Systematic and Applied Microbiology 38 (2015) 30–35
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Arcobacter ebronensis sp. nov. and Arcobacter aquimarinus sp. nov., two new species isolated from marine environment Arturo Levican a,b,c , Sara Rubio-Arcos a , Antonio Martinez-Murcia d , Luis Collado e , María José Figueras a,∗ a Unitat de Microbiologia, Departament de Ciències Mediques Bàsiques, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain b Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas Universidad Andrés Bello, Vi˜ na del Mar, Chile c Interdisciplinary Center for Aquaculture Research (INCAR) , Concepción, Chile d Area de Microbiología, EPSO, Universidad Miguel Hernández, Orihuela, Alicante 03312, Spain e Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile
a r t i c l e
i n f o
Article history: Received 18 May 2014 Received in revised form 28 October 2014 Accepted 31 October 2014 Keywords: Arcobacter A. ebronensis A. aquimarinus MLPA 16S rRNA MALDI-TOF
a b s t r a c t Two strains recovered from mussels (F128-2T ) and sea water (W63T ) were characterized as Arcobacter sp., but they could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR) and rpoB gene analysis. The 16S rRNA gene sequence similarity to the type strains of all Arcobacter species ranged from 92.2% to 96.7% with strain F128-2T , and from 94.1% to 99.4% with strain W63T , the most similar being A. bivalviorum (CECT 7835T ) and A. defluvii (CECT 7697T ), respectively. The phylogenetic analyses of 16S rRNA, and the concatenated sequences of gyrB, gyrA, rpoB, atpA and hsp60 genes confirmed that strains F128-2T and W63T belonged to two new lineages within the genus Arcobacter. Moreover, both strains showed differential phenotypic characteristics and MALDI-TOF mass spectra from all other Arcobacter species. Therefore, it has been demonstrated the existence of two new Arcobacter species and the proposed names are Arcobacter ebronensis (type strain F128-2T = CECT 8441T = LMG 27922T ), and Arcobacter aquimarinus (type strain W63T = CECT 8442T = LMG 27923T ). © 2014 Elsevier GmbH. All rights reserved.
Members of the genus Arcobacter (Ar’co.bac.ter. L. n. arcus, bow; Gr. n. bacter, rod; M. L. masc. n. Arcobacter, bow-shaped rod) are Gram negative, slightly curved rods positive for oxidase and usually motile and belong to the Epsilonproteobacteria and family Campylobacteraceae [4,38]. These bacteria had previously been classified as Campylobacter spp. because of their similar morphology, despite they differed from the latter genus because they are aerotolerant and able to grow at lower temperatures [4,38]. In fact, based on the latter characteristics Vandamme et al. created the genus Arcobacter in 1991 with 2 species formerly known as campylobacters, Arcobacter nitrofigilis and Arcobacter cryaerophilus [39]. Currently this genus includes 18 species that have been recovered from different hosts and environments [4,33]. Moreover, the analysis of the 16S rRNA gene sequences deposited in GenBank indicates that many other potential new Arcobacter species remain to be characterized [40]. Some Arcobacter spp. have been linked with gastroenteritis and bacteraemia in humans, and with abortions, mastitis and diarrhoea
∗ Corresponding author. Tel.: +34 977759321; fax: +34 977759322. E-mail address: mariajose.fi[email protected] (M.J. Figueras). http://dx.doi.org/10.1016/j.syapm.2014.10.011 0723-2020/© 2014 Elsevier GmbH. All rights reserved.
in animals, and they are considered as potential water and foodborne pathogens [4,5,17]. In this sense, it has been demonstrated that the presence of Arcobacter in water increases with the levels of faecal pollution [6]. It was suggested that Arcobacter spp, entered seawater with the contaminated inputs of freshwater despite some species could be autochthonous of the marine environment [6]. So far Arcobacter was recovered either from the water or from the stools of the patients in 3 drinking water outbreaks, 2 of them in USA and 1 in Slovenia [18,23,32]. However, in none of them the implication of this microbe was completely proven. Arcobacter spp. have also been found in association with food of animal origin, mainly meat products, but also in shellfish [4,5,28]. Shellfish could be an important reservoir and source of infection of these bacteria, as suggested in recent studies [5,28]. In a recent study on the prevalence of Arcobacter in different types of shellfish collected from the Ebro river delta [28], one strain recovered from mussels (F128-2T ) could not be assigned to any known species. The same occurred for another strain of our collection (W63T ) obtained from a seawater sample. The objective of the present study was to establish the taxonomic position of both strains (F128-2T and W63T ) using a polyphasic approach.
A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
Strain F128-2T was obtained from mussels collected from the Ebro delta, Catalonia (northeast Spain) in June 2011, while strain W63T was isolated from a seawater sample at the Garraf beach also in Catalonia, in September 2008. Both strains showed the typical colony morphology of arcobacters on blood agar (small, translucent, beige to off-white) and were Gram negative, slightly curved rods that produce oxidase activity, as previously described for the species of this genus [4,39]. The characterization of the strains was initially attempted using specific Arcobacter identification methods i.e. two Multiplex-PCR (m-PCR) [10,21] and the restriction fragment length polymorphism of the 16S rRNA gene (16S rDNA-RFLP) [12,15]. Both strains (F1282T and W63T ) produced an amplicon of the expected size described for A. cryaerophilus with the m-PCR of Houf et al. [21]. However, with the m-PCR of Douidah et al. [10] strain W63T produced the expected amplicon for Arcobacter butzleri while strain F128-2T , produced no amplification. On the other hand, with the recently updated16S rRNA RFLP identification method [15] strain F128-2T produced a new pattern after digestion with the endonuclease MseI (719, 138, 81, 52, 34 and 3 bp; Fig. S1), while W63T produced the same pattern described for Arcobacter defluvii when digested with MseI [7] or BfaI endonucleases [15] (Fig. S1). Considering these contradictory results, the rpoB (621 bp) and the 16S rRNA (1401 bp) genes of both strains (F128-2T and W63T ) were sequenced and analysed as described previously [3,25] and the constructed phylogenetic trees indicated that both strains formed independent phylogenetic lines within the genus (Fig. 1 and Fig. S2). In the 16S rRNA gene tree, strain W63T clustered with the species A. defluvii, Arcobacter cloacae and Arcobacter ellisii, whereas the most closest species for strain F128-2T were A. bivalviorum and A. anaerophilus (Fig. 1). The 16S rRNA gene similarity, calculated with the EzTaxon software [2], between strains F128-2T and W63T was 95.7%. Similarities between strain F128-2T and other Arcobacter spp. ranged from 92.2%, with the type strain of A. cryaerophilus (LMG 9904T ), to 96.7%, with the type strain of A. bivalviorum (CECT 7835T ). The similarities of strain W63T with other arcobacters ranged from 94.1%, with the type strain of Arcobacter mytili (CECT 7386T ), to 99.4%, with the type strain of A. defluvii (CECT 7697T ). These similarities