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Genetic stability of Lactobacillus paracasei subsp. paracasei F19

Article  in  Microbial Ecology in Health and Disease · July 2009

DOI: 10.1080/089106002760003297

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 ORIGINAL ARTICLE

Genetic stability of Lactobacillus paracasei subsp.

paracasei F19
L. Morelli and E. Campominosi

From the Istituto di Microbiologia, U.C.S.C., Via Emilia Parmense 84, 29100, Piacenza, Italy

Correspondence to: L. Morelli, Istituto di Microbiologia, U.C.S.C., Via Emilia Parmense 84, 29100, Piacenza,
Italy. Fax: »39 523 599246; E-mail: morelli@pc.unicatt.it

Microbial Ecology in Health and Disease 2002; Suppl 3: 14 – 16

Assessment of the genetic stability of strains that have to be reproduced at industrial scale and then freeze dried or incorporated into a
food matrix is extremely relevant, in order to guarantee consumers of the quality of probiotic products. Stability of plasmid complement
of Lactobacillus paracasei subsp. paracasei isolate F19 (Lactobacillus F19), a strain containing three extrachromosomal elements, was
carefully checked in each step of the industrial reproduction process. Results did not detect any selection of cured derivatives. The plasmid
proŽ le was unaltered when compared with the plasmid content determined in the same strain by our laboratory 6 years earlier. Attention
was then paid to the functional roles (if any) of the plasmid complement of Lactobacillus F19. Laboratory-induced cured derivatives of
the strain were obtained and analyzed in detail as regards their probiotic and microbiological features, namely sugar fermentation pattern,
drug and bile resistance and growth curves. Cured derivatives of L. paracasei F19 were obtained only after severe curing treatments.
These mutants have been shown to retain all the properties of the wild-type strain. Key words : Lactobacillus, probiotic, genetics, stability,
plasmid, curing.

INTRODUCTION MATERIALS AND METHODS

A general consensus among scientists exists on strain-spe-
ciŽ city of the probiotic features (1 –6), which are not
shared by all members of a given bacterial species.
Assessment of the genetic stability of these strain-related
traits is, therefore, essential, in order to provide consumers
a stable product, able to maintain health-promoting
properties.
Extrachromosomal elements are more likely to be af-
fected in their duplication and repartition mechanisms
during the whole manufacturing chain (7, 8), leading to the
appearance of cured, plasmid devoid derivatives. Repro-
duction of the bacterial cells in the stressing conditions of
industrial fermentation and freeze-drying could also select
for cured derivatives, which have generally shorter genera-
tion time than the wild types.
Aim of our investigation on Lactobacillus paracasei
subsp. paracasei F19 (thereafter Lactobacillus F19) was to
evaluate the Ž delity of transmission of its three extrachro-
mosomal elements from one generation to another during
industrial reproduction process. As an additional objec-
tive, we have investigated the potential role played by
these genetic elements by inducing their curing by means
of curing agents. Laboratory-induced cured derivatives of
Lactobacillus F19 were analyzed in detail as regards their
probiotic and microbiological features, namely sugar fer-
mentation, bile and drug resistance and growth curve.
Strains and culturing conditions
Lactobacillus F19 and its derivatives obtained during this
study were grown in MRS (Difco) agar or broth. Incuba-
tion conditions were 37°C for 48 h. Anaerobic conditions
provided by the gas Pack System (BBL) were used when
required.
Stability of plasmid complements in Lactobacillus F19
Freeze dried preparations and frozen samples of Lacto -
bacillus F19, taken during industrial reproduction, were
received from Arla Foods. These samples were diluted and
plated onto MRS agar plates. After growth, 50 CFUs
from each readable dilution (25– 250 CFUs per plate) were
checked for their plasmid content.
Curing experiments
Lactobacillus F19 broth cultures in triplicate were incu-
bated overnight at sub-inhibitory temperatures (48 and
16°C) or in the presence of 10 mg of novobiocin (Sigma) in
order to promote the loss of plasmids. After growth, 50
CFUs from each readable dilution were checked for their
plasmid content. Overnight cultures were diluted and
plated onto MRS agar plates. After growth, 50 CFUs
from each readable dilution were checked for their plasmid
content.

