Microbiological Research 166 (2011) 1—13
www.elsevier.de/micres
Properties of different lactic acid bacteria isolated
from Apis mellifera L. bee-gut
M. Carina Audisioa,b,c,, Marı́a J. Torresa,b, Daniela C. Sabatéa,b,
Carolina Ibargurena,b, Marı́a C. Apellab,d
a
Instituto de Investigaciones para la Industria Quı́mica (INIQUI-CONICET), A4402FDC- Salta, Salta, Argentina
Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET), Argentina
c
Facultad de Ingenierı́a, Universidad Nacional de Salta; Salta, Argentina
d
Centro de Referencia para Lactobacilos (CERELA-CONICET); 4000-San Miguel de Tucumán, Tucumán, Argentina.
b
Received 11 November 2009; received in revised form 30 December 2009; accepted 3 January 2010
KEYWORDS
Lactobacillus
johnsonii;
Apis mellifera L.;
Lactic acid bacteria;
Bee-gut microbiota
Summary
Eight strains belonging to Lactobacillus spp. and five to Enterococcus spp. were isolated
from the gut of worker Apis mellifera L. bees. Studies based on 16S rRNA sequencing
revealed that AJ5, IG9, A15 and CRL1647 strains had a 99% identity with Lactobacillus
johnsonii, while SM21 showed a 99% similarity with Enterococcus faecium. L. johnsonii
CRL1647, AJ5 and IG9 were high lactic acid producers (values were between 177 and
275 mM), and in vitro they inhibited different human food-borne pathogens and
Paenibacillus larvae, the American foulbrood agent. This bacterium was the most
sensitive to the lactic acid effect being inhibited by 44 mM of this metabolite.
L. johnsonii CRL1647, AJ5 and IG9 also presented important surface properties. These
cells showed between 77% and 93% of auto-aggregation. The preliminary study of the
chemical nature of the aggregating factors revealed that the molecules involved in the
surface of each L. johnsonii strain were quite complex; and something of a peptidic
nature was mainly involved. E. faecium SM21 produced bacteriocin-like compounds with
anti-Listeria effects. Furthermore, a band close to 6.0–7.5 kDA was detected by SDSPAGE studies, and the entA, B and P structural genes were amplified by PCR reactions.
For the first time, bee-gut associated L. johnsonii and E. faecium strains have been
isolated, identified, cultivated and some of their functional properties reported.
& 2010 Elsevier GmbH. All rights reserved.
Corresponding autor at: INIQUI (Instituto de Investigaciones
para la Industria Quı́mica), Universidad Nacional de Salta (UNSa),
Food Microbiology, Av. Bolivia 5150, A4402FDC-Salta, Argentina.
Tel./fax: þ54 387 4251006.
E-mail address:
[email protected] (M. Carina Audisio).
Introduction
The microbiota associated to the honeybee Apis
mellifera mellifera L. is complex and far from
being fully understood, or even known. It has been
0944-5013/$ - see front matter & 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2010.01.003
2
described as being mainly composed of yeasts, Grampositive bacteria (such as Lactobacillus rigidus apis,
L. constellatus, Bacillus spp., Streptococcus and
Clostridium), and Gram-negative or Gram variable
bacteria (Achromobacter, Citrobacter, Enterobacter,
Erwinia, Escherichia coli, Flavobacterium, Klebsiella,
Proteus and Pseudomonas) (Rousseau et al., 1969;
Gilliam and Morton, 1978; Gilliam and Prest, 1987;
Rada et al., 1997; Mohr and Tebbe, 2006). Gilliam
(1997) reported that these bacteria were likely to be
endemic in the alimentary tract of adult bees and
were neither dependent on seasonal nor food factors.
Glinski and Jarosz (1995), also suggests that this bees’
normal microbiota comes from pollen consumption or
through contact with older bees. The presence of
intestinal enterobacteria in hibernating honeybees
was also extensively discussed by Lyapunov et al.
(2008).
Interestingly, only five studies have been carried
out regarding the presence of lactic acid bacteria
associated with honeybee microbiota, and their
isolation and identification have yielded inconclusive results (Rada et al. 1997; Gilliam, 1997;
Máchová et al., 1997; Kačániova et al. 2004,
Olofsson and Vásquez 2008). In some cases, bacteria
considered as probable members of Lactobacillus
group, produced catalase and reduced nitrate
(Gilliam, 1997). These properties are negative for
this genus (Hammes et al. 1992). Kačániova et al.