were all within the range from 91.1% (for A. cryaerophilus and A. bivalviorum) to 99.6% (for A. cloacae and A. ellisii) described for the genus [16,25]. In the definition of several Arcobacter species the use of the concatenated sequences of housekeeping genes (gyrA, atpA, rpoB, gyrB and hsp60) has shown a better resolution, than DDH results [3,7,8,13,16,25,26]. In fact, the “ad hoc committee for the reevaluation of the species definition in bacteriology” has suggested that this approach could be used as an alternative to DDH if correlation with the latter method was demonstrated [34] as has recently been done for the genus Arcobacter [7,13,25,26]. This approach has been named multilocus sequence analysis (MLSA) or multilocus phylogenetic analysis (MLPA) [14,25,36]. In the Arcobacter studies, the MLPA has also provided a more robust overall phylogenetic relatedness (bootstrap values of 100% for all the species clusters) than 16S rRNA gene [3,7,8,13,16,25,26]. For strains F128-2T and W63T , apart from the rpoB, the sequences of the 4 remaining genes i.e. gyrB (618 bp), gyrA (686 bp), atpA (622 bp), and hsp60 (595 bp) were obtained as described previously [7,25]. In addition, in order to complete the MLPA with all currently accepted species, the sequences of the 5 genes (gyrA, atpA, rpoB, gyrB and hsp60) from the strain DSM 24636T of the recently described species A. anaerophilus [33] were obtained. Alignments were performed using the MEGA software version 5 [35] and CLUSTAL W [24] and clustering, using the neighbour-joining, maximum parsimony and maximum likelihood algorithms. The phylogenetic tree obtained with the sequences of the 5 concatenated genes (3142 bp), using
31
different the neighbour joining (Fig. 2) and other algorithms (data not shown) confirmed initial rpoB results that both strains belonged to two independent and unknown phylogenetic lines within the genus. Both strains (F128-2T and W63T ) were characterized as motile under the phase contrast microscope, and under the transmission electron microscope, they showed a single polar flagellum (Fig. S3). Further characterization was carried out using the test recommended in the minimal standards for the family Campylobacteraceae [37], as well as others tests previously used in the description of other new Arcobacter spp. [25]. All tests were carried out at least twice for the 2 new strains and for all the type strains of Arcobacter species with the exception of A. anaerophilus that could not be maintained alive despite repeated efforts using different culture conditions. The test that were able to differentiate strains F128-2T and W63T between them and also from all other Arcobacter spp. are shown in Table 1. Interestingly, strain F128-2T showed growth on media with 4% NaCl, as also did other species isolated in association with shellfish or starfish like A. nitrofigilis, Arcobacter skirrowii, Arcobacter halophilus, A. mytili, Arcobacter marinus, Arcobacter molluscorum and A. bivalviorum. However, F128-2T could be easily differentiated from all of these species by 7 to 12 tests (Table 1). On the other hand, the closest phylogenetic species to F128-2T was A. anaerophilus (Figs. 1 and 2). However, they could be differentiated by at least 3 tests (Table 1) and by the fact that A. anaerophilus is strictly anaerobe. The most similar species to strain W63T , was A. cloacae but they could be differentiated because, in contrast to W63T , the later species is able to grow on MacConkey but not on blood agar at 30 ◦ C under anaerobic conditions [25]. Additional characterization of the mussels (F128-2T ) and seawater (W63T ) strains included the analysis of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS). This analysis was carried out in parallel with the type strains of all Arcobacter species as previously described [16], with the only exception, as commented, of strain DSM 24636T of A. anaerophilus which could not be kept alive in our laboratory to carry out the MALDI-TOF analysis. The obtained MALDI-TOF profiles of strains were hierarchically clustered in a dendrogram using the SPECLUST web tool (http://bioinfo.thep.lu.se/speclust.html) [1]. In the obtained dendrogram, strain F128-2T clustered close to the type strain of A. halophilus (LA31BT ) and W63T , close to the type strain of A. cloacae, CECT 7834T (Fig. S4 and Table S1). Considering the origin of the studied strains (shellfish and water), they both were tested for the presence of 5 putative virulence genes (ciaB, irgA, hecA, cj1349 and cadF) as previously described [11]. Strains F128-2T and W63T possessed the ciaB gene that codifies for a major invasine protein in genus Campylobacter [11,30], while W63T also possessed the cj1349 gene, which codifies for a fibronectin binding protein in Campylobacter jejuni. Despite of this, strain W63T was not able to adhere or invade the human intestinal Caco-2 cell lines in a previous study [27]. These results warrant future studies on the potential pathogenic role of F128-2T and W63T for humans. In this study it has been demonstrated the existence of two new Arcobacter species, for which the names Arcobacter ebronensis (type strain F128-2T = CECT 8441T = LMG 27922T ), and Arcobacter aquimarinus (type strain W63T = CECT 8442T = LMG 27923T ) are proposed.
Description of Arcobacter ebronensis sp. nov. Arcobacter ebronises (e.bro.nen’sis. N.L. masc. adj. ebronensis, of or belonging to Ebro river delta Spain, where shellfish sample harbouring strain F128-2T was collected)
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A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
100 A. cryaerophilus LMG 9904T (L14624)
A. cryaerophilus LMG 9865(FR682113) A. skirrowii LMG 6621T (L14625) 99
A. skirrowii Houf 989 (GU300769) A. trophiarum CECT 7650 (FE2) (GU300768)
T 99 A. trophiarum LMG 25534 (FN650333) 73 A. trophiarum LMG 25535 (FN650332)
A. thereius LMG 24487 (AY314754)
95 98
A. thereius LMG 24486T (AY314753) A. cibarius CECT 7203T (AJ607391)
100
100 A. cibarius LMG 21997 (AJ607392)
A. butzleri F46 (GU300771) 100 A. butzleri LMG 10828T (AY621116)
A. venerupis F67-11T (HE565359)* A. suis F41T (FJ573216)* 76
100 91
91
A. defluvii SW 28-7 (HQ115597) A. defluvii SW30-2 (HQ115596) A. defluvii CECT 7697T (HQ115595) A. aquimarinus sp. nov. W63T (HG932574)
79
A. cloacae SW28-13T (HE565360)
78
A. cloacae F26 (HE565361) A. ellisii CECT 7837T (FR717550)
100 97
A. ellisii F79-7 (FR717552) A. ellisii F79-2 (FR717551) 100 A. bivalviorum F4T (FJ573217) 100
93
A. bivalviorum F118-2 (HE565357) A. bivalviorum F118-4 (HE565358)
77
A. ebronensis sp. nov. F128 2T (HG932573) A. anaerophilus JC84(FR686494)* 100
A. nitrofigilis CECT 7204T (L14627) A. nitrofigilis F2176 (EU106662) 100 A. mytili CECT 7385 (EU669906)
A. mytili CECT 7386T (EU669904) A. halophilus LA31BT (AF513455)*
86
A. marinus CECT 7727T (CL-S1T)(EU512920)*
84
A. molluscorum F101-1 (FR675875)
98 100
A. molluscorum F91 (FR675876) A. molluscorum F98-3T (FR675874)
0.005 Fig. 1. Neighbour joining tree based on 16S rRNA (1401 bp) sequences showing the phylogenetic position of A. ebronensis sp. nov. and A. aquimarinus sp. nov. within the genus Arcobacter. Bootstrap values (>70%) based on 1000 replications are shown at the nodes of the tree. Bar, 5 substitutions per 1000 nt. * Only type strain is available so far.