© Taylor & Francis 2002. ISSN 1403-417 4 Microbial Ecology in Health and Disease
 Stability of Lactobacillus F19 15

Table I In particular, it was possible to show the presence of

CFUs counted in the presence absence of Oxgall. Cured deri×ati×es isolate lacking:
of Lactobacillus F19 are unaffected by the presence of the bile salt
preparation 1. the 6.5 kb plasmid (four isolates);
2. the 9 kb plasmid (three isolates);
Strain MRS CFU MRS»Oxgall 3. both the 6.5 and the 9 kb plasmids (one isolate).
CFU
It was impossible to obtain a fully cured derivative,
Lactobacillus F19 wild type 2.2½10 9 2.1½109 lacking also the 2.2 kb plasmid (see Fig. 1). These results
Derivative lacking 9.0 kb plasmid 3.0½10 9 2.9½109 also indicate that plasmid harbored by Lactobacillus F19
Derivative lacking 6.5 plasmid 3.0½10 9 2.8½109
Derivative lacking both plasmids 1.8½10 9 1.6½109
are deŽ nitely stable genetic elements.

Phenotypic characterization
Sugar fermentation patterns, as determined by API
Plasmid isolation 50CHL were identical in the wild type and in the three
The alkaline lysis method for plasmid DNA isolation and types of cured derivatives. Positive reactions were detected
the agarose gel electrophoresis conditions employed have for: ribose, adonitol, galactose, glucose, fructose, mannose,
been previously described (9). sorbose, mannitol, sorbitol, N-acetyl glucosamine, amyg-
daline, arbutine, esculine, salicine, cellobiose, maltose, lac-
Phenotypic characterization tose, sucrose, threalose, inuline, melezitose, turanose,
tagatose. It is interesting to note that all cured derivatives
Fermentation patterns were determined by means of API
retained the ability to utilize lactose, suggesting that this
50CHL (API System, Moantalieu, France) galleries ac-
trait is not plasmid-linked in the studied strain.
cording to manufacturers’ s instruction and drug resistance
Growth curves were determined by RABIT. No sub-
was assayed by a disk diffusion test (Sensi Disks-Oxoid)
stantial differences were detected among wild type and
(10). Growth curves of wild type and cured derivatives
cured derivatives (Fig. 2).
were determined by a Rapid Automated Bacterial
Impedance Technique (RABIT). Bile resistance was as- Drug resistance
sayed by means of an agar plate method, in which cultures
This strain is intrinsically resistant, as all members of the
of the wild type and its derivatives were plated in MRS
L. paracasei species, to vancomycin. Sensi-disks detected
agar medium with or without the presence of 0.15% w v of
insensitivity to gentamycin, colistin, kanamycin strepto-
Oxgall (Sigma).
mycin and polymixin B. As a standard method to detect
drug resistance in lactobacilli does not exist, this method
RESULTS AND DISCUSSION
does not really indicate the presence of a drug resistance
Stability of plasmid complements of Lactobacillus F19
phenotype. All the three types of cured derivatives exhibit
Freeze dried preparations and samples taken from indus- the same pattern of insensitivity. This is an indication that
trial fermentation processes were plated. About 300 CFUs these phenotypes are not plasmid encoded.
were examined for their plasmid proŽ le. All of them
retained the typical plasmid proŽ le of the wild type. This
result suggests that industrial production processes do not
affect the plasmid content of Lactobacillus F19. It is to
note that the plasmid proŽ le was unaltered when com-
pared with plasmid content determined in the same strain
by our laboratory in 1992 (6 year between those studies),
during an EU-FLAIR project (AGRF056) devoted to the
selection and characterization of probiotic bacteria.

Curing experiments
All 300 CFUs assayed after growing Lactobacillus F19 at
stressing temperatures retained the original plasmid
proŽ le, consisting of three plasmid of 2.2, 6.5 and 9 kb,
respectively.
Among the 150 CFUs examined after incubation in the
Fig. 1. Lanes 1 and 5: plasmid complement of Lactobacillus F19
presence of 10 mg of novobiocin, it was possible to demon- (wild type); Lane 2: derivative cured from 9.0 kb plasmid; Lane 3:
strate the presence in the bacterial population of cured derivative cured from 6.5 kb plasmid; Lane 4: derivative cured
derivatives with a different plasmid proŽ le. from 9.0 and 6.5 kb plasmids; Lane 6: supercoiled M.W. marker.
16 L. Morelli et al.
Bile resistance
No differences in bile resistance are detected among the
studied strains (Table I). These results suggest that this
phenotype, which is relevant for the survival of the strain
into the human gut is not affected by the loss of the two
cured plasmids.
CONCLUSIONS
The plasmid complement of Lactobacillus F19 does not
seem to be altered by the industrial production processes.
Also, the plasmid proŽ le was unaltered when compared
with the plasmid content determined in the same strain by
our laboratory 6 year earlier. Curing is obtained in labora-
tory conditions only and the two curable plasmids do not
seem to be involved in the checked phenotypes.
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Fig. 2. Growth curves of Lacto-
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