(2004) could not detect Lactobacillus spp. at all.
Thus, only these scientific articles report the
presence of culturable bee-gut Lactobacillus,
Bifidobacterium or Enterococcus strains associated
with the intestinal tract of the honeybee.
Lactic acid bacteria are important inhabitants
of the intestinal tract of man and other mammalian
and vertebrate animals. Lactobacillus and
Enterococcus are members of this family and are
also present in food and fermentation processes.
These microorganisms disclose interesting properties not only for the food industry but also for
health (Naidu et al., 1999). The antimicrobial
potential of these bacteria includes, among others,
the synthesis of compounds such as lactic acid,
short-chain-volatile-fatty acids, and bacteriocinlike molecules (Jack et al. 1995; Wilson et al.
2005). Antagonistic studies are generally directed
towards food-spoilage and/or pathogenic microorganisms related to the host or product from which
the lactic acid bacteria were isolated. Different
microorganisms affect the honeybee and the most
serious bacterial agent may be Paenibacillus
larvae, a sporulated Gram-positive bacillus that
causes the American foulbrood disease in larvae
(Williams, 2000). An indiscriminate antibiotic use
against this bee pathogen has brought about not
M. Carina Audisio et al.
only chemoresistant strains, but also honey contamination (Evans, 2003).
The aim of this work was to isolate, characterize,
cultivate, preserve and pheno and genotypically
identify lactic acid bacteria associated to bee gut
in order to improve the understanding of this
microbial environment. Different properties were
also analyzed such as their surface characteristics
and in vitro antimicrobial spectra against the
bacterial bee pathogen, and in a broader sense,
against human foodborne pathogen bacteria.
Materials and methods
Honeybee intestinal tract sampling
Intestine samples of summer adult worker bees
from different hives of El Galpón (Salta, Argentina)
were used to obtain homogenates in physiological
solution (0.85% w/w NaCl). The whole intestinal
tracts (esophagus to rectum) were analyzed and
aseptically handled to avoid contamination with
the external surface of the bee body.
Culture media and growth conditions for
lactic acid bacteria isolation
Known aliquots of the different homogenates
were sown on the Streptococcus selective medium
(MSS agar) devised in our laboratory (tryptone 1.4%;
glucose 0.5%; sodium citrate 0.1%; NaCl 0.4%;
sodium azide 0.022%; meat peptone 0.5%; sodium
sulphite 0.022%; L-cysteine 0.022%; pH 6.5; Audisio
et al., 2005), on MRS agar (Britania, Argentina) and
on brain-heart infusion agar (BHI, Britania, Argentina). The plates were incubated at 37 1C for 24–48 h
except for the MRS plates which were incubated at
37 1C for 48–72 h under microaerophilic conditions. In
all cases, the incubation period was extended until
visible colonies appeared. White, small, round
colonies from the MSS and BHI agar were recovered
and placed into BHI broth and incubated at 37 1C.
Colonies with similar characteristics from MRS agar
were transferred to MRS broth.
Lactic acid bacteria phenotypic
characterization
Morphological observations were carried out
using Gram staining. Carbohydrate fermentation
patterns (APICH50, Biomérieux) and phenotypical
identification according to basic biochemical tests
were carried out for Lactobacillus (Hammes et al.,
1992). Enterococcus strains were also characterized
Properties of different lactic acid bacteria
according to typical biochemical tests for this genus
(Devriese et al., 1992).
Antimicrobial spectrum analyses
Antimicrobial substance synthesis by the isolated
lactic acid bacteria was analyzed in the cell-free
supernatants (CFS), obtained from cultures of the
lactobacilli (strains AJ5, A15, IG9 and CRL1647) and
enterococci (strains SM21 and CA12) in MRS and BHI
broths, respectively. After 24 h of incubation at
37 1C and under the appropiate atmosphere for
each strain, the CFS were recovered by centrifuging
(10,000g, 10 1C, 15 min), filter-sterilized (0.22 mm)
and kept at 4 1C until used. Listeria monocytogenes
99/287 was provided by Instituto ‘‘Dr. Carlos
Malbrán’’ (Buenos Aires, Argentina). Bacillus cereus
C1 was supplied by Dr. Morea (ISPA, Bari, Italy).