Cells of strains F128-2T are Gram-negative slightly curved rods, non-encapsulated, non-spore forming, 0.3–0.5 m wide and 1.5–2.5 m long. They are motile by a single polar flagellum. Colonies on blood agar incubated under aerobic conditions at 30 ◦ C for 48 h are 2–4 mm in diameter, beige to off-white, circular with entire margins, convex, and non-swarming. Pigments are not produced. This strain grows on blood agar at room temperature (18–22 ◦ C) and 30 ◦ C under both aerobic and microaerobic conditions with no significant differences. Growth of this strain is slightly enhanced by adding 1% NaCl to media. No growth is observed under anaerobic conditions as well as under aerobic or microaerobic conditions at 37 ◦ C or 42 ◦ C. No haemolysis is observed on TSA medium supplemented with 5% sheep blood. Strain F128-2T produce oxidase but not catalase activity. It hydrolyse indoxyl acetate and urea
but not casein, lecithin or starch. The strain is not able to produce acid from glucose by oxidization or fermentation, nor hydrogen sulphide, in triple-sugar iron agar medium, and it is not able to reduce nitrate. Under aerobic conditions at 30 ◦ C the strain grows on Marine Agar, minimal medium and on nutrient medium supplemented with 5% sheep blood containing 0.5–4% (w/v) NaCl; 0.1% sodium deoxycholate; 0.05% safranin; or 0.005% basic fuchsine. No growth occurs on, MacConkey agar, campylobacter charcoal deoxycholate agar (CCDA), and on nutrient medium supplemented with 5% sheep blood containing 1% glycine; 0.01–0.1% 2,3,5-triphenyl tetrazolium chloride (TTC); 64 mg l−1 cefoperazone;. 1% oxgall; 0.0005% crystal violet or 0.001% brilliant green A BLASTN analysis of the 16S rRNA sequence of strain F128-2T showed the closest match (98%) with strain sw026 isolated from
A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
33
A. cryaerophilus LMG9904T
100
A. cryaerophilus LMG9865 A. skirrowii LMG6621T 100
84
A. skirrowii HOUF989 A. thereius LMG24486T
100
A. thereius LMG24487
100
A. trophiarum LMG25534T 100 A. trophiarum CECT7650
76
A. cibarius CECT7203T 100 A. cibarius Houf 746
A. butzleri LMG 10828T 100
A. butzleri F46 A. cloacae SW28-13T (CECT 7834T)
100 100
A. cloacae F26 Arcobacter aquimarinus sp. nov. W63T A. venerupis F67-11T (CECT 7836T)*
100
100
A. suis F41T (CECT 7833T)* A. defluvii SW28-11T (CECT 7697T)
73 100
A. defluvii SW28-7 A. defluvii SW30-2
89
A. ellisii F79-2 100 98
A. ellisii F79-6T (CECT 7837T) A. ellisii F79-7 100 A. nitrofigilis CECT7204T 100
A. nitrofigilis LMG7547 A. nitrofigilis F2176 A. anaerophilus DMS 24636T*
99
Arcobacter ebronensis sp. nov. F128-2T 100 100
99
A. bivalviorum F4T (CECT 7835T) A. bivalviorum F118-2 A. bivalviorum F118-4 100 A. mytili CECT7386T
A. mytili CECT7385 98
100
A. halophilus DSM18005T* A. marinus CECT7727T*
80
A. molluscorum F98-3T (CECT 7696T) 100 A. molluscorum F91 82 A. molluscorum F101-1
0.02
Fig. 2. Neighbour joining tree based on the concatenated sequences of gyrA, gyrB, rpoB, atpA and hsp60 (3142 bp) sequences showing the phylogenetic position of A. ebronensis sp. nov. and A. aquamarinus sp. nov. within the genus Arcobacter. Bootstrap values (>70%) based on 1000 replications are shown at the nodes of the tree. Bar, 2 substitutions per 100 nt. * Only the type strain is available so far.
sea-surface water of the South Pacific (GenBank accession number JN118550). However, both strains form distinct phylogenetic lines (data not shown). The type strain is F128-2T (=CECT 8441T = LMG 27922T ), isolated from mussels collected at the Ebro river delta, Catalonia, Spain. Description of Arcobacter aquimarinus sp. nov. A. aquimarinus (a.qui.ma.rinus. L. fem. n. aqua water; L. adj. marinus of the sea). Cells of strains W63T are Gram-negative slightly curved rods, non-encapsulated, non-spore forming, 0.4–0.6 m wide and 1.0–2.0 m long. They are motile by a single polar flagellum.
Colonies on blood agar incubated in aerobic conditions at 30 ◦ C for 48 h are 2–4 mm in diameter, beige to off-white, circular with entire margins, convex, and non-swarming. Pigments are not produced. This strain grows on blood agar at room temperature (18–22 ◦ C), 30 ◦ C or 37 ◦ C under both aerobic and microaerobic conditions with no significant differences, and at 30 ◦ C under anaerobic conditions. No growth is observed under any incubation conditions at 42 ◦ C. No haemolysis is observed on TSA medium supplemented with 5% sheep blood. Strains produce oxidase and catalase activity, reduce nitrate, hydrolyse indoxyl acetate but not urea, casein, lecithin or starch. The strain is not able to produce acid from glucose by oxidization or fermentation, nor hydrogen sulphide, in triple-sugar iron agar medium. Under aerobic conditions at 30 ◦ C
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A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
Table 1 Differential characteristics of A. ebronensis sp. nov. and A. aquimarinus sp. nov. from other members of the genus arranged in order of description. Taxa: 1, A. ebronensis (n = 1); 2, A. aquimarinus (n = 1); 3, A. nitrofigilis (n = 4) [3,29,31]; 4, A. cryaerophilus (n = 19) [3,31]; 5, A. butzleri (n = 12) [31]; 6, A. skirrowii (n = 9) [31]; 7, A. cibarius (n = 15) [20]; 8, A. halophilus (n = 1) [9,16]; 9, A. mytili (n = 3) [3]; 10, A. thereius (n = 8) [19]; 11, A. marinus (n = 1) [22]; 12, A. trophiarum (n = 11) [8]; 13, A. defluvii (n = 8) [3]; 14, A. molluscorum (n = 3) [13]; 15, A. ellisii (n = 3) [16]; 16, A. bivalviorum (n = 3) [26]; 17, A. venerupis (n = 1) [26]; 18, A. cloacae (n = 2) [25]; 19, A. suis (n = 1) [25]; 20, A. anaerophilus (n = 1) [33]. The specific responses for type strains were coincidental or expressed in brackets. Unless otherwise indicated: +, ≥95% strains positive; −, ≤11% strains positive; V, 12–94% strains positive; CO2 indicates microaerobic conditions. Characteristics Growth in/on Air at 37 ◦ C CO2 at 37 ◦ C CO2 at 42 ◦ C 0.5% (w/v) NaCl 4% (w/v) NaCl 1% (w/v) glycine 0.05% safranin 0.1% sodium deoxycholate 1% (w/v) oxgall 0.04% TTC 0.01% TTC Minimal medium MacConkey CCDA Resistance to: Cefoperazone (64 mg l–1 ) Enzyme activity Catalase Urease Nitrate reduction Indoxyl acetate hydrolysis
1a
2
3
4
5
6
7
8a
9
10
11a
12
13
14
15
16
17
18
19
20
− − − + + − + + − − − + − −
+ + − + − − + + + − + − − +
V(−) − − + + − − V(−) − − − − − −
V(+) V(+) − + − − + V(+) + + + −c V(−) +
+ + V(+) + − − + + V(+) + + + + +
+ + − + + − + + + V(−) + − − +
− + − + − − + + + V(−) + + + V(−)
+ + − − + + − − − − − − − −
+ + + + + + − + + − − − + −
− − − + − + + V(−) − V(−) + + V(+) V(−)
+ + − − + + + − − − − − − −
− − − + − V(−) V(+) + + + + −d V(+)e +
+ + + + − − + + + − + + + +
+ + + + + − + + + − + − + −
+ + + + − − − +b − − − + V(+) +b
+ + − + + − − − − − − − − −
− + − + − − − − − − − + + +
+ + − + − − + + + − + V(+) + +
− − − + − − − + − − + + + −
− − − + + + ND ND ND ND ND ND ND ND
−
−
−
+
+
+
+
−
−
+
−
+
V(+)
+
−
−
−
−
−
−
− + − +
+ − + +
+ + + +
+ − +g +
V(+) − + +
+ − + +
V(−) − − +
− − + +
+f − +h −
+ − + +
− − + +
+ − − +
+f + + +
+ − +i −
+ V(−) + +
+ − − +
+ + + +
+ − + +
+ − + +
− − + +
a For these strains, the tests were carried out on media supplemented with 2% NaCl, with the exception of 0.5 and 4% (w/v) NaCl, catalase and indoxyl acetate hydrolysis [16]. b All strains grew weakly after 5 days of incubation. c Two (LMG 7537 and LMG 10241) of the four strains tested were positive [3]. d Test not evaluated by De Smet et al. [8] but tested by Figueras et al., [16]. e Strains LMG 25534T , LMG 25535 of A. trophiarum and strain FE2 (CECT 7650) of this species identified in our laboratory grew on MacConkey agar in contraposition with the 80% described for this species [8]. f Weak reaction [3,7]. g Two (LMG 9904 T and LMG 9065) of the four strains tested were negative [3]. h Nitrate reduction was positive for the 3 strains of A. mytili [13], contrary to our previously published data [3]. i Nitrate is reduced after 72 h and 5 days for all strains under microaerobic and aerobic conditions, respectively [16].
