
orf1 (rep)
resistance and/or virulence determinants (Borrego et al. 1991;
Majumdar et al. 2007). There are studies which reported Aeromonas
strains harboring one or more plasmids (Borrego et al. 1991; Son
et al. 1997; Boyd et al. 2003) but their exact role in virulence is
Fig. 2. Schematic map of pAhy2.5. Some restriction sites and their positions are yet to be established. The presence of plasmids in these poten-
shown. Closed arrows indicate the two ORFs. tially pathogenic bacteria may facilitate the exchange of genetic
information between different Aeromonas species and between the
metal-binding motif that is shared with Rep proteins of the pC194 human and aquaculture environments. Plasmids are considered the
family. Motif III contains two conserved histidine residues which predominant factors mediating horizontal gene transfer between
may act as ligands for the Mg2+ and Mn2+ ions required for the Rep bacteria in the environment (Halary et al. 2010). However, neither
function. Motif IV includes the conserved tyrosine residue that has the antibiotic resistance nor virulence of A. hydrophila has been
been proposed to be involved in DNA cleavage and linking. Motif found to be associated with the pAhy2.5 plasmid. Therefore, the
V, also known as the C-terminal motif, is involved in nucleophilic genes conferring resistance toward antibiotics could be located
attack of the newly synthesized leading strand near the replica- elsewhere in the chromosomal or in other endogenous plasmids.
tion termination site. In contrast to the above described motifs, the The role of other ORFs of pAhy2.5 is presently unknown but may
function of motif II, conserved among RCR plasmids, remains to be confer advantages to the isolate which carries them in a particular
elucidated. environment.
ORF2 spans positions 1973–2269 and encodes a hypothetical
protein of 98 amino acids, which shared 37–59% identity with 3.3. Characterization of restriction endonucleases
hypothetical proteins found in various Aeromonas spp. (Fig. 3) and
unrelated bacteria. In addition to the sequence similarity, the orf2- Numerous bacterial species harbor restriction-modification
like genes are all positioned in close proximity to the rep gene (R-M) systems as a prokaryotic defense mechanism against bac-
suggesting that orf2 may be involved in replication of these plas- teriophage infection (Roberts et al. 2007). The ability of the type II
mids. These two genes were organized similar to the corresponding restriction endonucleases to cleave DNA only at their recognition
genes for plasmid replication in the plasmids pAsa3, pAsa1 isolated sites makes these enzymes important tools in molecular biology
from A. salmonicida subsp. salmonicida A449 (Boyd et al. 2003) and and genetic engineering. Due to the virulence of pathogenic bacte-
pAQ2-1, pAQ2-2 isolated from Aeromonas sobria SNUFPC-A1 and A. ria only a few studies have been conducted to assess the presence of
hydrophila SNUFPC-A5, respectively (Han et al. 2012). restriction enzymes in the bacterial cell. Locally growing microor-
In addition to the five conserved motifs in the replication ganisms offer the chance to isolate new restriction endonucleases,
proteins, RC plasmids are characterized by the presence of dou- for this reason A. hydrophila was tested for the occurrence of these
ble (dso)- and single (sso)-stranded origins of replication. In the enzymes. A two-step purification protocol was used for isolation
pAhy2.5 sequence these elements were predicted in the intergenic of restriction enzymes from a culture of A. hydrophila. Phospho-
region upstream of the rep gene on the same strand. The non-coding cellulose P11 chromatography of the cell extract resulted in the
region between 2270 nt and 139 nt located upstream of the rep gene detection of two peaks of site specific endonucleolytic activities.
exhibits multiple inverted repeated sequences that are typical for The endonucleases named AehI and AehII were eluted at 0.2–0.35 M
the origin of replication of some RCR plasmids. This DNA fragment KCl and 0.35–0.5 M KCl, respectively. To separate proteins of both
contains two possible hairpin loops which are positioned from nt peaks the central fractions in each case were run on CM-Sephadex
2275 to nt 2333 and from nt 2355 to nt 2390. Usually, the nick C50. To determine the recognition sequences of enzymes, restric-
regions, important in the binding of the Rep protein and the nicking tion sites were mapped on ␭ DNA. The restriction data suggest
of the double strand, are highly conserved in the dsos of all plas- that ␭ DNA is cleaved by AehI at one site producing two frag-
mids belonging to the same family, while the binding sequences ments of approximate sizes 33,000 and 15,000 bp (Fig. 4). The
of the dsos are less well conserved (Khan 2005). Moreover, the dso AehII restriction endonuclease cleaved ␭ DNA into four fragments
is present in a plasmid region that has the potential to form sec- (approximately 19,000, 12,000, 8000, 7000 bp) (Fig. 4). The tar-
ondary structures. Having identified the gene encoding a potential get sequences of purified enzymes were inferred from restriction
Rep protein of the pC194 type, the search for the corresponding mapping of recognition sites on ␭ DNA using double digestions
origin of replication in the pAhy2.5 plasmid was performed. Such with restriction endonucleases EcoRI or HindIII (data not shown).
a sequence is located upstream of the rep gene and contains the Based on the restriction patterns obtained from single and double
highly conserved sequence 5 -CTTGATA-3 , identical to the origin digestions of ␭ DNA it was assumed that AehI and AehII rec-
described for the plasmid pC194 group (del Solar et al. 1998). ognize 5 -CTCGAG-3 and 5 -AGGCCT-3 sequences respectively.
The nucleotide sequences of the ssos of RCR plasmid are not Computer-generated fragment sizes of ␭ DNA after cleavage with
well conserved and generally exist within the region characterized XhoI and StuI prototypes of type II restriction endonucleases were
by inverted repeats that form strong secondary structures occur- in close agreement with the fragment sizes observed experimen-
ring in proximity to the dsos (del Solar et al. 1998). In the loop tally for AehI and AehII. The type of isoschizomer and the identity
of these hairpin structures it is often possible to find a conserved of the restriction profile were confirmed by double digestion of ␭
sequence termed CS-6 (5 -TAGCGt/a-3 ) that was shown by Kramer DNA by AehI-XhoI and AehII-StuI. To date, isoschizomer of AehI
 B. Furmanek-Blaszk / Microbiological Research 169 (2014) 547–552 551