Escherichia coli 0157:H7 was supplied by Dr. Raya
(CERELA, Argentina). Staphylococcus aureus 29213
from ATCC (American Type Culture Collection). Six
different P. larvae strains (I, II, lll, IV, Azul and C)
were provided by Dr. Terzolo and Engineer Borracci
(INTA-Balcarce, Argentina). P larvae 7 and 35 were
from Unidad de Bacteriologı́a of CIDEFI (La Plata,
Buenos Aires – Argentina). P. larvae strains were
activated in MPYGP (Dingman and Stahly, 1983)
agar at 37 1C for 72 h in a microaerophilic atmosphere. The other indicator strains were grown in
BHI broth at 37 1C for 24 h. All strains were kept at
20 1C in MRS or in BHI broth plus glycerol (20%).
The different CFS were split off in two fractions:
one was kept unmodified and the other was
adjusted to pH 6.0 with NaOH 2.0 N; both aliquots
were filter-sterilized (0.22 mm pore size cellulose
acetate filter) and kept at 4 1C until used. Different
lawns of the human pathogen strains were obtained
on BHI agar (1.5%) for the agar diffusion assay.
P. larvae cell suspensions were prepared by
recovering the colonies grown on MPYGP agar, with
a sterile cotton swab soaked in buffered-peptone
water. They were resuspended in 2 mL of MPYGP
broth (ca. 1 107 CFU/mL) and 300 mL were inoculated in 10 mL of molten MPYGP agar. Twenty five
microliters of each CFS were placed in wells (5 mm
diameter) cut in agar plates previously sown with
the indicator strains. The plates were incubated at
30 1C for 12–24 h and examined for inhibition halos.
For the microplaque technique, 100 mL of each CFS
fractions were placed into 96 wells microplaque
and 10 mL of the different indicator strain cell
suspensions (final concentration varies from 106 to
108 CFU/mL) were added. Then, the microtitulation plaques were incubated at 37 1C for 2, 4 and
24 h under the optimal conditions for the tested
3
bacteria. Lack of turbidity in the wells sown with
the pathogen indicated its inhibition by the sample
under analysis. Peptone–water (0.1%) dilutions
were made and viable indicator cell number was
determined by plate counting. In the particular
case of P. larvae, aliquots of 5 ml of those suspensions that presented slight turbidity were sown on
MPYGP agar and the plates were incubated in order
to determine P. larvae residual viability. All assays
were carried out in triplicate.
Phylogenetic analyses of selected bacteria
Lactobacilli CRL1647, A15, AJ5 and IG9 and the
Enterococcus faecium SM21 were selected for
genotypical characterization and further analyses.
Their total genomic DNA were extracted from
overnight MRS or BHI cultures by alkaline lysis
(Pospiech and Neumann 1995). Two oligonucleotides, based on the report of Daffonchio et al.
(1998), were used to determine 16S rDNA of the
isolate: S-D-BACT-1494-A-S-20 (GTCGTAACAAGGTAGCCGTA) and L-D-BACT-0035-A-A-15 (CAAGGCATCCACCGT). PCR amplification was performed
in a 25 mL reaction mix containing 2.5 mL 10X STR
reaction buffer (Promega), 20 ng total DNA, 0.5 mM
of each primer and 1 U of Taq DNA polymerase
(Promega). Amplification consisted of an initial
denaturation step at 94 1C for 5 min, followed by 35
cycles at 94 1C for 1 min, 50 1C for 2 min and 72 1C
for 2 min. A final extension step consisting of 7 min
at 72 1C was included (Sabaté et al., 2009). The
PCR amplification fragments were resolved by
agarose 0.8% gel electrophoresis at 75 V for 1 h
30 min. The gel was stained with ethidium bromide,
and the bands were visualized under UV illumination at 254 nm. The 16S rDNA amplified with PCR
was purified and the DNA sequencing carried out by
Macrogen Services. Sequences were compared and
aligned with sequences from the GenBank database
by using the BLAST program of the National Center
for Biotechnology Information (NCBI; http://www.
ncbi.nlm.nih.gov) network server. Phylogenetic trees,
based in the 16S-rRNA gen sequences (1,339 bp) of the
isolate and culturable lactic acid bacteria were
constructed using the Tree Builder program of the
Ribosomal Database Project II (http://rdp.cme.msu.
edu) (Cole et al. 2009). The neighbour-joining tree was
bootstrapped 1000 times. Lactobacillus acidophilus
(acc. Number 780913) was used as an outgroup.