the strain grows on Marine Agar, campylobacter charcoal deoxycholate agar (CCDA) and on nutrient medium supplemented with 5% sheep blood also containing 0.5–2% (w/v) NaCl; 0.1% sodium deoxycholate; 1% oxgall; 0.01% 2,3,5-triphenyl tetrazolium chloride (TTC); 0.05% safranin; 0.0005% crystal violet or 0.001% brilliant green. No growth occurs on minimal medium, MacConkey agar and on nutrient medium supplemented with 5% sheep blood containing 4% (w/v) NaCl; 1% glycine; 0.04–0.1% TTC; 64 mg l−1 cefoperazone; or 0.005% basic fuchsine. A BlastN analysis of the 16S rRNA sequence of strain W63T matched with 99% with several sequences of uncultured clones of arcobacters, as well as the strains of A. cloacae, A. defluvii and A. suis that are already included in this study. The type strain is W63T (=CECT 8442T = LMG 27923T ), isolated from a water sample collected from the Mediterranean Sea at the Garraf beach, Catalonia, Spain.
Acknowledgments We thank IRTA (Research and Technology Food and Agriculture) of Sant Carles de la Ràpita (Tarragona, Spain) for sampling the shellfish. We also thank Jean P. Euzéby for kindly reviewing the species names. A. L. thanks the Universitat Rovira i Virgili for a doctoral grant; and also CONICYT, Chile, for financial support through Becas Chile, FONDECYT postdoctoral research project number 3140296 and FONDAP grant number 15110027. This work was supported in part by the Ministerio de Ciencia e Innovación (MICINN), Spain, project number
AGL2011-30461-C02-02; and by funding from European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 311846. The GenBank/EMTBL/DDBJ accession numbers of the sequences of strains F128-2T and W63T , for the 16S rRNA gyrA, atpA, rpoB, gyrB and hsp60 genes are HG932563 to HG932574 and for the gyrA, atpA, rpoB, gyrB and hsp60 genes from the type strains of the species Arcobacter anaerophilus from LK392365 to LK392369. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm. 2014.10.011. References [1] Alm, R., Johansson, P., Hjernø, K., Emanuelsson, C., Ringnér, M., Häkkinen, J. (2006) Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra. J. Proteome Res. 5, 785–792. [2] Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K., Lim, Y.W. (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol. 57, 2259–2261. [3] Collado, L., Cleenwerck, I., Van Trappen, S., De Vos, P., Figueras, M.J. (2009) Arcobacter mytili sp. nov., an indoxyl acetate-hydrolysis-negative bacterium isolated from mussels. Int. J. Syst. Evol. Microbiol. 59, 1391–1396. [4] Collado, L., Figueras, M.J. (2011) Taxonomy, epidemiology and clinical relevance of the genus Arcobacter. Clin. Microbiol. Rev. 2011 (24), 174–192. [5] Collado, L., Guarro, J., Figueras, M.J. (2009) Prevalence of Arcobacter in meat and shellfish. J. Food Prot. 72, 1102–1106. [6] Collado, L., Inza, I., Guarro, J., Figueras, M.J. (2008) Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution. Environ. Microbiol. 10, 1635–1640.
A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35 [7] Collado, L., Levican, A., Perez, J., Figueras, M.J. (2011) Arcobacter defluvii sp. nov., isolated from sewage. Int. J. Syst. Evol. Microbiol. 61, 1895–1901. [8] De Smet, S., Vandamme, P., De Zutter, L., On, S.L., Douidah, L., Houf, K. (2011) Arcobacter trophiarum sp. nov. isolated from fattening pigs. Int. J. Syst. Evol. Microbiol. 63, 356–361. [9] Donachie, S.P., Bowman, J.P., On, S.L., Alam, M. (2005) Arcobacter halophilus sp. nov., the first obligate halophile in the genus Arcobacter. Int. J. Syst. Evol. Microbiol. 55, 1271–1277. [10] Douidah, L., De Zutter, L., Vandamme, P., Houf, K. (2010) Identification of five human and mammal associated Arcobacter species by a novel multiplex-PCR assay. J. Microbiol. Methods 80, 281–286. [11] Douidah, L., de Zutter, L., Baré, J., De Vos, P., Vandamme, P., Vandenberg, O., Van den Abeele, A.M., Houf, K. (2012) Occurrence of putative virulence genes in Arcobacter species isolated from humans and animals. J. Clin. Microbiol. 50, 735–741. [12] Figueras, M.J., Collado, L., Guarro, J. (2008) A new 16S rDNA-RFLP method for the discrimination of the accepted species of Arcobacter. Diagn. Microbiol. Infect. Dis. 62, 11–15. [13] Figueras, M.J., Collado, L., Levican, A., Perez, J., Solsona, M.J., Yustes, C. (2011) Arcobacter molluscorum sp. nov., new species isolated from shellfish. Syst. Appl. Microbiol. 34, 105–109. [14] Figueras, M.J., Beaz-Hidalgo, R., Collado, L., Martínez-Murcia, A. (2011) Recommendations for a new bacterial species description based on analyses of the unrelated genera Aeromonas and Arcobacter. Bull. BISMiS 2, 1–16. [15] Figueras, M.J., Levican, A., Collado, L. (2012) Updated 16S rDNA-RFLP method for the identification of all currently characterized Arcobacter spp. BMC Microbiol. 18 (12), 292, http://dx.doi.org/10.1186/1471-2180-12-292. [16] Figueras, M.J., Levican, A., Collado, L., Inza, M.I., Yustes, C. (2011) Arcobacter ellisii sp. nov., isolated from mussels. Syst. Appl. Microbiol. 34, 414–418. [17] Figueras, M.J., Levican, A., Pujol, I., Ballester, F., Rabada Quilez, M.J., GomezBertomeu, F. (2014) A severe case of persistent diarrhoea associated with Arcobacter cryaerophilus but attributed to Campylobacter sp. and a review of the clinical incidence of Arcobacter. New Microbe. New Infect. 2, 31–37, http://dx.doi.org/10.1002/2052-2975.35. [18] Fong, T.T., Mansfield, L.S., Wilson, D.L., Schwab, D.J., Molloy, S.L., Rose, J.B. (2007) Massive microbiological groundwater contamination associated with a waterborne outbreak in Lake Erie, South Bass Island, Ohio. Environ. Health Perspect. 115, 856–864. [19] Houf, K., On, S.L., Coenye, T., Debruyne, L., De Smet, S., Vandamme, P. (2009) Arcobacter thereius sp. nov., isolated from pigs and ducks. Int. J. Syst. Evol. Microbiol. 59, 2599–2604. [20] Houf, K., On, S.L., Coenye, T., Mast, J., Van Hoof, J., Vandamme, P. (2005) Arcobacter cibarius sp. nov., isolated from broiler carcasses. Int. J. Syst. Evol. Microbiol. 55, 713–717. [21] Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J., Vandamme, P. (2000) Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS Microbiol. Lett. 193, 89–94. [22] Kim, H.M., Hwang, C.Y., Cho, B.C. (2010) Arcobacter marinus sp. nov. Int. J. Syst. Evol. Microbiol. 60, 531–536. [23] Kopilovic, B., Ucakar, V., Koren, N., Krek, M., Kraigher, A. (2008) Waterborne outbreak of acute gastroenteritis in a coastal area in Slovenia in June and July 2008. Eurosurveillance 13, 1–3. [24] Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948.
