Download Characterization of Enterococcus species occurring on terrestrial

Characterization of Enterococcus species
occurring on terrestrial vegetation
Czech Collection of Microorganisms
http://www.sci.muni.cz/ccm/
Pavel Švec, Lucie Zátopková, Hana Večerková, Ivo Sedláček
Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
e-mail: [email protected]
Similarity (%)
Introduction
20
40
60
80
A13
100
B31/2
G40
C36
E. mundtii
CCM 4067
F18
F15/2
F19
B29
The genus Enterococcus presently contains 48 validly recognised species
occurring in a wide variety of habitats comprising humans, animals and the
environment. The vast majority of studies dealing with enterococci focus
mainly on clinical and/or food-associated strains due to the high importance
of these strains for humans in the view of their medical and economical
impacts. In contrast, much less attention has been paid to the taxonomy of
the environmental Enterococcus spp. strains.
F4/1
G19
D17
I20
CCM 7889
F6
T
E23/1
D15
D32
D27/2
CCM 7000T
C38/2
D17
I6
D25
F58
E. plantarum
CCM 2478T
I74
E4
B4
F15/1
G10/2
CCM 4239
G25
I10
B1
B4
B12
Particularly, enterococci associated with terrestrial vegetation have been
studied scantily. Studies published by Müller et al. (2001), Ott et al. (2001),
Ulrich and Müller (1998), and Švec et al. (2012) revealed high taxonomic
heterogeneity among the enterococcal strains isolated from terrestrial plants
and showed that E. faecium, E. faecalis, E. sulfureus, E. mundtii,
E. casseliflavus, and E. plantarum species are typically associated with plants.
Moreover, high numbers of unidentified strains representing hitherto
unknown species were isolated in the frame of the aforementioned studies.
B28
I67
F9
E37/1
H77
F60I
E30/2
H53
E35
G33
CCM 2401
E. ureilyticus
CCM 2498
D23/2
I78I
A2
I83I
CCM 4629T
E35
D18
CCM 4858
G96
C12
I52
I58
C5II
I46
A3
C37
E. faecium
CCM 4856T
B28
Material and methods
D31
CCM 7800
E27/1
E55
B21
D26
C23
CCM 7167T
Bacterial strains
B36
H19
B33
CCM 4632
Repetitive extragenic palindromic-PCR with the (GTG)5 primer
The rep-PCR fingerprinting using the (GTG)5 primer was performed as described
previously (Švec et al., 2010). Briefly, bacterial DNAs isolated by alkaline extraction
procedure were included in PCR reactions performed by using a Tpersonal
thermocycler (Biometra). Initial denaturation at 94°C for 7 min was followed by 30
cycles of denaturation at 94°C for 1 min, primer annealing at 40°C for 1 min, and
extension at 65°C for 8 min. The last cycle was followed by the final elongation at
65°C for 16 min. Obtained PCR products were separated in 1.5% agarose gels.
Resulting fingerprints were digitized and processed using BioNumerics v. 7.1
software (Applied Maths) and compared within an in-house CCM reference database
containing more than 5000 fingerprints representing multiple Gram-positive
bacterial species including a large set of Enterococcus spp. type and representative
strains.
Results
In total, 173 strains revealing enterococcus-like colony morphology on
Kanamycin Aesculin Azide agar and Gram-positive and catalase negative
characteristics were isolated.
Rep-PCR fingerprints revealed by 102 isolates were clustered with Enterococcus
spp. reference database entries (Fig 1.) and were assigned as E. faecalis (23
strains), E. haemoperoxidus (20), E. plantarum (14), E. casseliflavus (12),
E. faecium (9), E. ureilyticus (8), E. moraviensis (6), E. rotai (6), E. durans (2), and
E. mundtii (2).
In total, 47% of Enterococcus spp. strains were retrieved from flowers, 27% from
whole plants, 14% from fruits and 11% from leaves.
Remaining 15 strains were identified as members of other genera (e.g.
Lactobacillus, Lactococcus, Vagococcus) or were not clustered with any reference
fingerprint included in the database (56 strains).
A11
G17
Analysed plant samples were obtained from four different localities unaffected by
farming or other intensive human activities during the vegetation periods in 2009
and 2013 in the frame of two studies dealing with the investigation of
Enterococcus spp. populations occurring on plants.
