This document describes a study that isolated lactic acid bacteria (LAB) from various plant and vegetable matrices. 22 LAB strains were isolated belonging to Lactobacillus, Lactococcus, and Enterococcus genera. The predominant species were Lactobacillus brevis (57%) and Lactobacillus delbrueckii subsp. bulgaricus (14%). The strains were tested for their ability to produce bacteriocin-like substances (BLS) with antimicrobial activity. A conjugation experiment was then successfully performed between Enterococcus faecium and Lactobacillus acidophilus to demonstrate horizontal gene transfer of BLS production.

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ISOLATION AND IDENTIFICATION OF LACTIC
ACID BACTERIA FROM PLANTS AND OTHER
VEGETABLE MATRICES AND MICROBIAL
RECOMBINATION WITH ENTEROCOCCUS SPP.
Immacolata Anacarsoi
, Lara Bassoli, Carla Sabia, Ramona Iseppi,
Carla Condò
Dept. Life Sciences, University of Modena and Reggio Emilia, Italy
Abstract: Twenty-two lactic acid bacteria (LAB) belonging to Lactobacillus, Lactococcus and
Enterococcus genera were isolated from plants, flowers and other vegetable matrices. The predominant
LAB species were Lactobacillus brevis (57%) followed to Lactobacillus delbrueckii subsp. bulgaricus
(14%) and Lactococcus lactis (14%). Among enterococci E. faecalis (4 /50%), E. faecium (2 /25%), E.
hirae (2 /12,5%) were isolated. The strains were identified previously with biochemical kits and then
confirmed by PCR. Bacteria isolated were tested against nine strains selected among pathogenic and
opportunistic strains to evaluate their capacity to produce BLS (bacteriocin like substance).
Subsequently to demonstrate the horizontal transfer of genes coding for the production of bacteriocin
substances, a conjugation experiment was positively carried out between an Enterococcus faecium
used as donor and a Lactobacillus acidophilus used as recipient.
Key Words: Lactobacillus, Enterococcus, conjugation, bacteriocin.
INTRODUCTION
The lactic acid bacteria (LAB) are generally defined as a cluster of lactic acid-producing,
low %G + C, non-spore-forming, Gram-positive rods and cocci, catalase negative
bacteria which share many biochemical, physiological, and genetic properties. LAB
i Dr. Immacolata Anacarso – Dept. Life Sciences University of Modena and Reggio Emilia
Via Campi, 287 – 41125 - Modena (Italy)
Tel. +39 0592055795, Fax +39 0592055483.
E-mail: immacolata.anacarso@unimore.it

2. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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group include the most important groups of microorganisms like Lactobacillus,
Lactococcus, Enterococcus, usually used in food fermentations. These microorganisms in
fact contribute to the taste and to the texture of fermented products and inhibit the food
spoilage and pathogenic bacteria by producing an array of antimicrobial substances
such as large amounts of lactic acid (and other organic acids), hydrogen peroxide,
antifungal peptides, and bacteriocins (Daeschel, 1989; Dobrogosz and Lindgren, 1990).
Bacteriocins are the best-known antimicrobial compounds isolated from LAB.
These antimicrobial compounds show bactericidal activity against species closely
related to producers (Paker et al., 1989; Cintas et al., 2001) and they can exhibit
heterogeneous characteristics such as resistance to very low pH values, stability at a
very wide range of temperatures, resistance to organic acids, salts and enzymes. Some
studies show the potential use of bacteriocins as food preservatives or additives to
packaging for extend the products shelf life.
The importance of the LAB is because these bacteria have beneficial effects to the
consumers in different ways. Probiotic lactobacilli, for example, are known to confer an
array of health promoting activities on their host after either parenteral or oral
administration (Oyetayo and Osho, 2004). The other beneficial effects of the LAB
include prevention of intestinal infections (Casas and Dobrogosz, 2000),
anticarcinogenic activity (Kumari et al., 2011), control of serum cholesterol, immunity
promotion (Aattouri et al., 2001), and growth enhancement of animals (Chang et al.,
2001). The mechanism by which these probiotics affect their host and bring an
improvement in the gut barrier can be thanks to competition for the adhesion site, the
production of inhibitory compounds, the rebalancing of disturbed gastrointestinal
microbial composition and of the metabolism (FAO/WHO, 2001). Many are the study
about the new isolated LAB capable to produce natural bacteriocin substances, with a
broad spectrum. Many times the best bacteriocins producers belonging to the
Enterococcus genera. Though enterococci are LAB, and are the natural flora of some
fermented foods (cheese, salami etc.) they are however opportunistic bacteria capable to
produce many virulence factors and able to cause important pathologies (Sabia et al.,
2002); for these reasons the use of enterococci or bacteriocins produced from these
bacteria not always is well accepted. Always actual is the research of new bacteriocin
substances product by LAB as Lactobacillus or Lactococcus. Different are the matrices
from which these LAB were isolated, like kefir grains (Leite et al., 2015), fermented

3. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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sausages (Golneshin et al., 2015), wine (Ndlovu et al., 2015), olive (Hikmate et al., 2015)
usually starting to a fermented substrates.
In this study we have isolated different bacterial strains from plants, flowers and
other vegetable matrices, showing that also in these sample it was possible to isolate
LAB as Lactobacillus and Lactococcus plus Enterococcus. The strains isolated were
interesting because in large part were good BLS (bacteriocins like substances)
producers. The study here presented has also shown a conjugation experiment between
an Enterococcus donor and a Lactobacillus recipient to obtain a best bacteriocins
producers with an broad antimicrobial activity typically of a Enterococcus but from
Lactobacillus producers.
MATERIAL AND METHODS
Sample analysis
Forty different vegetable matrices derived from houseplants (including cacti, potted
flowers, bushes flowers, climber plants, seeds), leaves and flowers trees, were subjected
to microbiological analysis for the research of LAB.
Samples were weighted and diluted in a 1:10 ratio with physiological solution
and then homogenized by Stomacher for 1 minute. The obtained solutions, after
appropriate dilutions in physiological solution, were plated in Kanamycin Aesculin
Azide agar and MRS agar (Biomerieux, Milan-Italy) and incubated at 30°C in aerobic
and anaerobic conditions respectively for 48-72 h.
Bacterial identifications
For a preliminary recognition of isolated strains, several preliminary tests like Gram
stain, microscopic view and catalase analysis were carried out.
All strains grown on MRS agar that were Gram-positive, catalase negative and
cocco- or rod-shaped were before identified with API 50 CHL (Biomerieux, Milan-Italy)
and lastly identified by PCR. All strains grown on Kanamycin Aesculin Azide agar with
a typical aspect for enterococci were before identified with API 20 Strep (Biomerieux,
Milan-Italy) and lastly identified by PCR. DNA was extracted according to the method
of Marzotto et al., (2006). The PCR reactions were carried out by Techne Termal cycler
TC312 (Bibby Scientific, Italy). The following conditions were used: 2 min at 94°C, 30
cycles of 1 min at 92°C, 1 min at optimum temperature based on primers used, 1 min at

4. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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72°C and finally 10 min at 72°C. PCR primers and conditions used are listed in the Table
1. Also, negative controls containing no DNA template were included in the
experiment. All PCR products were electrophoresed in a 1% (w/v) agarose gel with 1 X
Tris/borate/EDTA buffer and viewed under UV light.
Evaluation of BLS (Bacteriocins like Substances) production
BLS production was performed by using the agar-spot-test method (Todorov and Dicks,
2005) on MRS (Man Rogosa Sharp) (Oxoid, Italy) for the LAB, correcting the cell-free
supernatant to pH 6.0 with 1M NaOH to prevent the inhibitory effect of lactic acid, and
on TSA (tryptic soy agar) (Oxoid, Italy) for enterococci. Nine strains were used as
indicators selected among pathogenic and opportunistic strains from registered
collections (ATCC - American Type Culture Collection; NCTC - National Collection of
Type Cultures): Listeria monocytogenes ATCC 13932, Enterococcus casseliflavus 416/K1
(Sabia et al., 2002), Listeria monocytogenes NCTC 10888, Staphylococcus aureus ATCC 6538,
Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Candida albicans ATCC
10231, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853.
Conjugation experiment
This experiment was performed with two of the strains isolated to demonstrate the
horizontal transfer of genes coding for the production of bacteriocins. Twelve different
conjugation experiments to obtain a positive result were done.
A strain of Enterococcus faecium EN313 (KanamycinR, TeicoplaninS), was selected
as donor, because producer of a bacteriocin with high antibacterial activity against
Listeria monocytogenes NCTC 10888, that was used as indicator. A strain of Lactobacillus
acidophilus LB813 (KanamicynS
, TeicoplaninR
), was selected as recipient, totally
incapable to produce any antibacterial substances vs Listeria monocytogenes NCTC 10888.
Overnight MRS broths of both strains were then combined in a ratio of 1:2 and
maintained under agitation for 48-72 h at 30°C and anaerobic conditions.
Conjugation broths were filtered on cellulose acetate filters with 0.45 µM pores
(Millipore, Italy), which were laid on MRS agar with the selective agents (Kanamycin 64
mg ml-1 and Teicoplanin 32 mg ml-1) and incubated at 30°C for 24h under anaerobic
conditions.
The donor and recipient were subjected to plasmid-DNA extraction by
O’Sullivan-Klaenhammer method (1993), to evaluate the presence of plasmids; the

5. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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extracts were electrophoresed in 0.7% (w/v) agarose gel with 1% Tris/borate/EDTA
buffer at 100 mV for 35 min and viewed under UV light.
Isolation of transconjugant
The colonies grown on filter surface were collected, suspended in 1 ml of MRS broth
and then aliquots of 100 µl were plated on MRS agar plates with the addition of both
selective agents (Kanamycin 64 mg ml-1 and Teicoplanin 32 mg ml-1). Plates were
incubated for 48-72 h at 30°C under anaerobic conditions and the bacteria colonies
grown after the incubation were used for a subsequent Gram stain and morphological
viewing under the microscope. Those with a rod-shape (easily distinguishable from the
cocco-shape of the donor strain) were isolated and extracted by O’Sullivan-
Klaehnammer method to evaluate the plasmidic bands. To confirm the belonging of
trasconjugats at the Lactobacillus specie, PCR with L. acidophilus primers listed in the
Table 1 was performed. Lastly, to exclude the contamination by E. faecium a PCR for the
research of a specie-specific ligase ddl E. faecium was performed (Dutka-Malen and al.,
1995). Transconjugant strains were tested by agar-spot-test method; previously
indicated, using L. monocytogenes NCTC 10888 as indicator, for evaluate the presence of
antimicrobial activity against the indicator.
RESULTS
Bacterial identification
Only from 6 vegetable matrices (Pteridium aquilinum leaves, Linum usitatissimum seeds,
Daucus carota root, Ficus benjamin leaves, Aloe Barbadensis leaves, Prunus cerasifera
flowers), of the 40 tested it was possible to isolate 14 different LAB including
Lactobacillus and Lactococcus and 8 different enterococci. The strains identification was
made in first time using API kits and lastly by PCR analysis, confirming the biochemical
results. For the LAB strains, 8 (57%) Lactobacillus brevis, 2 (14%) Lactobacillus delbrueckii
subsp. bulgaricus, 2 (14%) Lactococcus lactis, 1 (7%) Lactobacillus acidophilus and 1 (7%)
Leuconostoc were recovered. For the strains grown with the typical enterococci aspect,
on Kanamycin Aesculin Azide agar, 4 (50%) E. faecalis, 2 (25%) E. faecium, 2 (12,5%) E.
hirae were identified.
In Table 2 are listed the bacterial identifications and vegetable matrices from
which they derived.

6. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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Evaluation of BLS (Bacteriocin like Substance) production
The strains were tested for BLS production by agar spot method earlier indicated, using
9 different indicators. Most of the LAB strains showed an antibacterial activity against
L. monocytogenes NCTC 10888, and 3 strains in particular (L. lactis LC214, L. brevis
LB1013, L. brevis LB1113 demonstrated a broad antimicrobial activity against the
majority of the indicator strains. Regarding the enterococci only one not showed
antimicrobial activity vs L. monocytogenes NCTC 10888 and 2 in particular EN313,
EN317 showed a broad antimicrobial activity (Table 3).
Conjugation experiment
Out of 12 matings performed, the successful transfer was obtained in one conjugation,
subsequently confirmed, between Enterococcus faecium EN313 (KanamycinR
,
TeicoplaninS
), used as donor and Lactobacillus acidophilus LB813 (KanamicynS
,
TeicoplaninR
), used as recipient. The plasmid-DNA analysis of parental strains showed
a high weight molecular plasmid for E. faecium EN313 and none plasmid line for
Lactobacillus acidophilus LB813 (data not shown).
Isolation of transconjugants
Transconjugant strains with the following characteristics: rod-shaped, KanamicinR
and
TeicoplaninR with a high antimicrobial activity against L. monocytogenes NCTC 10888,
were isolated. The plasmid-DNA analysis of the trasconjugants showed the passage of
plasmid from donor to recipient. The PCR results confirmed the belonging of
transconjugant strains to the L. acidophlilus species. None amplifications was performed
with specie-specific ligase ddl E. faecium, excluding the enterococci cells presence as
contamination and confirming definitively that the conjugation experiment was
successfully performed.
DISCUSSION
Lactobacilli and other bacteria close related have high potentialities. Many could be
their applications in food as well as in medical fields, remembering that the
effectiveness of LAB is strain-dependent. For all these reasons, new strains are always
researched with characteristics that can help the human health, searching the new
desirable strains in natural niches as plants, foods, fermented products, animals and

7. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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humans that constitute natural ecological systems and good sources for LAB. The
beneficial characteristics may be also obtained by genetic manipulation for example to
improve the pH resistance or the antibacterial capacity, tied to capacity to inhibit the
pathogens, in particular in the human gut, that is a typical characteristic recognized for
many LAB. In this study were isolated some LAB, including bacteria belonging to
Lactobacillus, Lactococcus and Enterococcus genera, from different plants or other
vegetable matrices. In particular from 40 vegetable matrices only from 6 of these
(Pteridium aquilinum leaves, Linum usitatissimum seeds, Daucus carota root, Ficus benjamin
leaves, Aloe Barbadensis leaves, Prunus cerasifera flowers), it was possible to isolate 14
different LAB including Lactobacillus and Lactococcus and 8 different enterococci.
Plants may be a good source of LAB but as Stirling and Whittenburg (1963)
suggest, the LAB are not usually part of the normal microflora of the growing plant
indicating the role of insects in the spread of these organisms. Other studies reported
the occurrence of LAB on leaving or decayed plants (Mundt and Hammer, 1968; Mundt
et al., 1969) but not so recent, as well as part of the accumulated information about the
occurrence of lactobacilli (and other LAB members) on plants is derived from
microbiological studies of the fermentation process. The microbial population of LAB is
known in several vegetable products (cabbage, silage raw materials, carrots and beets,
olives and fruits such as grapes and pears, etc.). However, scarce information about the
occurrence of LAB on flowers and other parts of plants is available in the literature.
Moreover, LAB are well known for their antagonism towards other Gram-positive
bacteria, especially taxonomically related species (Listeria spp., Bacillus spp. Micrococcus
spp., etc.), but also against Escherichia coli, Salmonella spp., Helicobacter pylori and
Pseudomonas aeruginosa. It is well known that the presence of lactobacilli is important for
maintenance of the intestinal microbial ecosystem, thanks the inhibitory activity against
pathogenic bacteria. This inhibition could be due to the production of inhibitory
compounds such as organic acids, hydrogen peroxide, and bacteriocins (Todorov and
Dicks, 2005; Kumari et al., 2011; Anacarso et al., 2014).
The results reported here indicate that 20 LAB were isolated and most of these
strains have shown an antibacterial activity against L. monocytogenes NCTC 10888 and
other eight pathogenic/opportunistic bacteria, including the Gram-negative indicator
strains. Inhibition caused by hydrogen peroxide and organic acids was ruled out, in fact
the producer strains were cultured anaerobically and the culture supernatants were
neutralized (pH 6.0) before assaying the antimicrobial activity. These results are in

8. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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accordance with earlier results reported by Trias et al. (2008), who showed that most
LAB originating from fruits and other vegetables displayed good antagonistic activity
against foodborne pathogens, such as, Listeria monocytogenes, Salmonella typhimurium
and Escherichia coli.
In this study was also performed a conjugation to pass the antimicrobial activity
showed against L. monocytogenes from a donor strain, Enterococcus faecium EN313 and a
recipient, Lactobacillus acidophilus LB813. Enterococcus spp. is the most controversial
genus of LAB group and if these bacteria are frequently isolated from several fermented
foods, they are always seen with fear owing to their potential risk in human health
(Klein et al., 1998). In many studies, different enterococci are great bacteriocin
producers with a high activity against pathogenic bacteria as Listeria or others. In our
conjugation experiment it was formed a strain with a high antibacterial activity against
L. monocytogenes typical of a Enterococcus strain but produced from a L. acidophilus,
eliminating the apprehension dated to potential risk for human health of the
enterococci. The positive result of this experiment was displayed by a plasmidic band in
trasconjugant and confirmed with a PCR reaction where the absence of a specie-specific
ligase ddl E. faecium it excluded the enterococci cells presence as contamination. Our
results are of interest considering the important probiotic characteristics of lactobacilli
and their possible use, in particular during antimicrobial therapy and more interesting
is the possibility of to have a probiotic capable to produce a bacteriocin with a high
antimicrobial activity against the main food pathogenic bacteria.
Table 1: Primer sequences used for the identifications
Specie
product
size
(pb)
oligonucleotide
primer
sequence ( 5’-3’) Tm (°C)
Leuconostoc 976 LeuF / LeuR
CGA AAG GTG CTT GCA CCT TTC
AAG
TTT GTC TCC GAA GAG AAC A
55
L. brevis 1340
BREVDIR /
BREVREV
CTT GCA CTG ATT TTA ACA
CCC ACT GCT GGG CGG TGT GTA
CAA GGC
40
L. delbrueckii 450 LDEL7 / Lac2
ACA GAT GGA TGG AGA GCA GA
CCT CTT CGC TCG CCG CTA CT
50

9. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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L. acidophilus 1000 Acido / Lac
TGA ACC AAC AGA TTC ACT TC
TGA CGA CAG CCA TGC ACC A 45
L. lactis 380 G1 / L1
GAA GTC GTA ACA AGG
CAA GGC ATC CAC CGT 40
E. faecium 941 E1 / E2
ATCAAGTACAGTTAGTCT
ACGATTCAAAGCTAACTG 45
E. faecium 190 Efm1 / Efm2
TKCAGCAATTGAGAAATAC
CTTCTTTTATTTCTCCTGTA 50
E. faecalis 209 Efs1 / Efs2
CTGTAGAAGACCTAATTTCA
CAGCTGTTTTGAAAGCAG 50
E. hirae 263 Eh1 / Eh2
AAACAATCGAAGAACTACTT
TAAATTCTTCCTTAAATGTTG 50
Table 2: Bacterial Identifications and vegetable matrices of origins
Matrix
Strain identification
LAB
enterococci
Pteridium aquilinum LB113 - L. brevis EN313 - E. faecium
Linum usitatissimum
LB213 - L. delbrueckii
LB313 - L. brevis
LB413 - L. brevis
Daucus carota
LC114 - L. lactis
LC214 - L. lactis
LC314 - Leuconostoc
EN315 - E. faecium
EN316 - E. hirae
Ficus benjamin LB513 - L. brevis
EN314 - E. faecalis
EN319 - E. faecalis
EN320 - E. hirae
Aloe Barbadensis
LB613 - L. brevis
LB713 - L. brevis

10. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
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LB813 - L. acidophilus
Prunus cerasifera
LB913 - L. delbrueckii
LB1013 - L. brevis
LB1113 - L. brevis
EN317 - E. faecalis
EN318 - E. faecalis
Table 3: Antibacterial activity of isolated LAB
Indicators
L. i. E. c. 1 C. a E. c. 2 S. a. E. f. L. m. P. a. B. s.
LB113 ++ - - - - - - - -
LB213 ++ - - - - - +++ - -
LB313 ++ - - - - - +++ - -
LB413 + - - - - + - - -
LB513 + - - - - - - - +
LB613 + - - - - - - - +
LB713 + - - - - - + - -
LB813 - - - - - - - - -
LB913 + - - - - - - - +
LB1013 +++ + - + + + +++ + +
LB1113 + + - + + + ++ + -
LC114 ++ - - - - - +++ - -
LC214 ++ + - + ++ + ++ + +
LC314 ++ - - - - - ++ - -
EN313 ++ - - + + - ++ - +
EN314 + - - - - - + - -
EN315 + - - - - - - + -
EN316 + - - - - - + - -
EN317 +++ - - + + - +++ - ++
EN318 + - - - - - + - -
EN319 ++ - - - - - + - +
EN320 + - - - - - + - -
L .i.: L. ivanovii ATCC 13932; E. c. 1: E. casseliflavus 416/k1; C. a.: C. albicans ATCC 10231;
E. c. 2: E. coli ATCC 6538; E. f.: E. faecalis ATCC 29212; L. m.: L. monocytogenes NCTC 10888;
P. a.: P. aeruginosa ATCC 27853; B. s.: B.subtilis ATCC 6633

11. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
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AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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13. Immacolata Anacarso, Lara Bassoli, Carla Sabia, Ramona Iseppi, Carla Condò- ISOLATION AND
IDENTIFICATION OF LACTIC ACID BACTERIA FROM PLANTS AND OTHER VEGETABLE MATRICES
AND MICROBIAL RECOMBINATION WITH ENTEROCOCCUS SPP.
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