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[25] Levican, A., Collado, Figueras, M.J. (2013) Arcobacter cloacae sp. nov. and Arcobacter suis sp. nov., two new species isolated from food and sewage. Syst. Appl. Microbiol. 36, 22–27. [26] Levican, A., Collado, L., Aguilar, C., Yustes, C., Diéguez, A.L., Romalde, J.L., Figueras, M.J. (2012) Arcobacter bivalviorum sp. nov. and Arcobacter venerupis sp. nov., new species isolated from shellfish. Syst. Appl. Microbiol. 35, 133–138. [27] Levican, A., Alkeskas, A., Günter, C., Forsythe, S.J., Figueras, M.J. (2013) Adherence to and invasion of human intestinal cells by Arcobacter species and their virulence genotypes. Appl. Environ. Microbiol. 79, 4951–4957. [28] Levican, A., Collado, L., Yustes, C., Aguilar, C., Figueras, M.J. (2014) Higher water temperature and incubation under aerobic and microaerobic conditions increase the recovery and diversity of Arcobacter spp. from shellfish. Appl. Environ. Microbiol. 80, 385–391. [29] McClung, C.R., Patriquin, D.G., Davis, R.E. (1983) Campylobacter nitrofigilis sp. nov., a nitrogen fixing bacterium associated with roots of Spartina alterniflora Loisel. Int. J. Syst. Bacteriol. 33, 605–612. [30] Miller, W.G., Wesley, I.V., On, S.L., Houf, K., Mégraud, F., Wang, G., Yee, E., Srijan, A., Mason, C.J. (2007) The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri. PLoS One 2, e1358. [31] On, S.L.W., Holmes, B. (1995) Classification and identification of campylobacters, helicobacters and allied taxa by numerical analysis of phenotypic characters. Syst. Appl. Microbiol. 18, 374–390. [32] Rice, E.W., Rodgers, M.R., Wesley, I.V., Johnson, C.H., Tanner, S.A. (1999) Isolation of Arcobacter butzleri from ground water. Lett. Appl. Microbiol. 28, 31–35. [33] Sasi Jyothsna, T.S., Rahul, K., Ramaprasad, E.V., Sasikala, Ch., Ramana, Ch.V. (2013) Arcobacter anaerophilus sp. nov., isolated from an estuarine sediment and emended description of the genus Arcobacter. Int. J. Syst. Evol. Microbiol. 63, 4619–4625. [34] Stackebrandt, E., Frederiksen, W., Garrity, G.M., Grimont, P.A.D., Kämpfer, P., Maiden, M.C.J., Nesme, X., Rosselló- Mora, R., Swings, J., Trüper, H.J., Vauterin, L., Ward, A.C., Whitman, W.B. (2002) Report of the ad hoc committee for the reevaluation of the species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 52, 1043–1047. [35] 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. Mol. Biol. Evol. 28, 2731–2739. [36] Tindall, B.J., Rosselló-Mora, R., Busse, H.J., Ludwig, W., Kämpfer, P. (2010) Notes on the characterization of prokaryote strains for taxonomic purposes. Int. J Syst. Evol. Microbiol. 60, 249–266. [37] Ursing, J., Lior, H., Owen, R. (1994) Proposal of minimal standards for describing new species of the family Campylobacteraceae. Int. J. Syst. Evol. Microbiol. 44, 842–845. [38] Vandamme, P., Dewhirst, F.E., Paster, B.J., On, S.L.W. (2005) Genus II. Arcobacter Vandamme, Falsen, Rossau, Segers, Tytgat and De Ley 1991, 99VP. In: Brenner, D.J., Kreig, N.P., Staley, J.T., Garrity, G.M. (Eds.), Bergey’s Manual of Systematic Bacteriology, second ed., Springer, New York, NY, pp. 1161–1165. [39] Vandamme, P., Falsen, E., Rossau, R., Hoste, B., Segers, P., Tytgat, R., De Ley, J. (1991) Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int. J. Syst. Bacteriol. 41, 88–103. [40] Wesley, I.V., Miller, G.W. (2010) Arcobacter: an opportunistic human foodborne pathogen? In: Scheld, W.M., Grayson, M.L., Hughes, J.M. (Eds.), Emerging Infections 9, ASM Press, Washington, DC, pp. 185–211.
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Arcobacter ebronensis sp. nov. and Arcobacter aquimarinus sp. nov., two new species isolated from marine environment Arturo Levican a,b,c , Sara Rubio-Arcos a , Antonio Martinez-Murcia d , Luis Collado e , María José Figueras a,∗ a Unitat de Microbiologia, Departament de Ciències Mediques Bàsiques, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain b Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas Universidad Andrés Bello, Vi˜ na del Mar, Chile c Interdisciplinary Center for Aquaculture Research (INCAR) , Concepción, Chile d Area de Microbiología, EPSO, Universidad Miguel Hernández, Orihuela, Alicante 03312, Spain e Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile
a r t i c l e
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Article history: Received 18 May 2014 Received in revised form 28 October 2014 Accepted 31 October 2014 Keywords: Arcobacter A. ebronensis A. aquimarinus MLPA 16S rRNA MALDI-TOF
a b s t r a c t Two strains recovered from mussels (F128-2T ) and sea water (W63T ) were characterized as Arcobacter sp., but they could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR) and rpoB gene analysis. The 16S rRNA gene sequence similarity to the type strains of all Arcobacter species ranged from 92.2% to 96.7% with strain F128-2T , and from 94.1% to 99.4% with strain W63T , the most similar being A. bivalviorum (CECT 7835T ) and A. defluvii (CECT 7697T ), respectively. The phylogenetic analyses of 16S rRNA, and the concatenated sequences of gyrB, gyrA, rpoB, atpA and hsp60 genes confirmed that strains F128-2T and W63T belonged to two new lineages within the genus Arcobacter. Moreover, both strains showed differential phenotypic characteristics and MALDI-TOF mass spectra from all other Arcobacter species. Therefore, it has been demonstrated the existence of two new Arcobacter species and the proposed names are Arcobacter ebronensis (type strain F128-2T = CECT 8441T = LMG 27922T ), and Arcobacter aquimarinus (type strain W63T = CECT 8442T = LMG 27923T ). © 2014 Elsevier GmbH. All rights reserved.
Members of the genus Arcobacter (Ar’co.bac.ter. L. n. arcus, bow; Gr. n. bacter, rod; M. L. masc. n. Arcobacter, bow-shaped rod) are Gram negative, slightly curved rods positive for oxidase and usually motile and belong to the Epsilonproteobacteria and family Campylobacteraceae [4,38]. These bacteria had previously been classified as Campylobacter spp. because of their similar morphology, despite they differed from the latter genus because they are aerotolerant and able to grow at lower temperatures [4,38]. In fact, based on the latter characteristics Vandamme et al. created the genus Arcobacter in 1991 with 2 species formerly known as campylobacters, Arcobacter nitrofigilis and Arcobacter cryaerophilus [39]. Currently this genus includes 18 species that have been recovered from different hosts and environments [4,33]. Moreover, the analysis of the 16S rRNA gene sequences deposited in GenBank indicates that many other potential new Arcobacter species remain to be characterized [40]. Some Arcobacter spp. have been linked with gastroenteritis and bacteraemia in humans, and with abortions, mastitis and diarrhoea
∗ Corresponding author. Tel.: +34 977759321; fax: +34 977759322. E-mail address: mariajose.fi[email protected] (M.J. Figueras). http://dx.doi.org/10.1016/j.syapm.2014.10.011 0723-2020/© 2014 Elsevier GmbH. All rights reserved.
in animals, and they are considered as potential water and foodborne pathogens [4,5,17]. In this sense, it has been demonstrated that the presence of Arcobacter in water increases with the levels of faecal pollution [6]. It was suggested that Arcobacter spp, entered seawater with the contaminated inputs of freshwater despite some species could be autochthonous of the marine environment [6]. So far Arcobacter was recovered either from the water or from the stools of the patients in 3 drinking water outbreaks, 2 of them in USA and 1 in Slovenia [18,23,32]. However, in none of them the implication of this microbe was completely proven. Arcobacter spp. have also been found in association with food of animal origin, mainly meat products, but also in shellfish [4,5,28]. Shellfish could be an important reservoir and source of infection of these bacteria, as suggested in recent studies [5,28]. In a recent study on the prevalence of Arcobacter in different types of shellfish collected from the Ebro river delta [28], one strain recovered from mussels (F128-2T ) could not be assigned to any known species. The same occurred for another strain of our collection (W63T ) obtained from a seawater sample. The objective of the present study was to establish the taxonomic position of both strains (F128-2T and W63T ) using a polyphasic approach.