pAhy2.5 ---------MKDAHDNQTPDLLPIKRPRGRPRTGKAMTQAERQAKYRAKVAQNNVTVTVN 51
contig 121 ---------MKDSTDKGTGDMFGEKRGRGRPKTGNAKTGAERQAAFRAKQQESNVTVTIN 51
pAsa3 ---------MKDSTDKGTGDMFGEKRGRGRPKTGTAKTGAERQAAFRAKQQENNVTVTIS 51
pAsa1 ---------MKDTTDQGTGDMFGIKRGRGRPKTGAAKSGAERQAAYRAKLADINVTVTIN 51
pAQ2-1 MVTVTGIRAMKDNADKGTGDMFGEKRGRGRPKTGNAKTGAERQAAYRAKQQDNNVTVTIN 60
pAQ2-2 MVTVTGIRAMKDNADKGTGDMFGEKRGRGRPKTGNAKTGAERQAAYRAKQQDNNVTVTIN 60
pAHH01 --------------------MFCVKRRRGRPKTGGAKSGAERQAAYRAIQQGISVTVTIN 40
supercontig 1.1 ---------MRDPHDDSTSDLLPMPRRRGRPSTGTAFTPAQKQARYRERQRARTVTVTFN 51
pAsal2 ---------MKDERDDQTLDLLPSGKRRGLPPTGKALSAAAKQAAYRARQREKTVTVTLN 51
:: : ** * ** * : * :** :* .****..

pAhy2.5 RGLLERLDAHMQALRDGSTVQILTPDEAGQVLEAIRRAQLVQLRHSA--------- 98
contig 121 RELLEGLDAQMQAIRDGGSVLVLTSEQAGESLKAIRKSSLKQLRGQRPRPQGKPTA 107
pAsa3 RELLDGLDAQMQAIRDRGSVVVLTPEQAGEILKAIRKSSLKQLRGQRPRPQAKPTA 107
pAsa1 RELIDGLSVHLQAIRDGWSEVPLTPEQAGEILKALRTAELKQLRGQHPRPQAKPTA 107
pAQ2-1 RALVEGLDAQMQAIRDGGSAVVLTPEQAGEILRALRTAENKQLRG----------- 105
pAQ2-2 RALVEGLDAQMQAIRDGGSAVVLTPEQAGEILRALRTAENKQLRG----------- 105
pAHH01 RAHLDGLSAQLQAIRDGGSAVVLTPGAGRGDLAGDPIRSKETAPGITAAAHRGTSP 96
supercontig 1.1 RSAIDALDSHIRGLVAG-LDVPIPPEHAASILESIRSATLSQLAPLAES------- 99
pAsal2 RLDCGELEIFLRNLRDG-RTSTLAPEVVARLHEAVRSAWRGQLHSGNGDQK----- 101
* *. :: : :.. .
Fig. 3. Alignment of the deduced amino acid sequences of pAhy2.5 ORF2 and putative replication proteins from Aeromonas spp. The GenBank accession numbers of protein
sequences are given in parenthesis. A. salmonicida 01-B256 (EHI50146) contig 121, A. salmonicida A449 (AAP69900) pAsa3, A. salmonicida A449 (AAP69885) pAsa1, A. sobria
SNUFPC-A1 (AEW70680) pAQ2-1, A. hydrophila SNUFPC-A5 (AEW70684) pAQ2-2, A. hydrophila SNUFPC-A8 (AEW70672) pAHH01, A. veronii AER39 (EKB18347) supercontig
1.1, A. salmonicida (CAD48426) pAsal2.

has been detected in the A. hydrophila AH63 strain (Miyahara et al. The increasing interest in bacteria belonging to the genus
1996), however an AehII counterpart in aeromonads has not yet Aeromonas has grown over the past two decades due its path-
been reported. ogenicity for aquatic organisms and humans. The potential risk
Frequently, newly discovered enzymes are found to cut DNA from Aeromonas spp. isolated from aquatic environments to human
in patterns identical to those of previously isolated enzymes. The health has been noted by several researchers (Bello-Lopez et al.
data presented shows that restriction endonucleases AehI and AehII 2010; Janda and Abbott 2010). Cases of infections in healthy people
display an identical cleavage pattern as XhoI and StuI respectively. associated with recreational water activities have been described,
Thus, it is reasonable to postulate the existence of similar type IIR-M as have cases of pneumonia following aspiration of contaminated
systems in A. hydrophila. The occurrence of relatively rarely cutting recreational water. The fact that virulence factors are present in the
restriction endonucleases is an interesting but not unusual obser- environmental A. hydrophila strain suggests that the aquatic envi-
vation and could be attributed to the horizontal transfer of genes ronment may act as a reservoir of potentially virulent Aeromonas
coding for these systems. In complex microbial ecosystems such as spp.
the reservoir, rich in bacteriophages and free DNA, possession of
R-M systems might provide important advantages to the host pro-
tecting it from genome subversion through any invading foreign Acknowledgments
DNA.
The author thanks Agnieszka Kobylska for technical assistance
and Martin Blaszk for excellent help with editing this manuscript.

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