In total 862 plant specimens (flowers, fruits, leaves or whole plants) were
aseptically sampled and initially cultivated in Brain Heart Infusion broth (Oxoid) at
37°C for 48 h. Subsequently, the grown cultures were inoculated on Kanamycin
Aesculin Azide agar (Merck) and cultivated at 37°C for 48 h. Individual colonies
revealing typical black enterococcal colony morphology were picked up and purified
on Brain Heart Infusion agar (Oxoid).
In total, a group of 173 presumptive plant-associated enterococcal strains
revealing Gram-positive cocci and/or coccobacilli and negative catalase reaction
were isolated and investigated.
G13
G8
CCM 4630T
F29
E. rotai
B6
CCM 4633
E25
E40
G38
I80
J43II
E38
E13
F28
C18
E37/2
D1/2
B25
E27/2
F10
E24
F14
G25
CCM 2542
CCM 4851T
E3/1
CCM 4852
C5III
CCM 7263
C7
D3
B21
D9
B22
E. faecalis
E. durans
T
200
(bp)
500
1000
1500
2000
Molecular Size Marker
3000
A13
CCM 5612
5000
CCM 4224
E. haemoperoxidus
J43
G38
G24
E. moraviensis
I96
F56
D19I
E. casseliflavus
E13
Fig. 1. Cluster analysis of plant-associated enterococcal strains identified in this study. The dendrogram was calculated with
Pearson's correlation coefficients using the unweighted pair group method using arithmetic averages (UPGMA) clustering.
Conclusions
Cultivation of about 20% plant samples yielded typical black enterococcal
colonies on Kanamycin Aesculin Azide agar resulting in isolation of 173
suspected Enterococcus strains. However, only 102 strains were assigned as
known Enterococcus spp. Remaining 71 isolates were shown to be
representatives of other genera (15 strains) or were not identified (56
strains).
Plants are inhabited by diverse and taxonomically rich Enterococcus spp.
populations. Enterococcus faecalis and E. haemoperoxidus were the most
abundant species isolated in this study.
Majority of Enterococcus spp. strains (47%) were isolated from flowers
which supports the theory proposed by Mundt (1961, 1962) suggesting that
insects are involved in the dissemination of enterococci between plants. Also
the nectar produced in flowers may represent an important source of nutrients
supporting propagation of enterococci requiring complex nutrition.
Isolation of recently described Enterococcus spp. (e.g. E. plantarum,
E. rotai, E. ureilyticus) representatives and a high proportion of the
unidentified strains indicate that the populations associated with plants may
contain hitherto unknown enterococcal species.
References
Mundt J.O. (1961) Occurence of enterococci: bud, blossom, and soil studies. Appl Microbiol 9, 541-544.
Mundt J.O. (1962) Occurence of enterococci on plants in a wild environment. Appl Microbiol 11, 141-144.
Müller, T., Ulrich, A., Ott, E.M., Müller, M. (2001) Identification of plant-associated enterococci. J Appl Microbiol 91, 268-278.
Ott, E.M., Müller, T., Müller, M., Franz, C.M.A.P., Ulrich, A., Gabel, M., Seyfarth, W. (2001) Population dynamics and antagonistic potential of
enterococci colonizing the phyllosphere of grasses. J Appl Microbiol 91, 54-66.
Švec, P., Pantůček, R., Petráš, P., Sedláček, I., Nováková, D. (2010). Identification of Staphylococcus spp. using (GTG)5-PCR fingerprinting. Syst Appl
Microbiol 33, 451-456.
Švec, P., Vandamme, P., Bryndová, H., Holochová, P., Kosina, M., Mašlaňová, I., Sedláček, I. (2012). Enterococcus plantarum sp. nov., isolated from
plants. Int J Syst Evol Microbiol 62, 1499-1505.
Acknowledgements
This work was supported by the project CZ.1.07/2.3.00/20.0183.
Ulrich, A., Müller, T. (1998) Heterogeneity of plant-associated streptococci as characterized by phenotypic features and restriction analysis of PCRamplified 16S rDNA. J Appl Microbiol 84, 293-303.