A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
Strain F128-2T was obtained from mussels collected from the Ebro delta, Catalonia (northeast Spain) in June 2011, while strain W63T was isolated from a seawater sample at the Garraf beach also in Catalonia, in September 2008. Both strains showed the typical colony morphology of arcobacters on blood agar (small, translucent, beige to off-white) and were Gram negative, slightly curved rods that produce oxidase activity, as previously described for the species of this genus [4,39]. The characterization of the strains was initially attempted using specific Arcobacter identification methods i.e. two Multiplex-PCR (m-PCR) [10,21] and the restriction fragment length polymorphism of the 16S rRNA gene (16S rDNA-RFLP) [12,15]. Both strains (F1282T and W63T ) produced an amplicon of the expected size described for A. cryaerophilus with the m-PCR of Houf et al. [21]. However, with the m-PCR of Douidah et al. [10] strain W63T produced the expected amplicon for Arcobacter butzleri while strain F128-2T , produced no amplification. On the other hand, with the recently updated16S rRNA RFLP identification method [15] strain F128-2T produced a new pattern after digestion with the endonuclease MseI (719, 138, 81, 52, 34 and 3 bp; Fig. S1), while W63T produced the same pattern described for Arcobacter defluvii when digested with MseI [7] or BfaI endonucleases [15] (Fig. S1). Considering these contradictory results, the rpoB (621 bp) and the 16S rRNA (1401 bp) genes of both strains (F128-2T and W63T ) were sequenced and analysed as described previously [3,25] and the constructed phylogenetic trees indicated that both strains formed independent phylogenetic lines within the genus (Fig. 1 and Fig. S2). In the 16S rRNA gene tree, strain W63T clustered with the species A. defluvii, Arcobacter cloacae and Arcobacter ellisii, whereas the most closest species for strain F128-2T were A. bivalviorum and A. anaerophilus (Fig. 1). The 16S rRNA gene similarity, calculated with the EzTaxon software [2], between strains F128-2T and W63T was 95.7%. Similarities between strain F128-2T and other Arcobacter spp. ranged from 92.2%, with the type strain of A. cryaerophilus (LMG 9904T ), to 96.7%, with the type strain of A. bivalviorum (CECT 7835T ). The similarities of strain W63T with other arcobacters ranged from 94.1%, with the type strain of Arcobacter mytili (CECT 7386T ), to 99.4%, with the type strain of A. defluvii (CECT 7697T ). These similarities were all within the range from 91.1% (for A. cryaerophilus and A. bivalviorum) to 99.6% (for A. cloacae and A. ellisii) described for the genus [16,25]. In the definition of several Arcobacter species the use of the concatenated sequences of housekeeping genes (gyrA, atpA, rpoB, gyrB and hsp60) has shown a better resolution, than DDH results [3,7,8,13,16,25,26]. In fact, the “ad hoc committee for the reevaluation of the species definition in bacteriology” has suggested that this approach could be used as an alternative to DDH if correlation with the latter method was demonstrated [34] as has recently been done for the genus Arcobacter [7,13,25,26]. This approach has been named multilocus sequence analysis (MLSA) or multilocus phylogenetic analysis (MLPA) [14,25,36]. In the Arcobacter studies, the MLPA has also provided a more robust overall phylogenetic relatedness (bootstrap values of 100% for all the species clusters) than 16S rRNA gene [3,7,8,13,16,25,26]. For strains F128-2T and W63T , apart from the rpoB, the sequences of the 4 remaining genes i.e. gyrB (618 bp), gyrA (686 bp), atpA (622 bp), and hsp60 (595 bp) were obtained as described previously [7,25]. In addition, in order to complete the MLPA with all currently accepted species, the sequences of the 5 genes (gyrA, atpA, rpoB, gyrB and hsp60) from the strain DSM 24636T of the recently described species A. anaerophilus [33] were obtained. Alignments were performed using the MEGA software version 5 [35] and CLUSTAL W [24] and clustering, using the neighbour-joining, maximum parsimony and maximum likelihood algorithms. The phylogenetic tree obtained with the sequences of the 5 concatenated genes (3142 bp), using
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different the neighbour joining (Fig. 2) and other algorithms (data not shown) confirmed initial rpoB results that both strains belonged to two independent and unknown phylogenetic lines within the genus. Both strains (F128-2T and W63T ) were characterized as motile under the phase contrast microscope, and under the transmission electron microscope, they showed a single polar flagellum (Fig. S3). Further characterization was carried out using the test recommended in the minimal standards for the family Campylobacteraceae [37], as well as others tests previously used in the description of other new Arcobacter spp. [25]. All tests were carried out at least twice for the 2 new strains and for all the type strains of Arcobacter species with the exception of A. anaerophilus that could not be maintained alive despite repeated efforts using different culture conditions. The test that were able to differentiate strains F128-2T and W63T between them and also from all other Arcobacter spp. are shown in Table 1. Interestingly, strain F128-2T showed growth on media with 4% NaCl, as also did other species isolated in association with shellfish or starfish like A. nitrofigilis, Arcobacter skirrowii, Arcobacter halophilus, A. mytili, Arcobacter marinus, Arcobacter molluscorum and A. bivalviorum. However, F128-2T could be easily differentiated from all of these species by 7 to 12 tests (Table 1). On the other hand, the closest phylogenetic species to F128-2T was A. anaerophilus (Figs. 1 and 2). However, they could be differentiated by at least 3 tests (Table 1) and by the fact that A. anaerophilus is strictly anaerobe. The most similar species to strain W63T , was A. cloacae but they could be differentiated because, in contrast to W63T , the later species is able to grow on MacConkey but not on blood agar at 30 ◦ C under anaerobic conditions [25]. Additional characterization of the mussels (F128-2T ) and seawater (W63T ) strains included the analysis of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS). This analysis was carried out in parallel with the type strains of all Arcobacter species as previously described [16], with the only exception, as commented, of strain DSM 24636T of A. anaerophilus which could not be kept alive in our laboratory to carry out the MALDI-TOF analysis. The obtained MALDI-TOF profiles of strains were hierarchically clustered in a dendrogram using the SPECLUST web tool (http://bioinfo.thep.lu.se/speclust.html) [1]. In the obtained dendrogram, strain F128-2T clustered close to the type strain of A. halophilus (LA31BT ) and W63T , close to the type strain of A. cloacae, CECT 7834T (Fig. S4 and Table S1). Considering the origin of the studied strains (shellfish and water), they both were tested for the presence of 5 putative virulence genes (ciaB, irgA, hecA, cj1349 and cadF) as previously described [11]. Strains F128-2T and W63T possessed the ciaB gene that codifies for a major invasine protein in genus Campylobacter [11,30], while W63T also possessed the cj1349 gene, which codifies for a fibronectin binding protein in Campylobacter jejuni. Despite of this, strain W63T was not able to adhere or invade the human intestinal Caco-2 cell lines in a previous study [27]. These results warrant future studies on the potential pathogenic role of F128-2T and W63T for humans. In this study it has been demonstrated the existence of two new Arcobacter species, for which the names Arcobacter ebronensis (type strain F128-2T = CECT 8441T = LMG 27922T ), and Arcobacter aquimarinus (type strain W63T = CECT 8442T = LMG 27923T ) are proposed.
Description of Arcobacter ebronensis sp. nov. Arcobacter ebronises (e.bro.nen’sis. N.L. masc. adj. ebronensis, of or belonging to Ebro river delta Spain, where shellfish sample harbouring strain F128-2T was collected)
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A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
100 A. cryaerophilus LMG 9904T (L14624)
A. cryaerophilus LMG 9865(FR682113) A. skirrowii LMG 6621T (L14625) 99
A. skirrowii Houf 989 (GU300769) A. trophiarum CECT 7650 (FE2) (GU300768)
T 99 A. trophiarum LMG 25534 (FN650333) 73 A. trophiarum LMG 25535 (FN650332)
A. thereius LMG 24487 (AY314754)
95 98
A. thereius LMG 24486T (AY314753) A. cibarius CECT 7203T (AJ607391)
100
100 A. cibarius LMG 21997 (AJ607392)
A. butzleri F46 (GU300771) 100 A. butzleri LMG 10828T (AY621116)
A. venerupis F67-11T (HE565359)* A. suis F41T (FJ573216)* 76
100 91
91
A. defluvii SW 28-7 (HQ115597) A. defluvii SW30-2 (HQ115596) A. defluvii CECT 7697T (HQ115595) A. aquimarinus sp. nov. W63T (HG932574)
79
A. cloacae SW28-13T (HE565360)
78
A. cloacae F26 (HE565361) A. ellisii CECT 7837T (FR717550)
100 97
A. ellisii F79-7 (FR717552) A. ellisii F79-2 (FR717551) 100 A. bivalviorum F4T (FJ573217) 100
93
A. bivalviorum F118-2 (HE565357) A. bivalviorum F118-4 (HE565358)
77
A. ebronensis sp. nov. F128 2T (HG932573) A. anaerophilus JC84(FR686494)* 100
A. nitrofigilis CECT 7204T (L14627) A. nitrofigilis F2176 (EU106662) 100 A. mytili CECT 7385 (EU669906)
A. mytili CECT 7386T (EU669904) A. halophilus LA31BT (AF513455)*
86
A. marinus CECT 7727T (CL-S1T)(EU512920)*
84
A. molluscorum F101-1 (FR675875)
98 100
A. molluscorum F91 (FR675876) A. molluscorum F98-3T (FR675874)
0.005 Fig. 1. Neighbour joining tree based on 16S rRNA (1401 bp) sequences showing the phylogenetic position of A. ebronensis sp. nov. and A. aquimarinus sp. nov. within the genus Arcobacter. Bootstrap values (>70%) based on 1000 replications are shown at the nodes of the tree. Bar, 5 substitutions per 1000 nt. * Only type strain is available so far.
Cells of strains F128-2T are Gram-negative slightly curved rods, non-encapsulated, non-spore forming, 0.3–0.5 m wide and 1.5–2.5 m long. They are motile by a single polar flagellum. Colonies on blood agar incubated under aerobic conditions at 30 ◦ C for 48 h are 2–4 mm in diameter, beige to off-white, circular with entire margins, convex, and non-swarming. Pigments are not produced. This strain grows on blood agar at room temperature (18–22 ◦ C) and 30 ◦ C under both aerobic and microaerobic conditions with no significant differences. Growth of this strain is slightly enhanced by adding 1% NaCl to media. No growth is observed under anaerobic conditions as well as under aerobic or microaerobic conditions at 37 ◦ C or 42 ◦ C. No haemolysis is observed on TSA medium supplemented with 5% sheep blood. Strain F128-2T produce oxidase but not catalase activity. It hydrolyse indoxyl acetate and urea
but not casein, lecithin or starch. The strain is not able to produce acid from glucose by oxidization or fermentation, nor hydrogen sulphide, in triple-sugar iron agar medium, and it is not able to reduce nitrate. Under aerobic conditions at 30 ◦ C the strain grows on Marine Agar, minimal medium and on nutrient medium supplemented with 5% sheep blood containing 0.5–4% (w/v) NaCl; 0.1% sodium deoxycholate; 0.05% safranin; or 0.005% basic fuchsine. No growth occurs on, MacConkey agar, campylobacter charcoal deoxycholate agar (CCDA), and on nutrient medium supplemented with 5% sheep blood containing 1% glycine; 0.01–0.1% 2,3,5-triphenyl tetrazolium chloride (TTC); 64 mg l−1 cefoperazone;. 1% oxgall; 0.0005% crystal violet or 0.001% brilliant green A BLASTN analysis of the 16S rRNA sequence of strain F128-2T showed the closest match (98%) with strain sw026 isolated from
A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
33
A. cryaerophilus LMG9904T
100
A. cryaerophilus LMG9865 A. skirrowii LMG6621T 100
84
A. skirrowii HOUF989 A. thereius LMG24486T
100
A. thereius LMG24487
100
A. trophiarum LMG25534T 100 A. trophiarum CECT7650
76
A. cibarius CECT7203T 100 A. cibarius Houf 746
A. butzleri LMG 10828T 100
A. butzleri F46 A. cloacae SW28-13T (CECT 7834T)
100 100
A. cloacae F26 Arcobacter aquimarinus sp. nov. W63T A. venerupis F67-11T (CECT 7836T)*
100
100
A. suis F41T (CECT 7833T)* A. defluvii SW28-11T (CECT 7697T)
73 100
A. defluvii SW28-7 A. defluvii SW30-2
89
A. ellisii F79-2 100 98
A. ellisii F79-6T (CECT 7837T) A. ellisii F79-7 100 A. nitrofigilis CECT7204T 100
A. nitrofigilis LMG7547 A. nitrofigilis F2176 A. anaerophilus DMS 24636T*
99
Arcobacter ebronensis sp. nov. F128-2T 100 100
99
A. bivalviorum F4T (CECT 7835T) A. bivalviorum F118-2 A. bivalviorum F118-4 100 A. mytili CECT7386T
A. mytili CECT7385 98
100
A. halophilus DSM18005T* A. marinus CECT7727T*
80
A. molluscorum F98-3T (CECT 7696T) 100 A. molluscorum F91 82 A. molluscorum F101-1
0.02
Fig. 2. Neighbour joining tree based on the concatenated sequences of gyrA, gyrB, rpoB, atpA and hsp60 (3142 bp) sequences showing the phylogenetic position of A. ebronensis sp. nov. and A. aquamarinus sp. nov. within the genus Arcobacter. Bootstrap values (>70%) based on 1000 replications are shown at the nodes of the tree. Bar, 2 substitutions per 100 nt. * Only the type strain is available so far.
sea-surface water of the South Pacific (GenBank accession number JN118550). However, both strains form distinct phylogenetic lines (data not shown). The type strain is F128-2T (=CECT 8441T = LMG 27922T ), isolated from mussels collected at the Ebro river delta, Catalonia, Spain. Description of Arcobacter aquimarinus sp. nov. A. aquimarinus (a.qui.ma.rinus. L. fem. n. aqua water; L. adj. marinus of the sea). Cells of strains W63T are Gram-negative slightly curved rods, non-encapsulated, non-spore forming, 0.4–0.6 m wide and 1.0–2.0 m long. They are motile by a single polar flagellum.
Colonies on blood agar incubated in aerobic conditions at 30 ◦ C for 48 h are 2–4 mm in diameter, beige to off-white, circular with entire margins, convex, and non-swarming. Pigments are not produced. This strain grows on blood agar at room temperature (18–22 ◦ C), 30 ◦ C or 37 ◦ C under both aerobic and microaerobic conditions with no significant differences, and at 30 ◦ C under anaerobic conditions. No growth is observed under any incubation conditions at 42 ◦ C. No haemolysis is observed on TSA medium supplemented with 5% sheep blood. Strains produce oxidase and catalase activity, reduce nitrate, hydrolyse indoxyl acetate but not urea, casein, lecithin or starch. The strain is not able to produce acid from glucose by oxidization or fermentation, nor hydrogen sulphide, in triple-sugar iron agar medium. Under aerobic conditions at 30 ◦ C
34
A. Levican et al. / Systematic and Applied Microbiology 38 (2015) 30–35
Table 1 Differential characteristics of A. ebronensis sp. nov. and A. aquimarinus sp. nov. from other members of the genus arranged in order of description. Taxa: 1, A. ebronensis (n = 1); 2, A. aquimarinus (n = 1); 3, A. nitrofigilis (n = 4) [3,29,31]; 4, A. cryaerophilus (n = 19) [3,31]; 5, A. butzleri (n = 12) [31]; 6, A. skirrowii (n = 9) [31]; 7, A. cibarius (n = 15) [20]; 8, A. halophilus (n = 1) [9,16]; 9, A. mytili (n = 3) [3]; 10, A. thereius (n = 8) [19]; 11, A. marinus (n = 1) [22]; 12, A. trophiarum (n = 11) [8]; 13, A. defluvii (n = 8) [3]; 14, A. molluscorum (n = 3) [13]; 15, A. ellisii (n = 3) [16]; 16, A. bivalviorum (n = 3) [26]; 17, A. venerupis (n = 1) [26]; 18, A. cloacae (n = 2) [25]; 19, A. suis (n = 1) [25]; 20, A. anaerophilus (n = 1) [33]. The specific responses for type strains were coincidental or expressed in brackets. Unless otherwise indicated: +, ≥95% strains positive; −, ≤11% strains positive; V, 12–94% strains positive; CO2 indicates microaerobic conditions. Characteristics Growth in/on Air at 37 ◦ C CO2 at 37 ◦ C CO2 at 42 ◦ C 0.5% (w/v) NaCl 4% (w/v) NaCl 1% (w/v) glycine 0.05% safranin 0.1% sodium deoxycholate 1% (w/v) oxgall 0.04% TTC 0.01% TTC Minimal medium MacConkey CCDA Resistance to: Cefoperazone (64 mg l–1 ) Enzyme activity Catalase Urease Nitrate reduction Indoxyl acetate hydrolysis
1a
2
3
4
5
6
7
8a
9
10
11a
12
13
14
15
16
17
18
19
20
− − − + + − + + − − − + − −
+ + − + − − + + + − + − − +
V(−) − − + + − − V(−) − − − − − −
V(+) V(+) − + − − + V(+) + + + −c V(−) +
+ + V(+) + − − + + V(+) + + + + +
+ + − + + − + + + V(−) + − − +
− + − + − − + + + V(−) + + + V(−)
+ + − − + + − − − − − − − −
+ + + + + + − + + − − − + −
− − − + − + + V(−) − V(−) + + V(+) V(−)
+ + − − + + + − − − − − − −
− − − + − V(−) V(+) + + + + −d V(+)e +
+ + + + − − + + + − + + + +
+ + + + + − + + + − + − + −
+ + + + − − − +b − − − + V(+) +b
+ + − + + − − − − − − − − −
− + − + − − − − − − − + + +
+ + − + − − + + + − + V(+) + +
− − − + − − − + − − + + + −
− − − + + + ND ND ND ND ND ND ND ND
−
−
−
+
+
+
+
−
−
+
−
+
V(+)
+
−
−
−
−
−
−
− + − +
+ − + +
+ + + +
+ − +g +
V(+) − + +
+ − + +
V(−) − − +
− − + +
+f − +h −
+ − + +
− − + +
+ − − +
+f + + +
+ − +i −
+ V(−) + +
+ − − +
+ + + +
+ − + +
+ − + +
− − + +
a For these strains, the tests were carried out on media supplemented with 2% NaCl, with the exception of 0.5 and 4% (w/v) NaCl, catalase and indoxyl acetate hydrolysis [16]. b All strains grew weakly after 5 days of incubation. c Two (LMG 7537 and LMG 10241) of the four strains tested were positive [3]. d Test not evaluated by De Smet et al. [8] but tested by Figueras et al., [16]. e Strains LMG 25534T , LMG 25535 of A. trophiarum and strain FE2 (CECT 7650) of this species identified in our laboratory grew on MacConkey agar in contraposition with the 80% described for this species [8]. f Weak reaction [3,7]. g Two (LMG 9904 T and LMG 9065) of the four strains tested were negative [3]. h Nitrate reduction was positive for the 3 strains of A. mytili [13], contrary to our previously published data [3]. i Nitrate is reduced after 72 h and 5 days for all strains under microaerobic and aerobic conditions, respectively [16].
the strain grows on Marine Agar, campylobacter charcoal deoxycholate agar (CCDA) and on nutrient medium supplemented with 5% sheep blood also containing 0.5–2% (w/v) NaCl; 0.1% sodium deoxycholate; 1% oxgall; 0.01% 2,3,5-triphenyl tetrazolium chloride (TTC); 0.05% safranin; 0.0005% crystal violet or 0.001% brilliant green. No growth occurs on minimal medium, MacConkey agar and on nutrient medium supplemented with 5% sheep blood containing 4% (w/v) NaCl; 1% glycine; 0.04–0.1% TTC; 64 mg l−1 cefoperazone; or 0.005% basic fuchsine. A BlastN analysis of the 16S rRNA sequence of strain W63T matched with 99% with several sequences of uncultured clones of arcobacters, as well as the strains of A. cloacae, A. defluvii and A. suis that are already included in this study. The type strain is W63T (=CECT 8442T = LMG 27923T ), isolated from a water sample collected from the Mediterranean Sea at the Garraf beach, Catalonia, Spain.
Acknowledgments We thank IRTA (Research and Technology Food and Agriculture) of Sant Carles de la Ràpita (Tarragona, Spain) for sampling the shellfish. We also thank Jean P. Euzéby for kindly reviewing the species names. A. L. thanks the Universitat Rovira i Virgili for a doctoral grant; and also CONICYT, Chile, for financial support through Becas Chile, FONDECYT postdoctoral research project number 3140296 and FONDAP grant number 15110027. This work was supported in part by the Ministerio de Ciencia e Innovación (MICINN), Spain, project number
AGL2011-30461-C02-02; and by funding from European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 311846. The GenBank/EMTBL/DDBJ accession numbers of the sequences of strains F128-2T and W63T , for the 16S rRNA gyrA, atpA, rpoB, gyrB and hsp60 genes are HG932563 to HG932574 and for the gyrA, atpA, rpoB, gyrB and hsp60 genes from the type strains of the species Arcobacter anaerophilus from LK392365 to LK392369. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm. 2014.10.011. References [1] Alm, R., Johansson, P., Hjernø, K., Emanuelsson, C., Ringnér, M., Häkkinen, J. (2006) Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra. J. Proteome Res. 5, 785–792. [2] Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K., Lim, Y.W. (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol. 57, 2259–2261. [3] Collado, L., Cleenwerck, I., Van Trappen, S., De Vos, P., Figueras, M.J. (2009) Arcobacter mytili sp. nov., an indoxyl acetate-hydrolysis-negative bacterium isolated from mussels. Int. J. Syst. Evol. Microbiol. 59, 1391–1396. [4] Collado, L., Figueras, M.J. (2011) Taxonomy, epidemiology and clinical relevance of the genus Arcobacter. Clin. Microbiol. Rev. 2011 (24), 174–192. [5] Collado, L., Guarro, J., Figueras, M.J. (2009) Prevalence of Arcobacter in meat and shellfish. J. Food Prot. 72, 1102–1106. [6] Collado, L., Inza, I., Guarro, J., Figueras, M.J. (2008) Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution. Environ. Microbiol. 10, 1635–1640.
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