Physicochemical characterization of the
antagonistic compounds
The concentration of all organic acids was
determined by HPLC. Prior to the assay, CFS from
4
MRS (lactobacilli) and BHI (enterococci) cultures
were deproteinized. In brief, 1 mL of sample was
added to 2 mL of a solution in 1.8% Ba(OH)2, adding
2 mL of a solution of 2% ZnSO4. The mixture was
vortex shaken, allowed to settle for 10 min and
centrifuged at 3500g for 5 min. Each supernatant
was filter sterilized (0.45 mm) before the HPLC
analyses. The sample amount injected was 20 mL.
The column temperature was 55 1C and the flow rate
of the H2SO4 10 mM mobile phase was 0.6 mL/min.
Detection was carried out by determining the
refraction index using a 2142 LKB Differential
Refractometer. The chromatography column (Rezec
Organic Acid, Phenomenex) had a diameter of
7.8 mm and a length of 300 mm. Peak profiles,
integration, and quantification were obtained with a
CR601 Shimadzu chromatopac integrator (Shimadzu
Corporation, Analytical Instrument Division, Kyoto,
Japan). All tests were carried out in duplicate.
Bacteriocin-like substances production
Bacteriocin-like substances production was determined by the well-diffusion assay with
L. monocytogenes 99/287 as the indicator strain.
Trypsin, proteinase K, pronase E, a-chymotrypsin
and pepsin (final concentration of 1 mg/L in
phosphate buffer pH 7.00, 0.05 M), were assayed
to test the proteinaceous nature of the inhibitors.
Catalase, a-amylase and lipase effects (final concentration of 1 mg/L in phosphate buffer pH 7.00,
0.05 M), were also analyzed. Heat resistance was
studied after CFSs were heated to 70 1C for 30 min,
100 1C for 15 min and 121 1C for 15 min (in an
autoclave) while another aliquot without thermal
treatment was used as control. Then, they were
cooled and tested for antimicrobial activity.
To determine bacteriocin molecular size, the
peptides present in the CFS of E. faecium SM21
were separated by Tricine-SDS-PAGE as described
by Schäger (2006). Polyacrylamide concentration
on the separating gel was 16% (w/v). Electrophoresis was conducted at a constant voltage of 50 V for
1 h, 75 V for 2 h and 150 V for 1 h. A low rainbow
molecular weight marker with sizes ranging from
3.5 to 38 kDa (Amersham Pharmacia, Germany)
was used. The gel was fixed with an isopropanol
(20% v/v) – acetic acid (10% v/v) solution, exhaustively washed with distilled water, and overlaid on
BHI agar inoculated with L. monocytogenes 99/287.
The presence of enterocin-encoding genes was
studied by PCR amplification with primers for the
following well-known enterococcal bacteriocins:
enterocin A; enterocin B; enterocin P; enterocin
31 and enterocin L50A/B (du Toit et al., 2000).
M. Carina Audisio et al.
Total genomic DNA from strains was used. PCR
products were analyzed by electrophoresis (85 V for
1 h 5 min) on 2% (w/v) agarose gels in 1 TAE
buffer. Gels were stained GelRed (Invitrogen) and
observed under UV light. The sizes of the amplified
fragments were determined using 100-bp DNA
Ladder (Invitrogen) as a molecular weight marker.
As a positive control, the entA, B and P bacteriocingene sequenced of E. faecium CRL1385 were used
(accession numbers to the GeneBank database
GQ369790, GQ369791 and FJ57726, respectively).
Lactobacillus cell surface properties
When AJ5, IG9 and CRL1647 cells were grown in
an MRS (Britania, Argentina) medium, discernible
cell grouping appeared to the naked-eye, which
adhered to the tube walls or sedimented to the
bottom of the tube, due to this, their aggregation
properties were studied. These properties were
analyzed according to Handley et al. (1987) with
some modifications. Lactobacilli cells were washed
twice with PBS (NaCl 0.8; KH2PO4, 0.034; K2HPO4,
0.121, pH 7.4), resuspended in the same buffer and
adjusted to OD600=0.6070.05. Cell suspensions
(2 mL) were centrifuged at 2000g for 15 min and
optic density (OD) at 600 nm was measured, without agitation, at room temperature every hour for
4 h. The percentage of autoaggregation was expressed as: A%=(A0At)/A0 100, where A0 represents the absorbance (A600) at 0 h and At represents
the absorbance (A600) taken at 1, 2 and 3 hours.
Preliminary physic-chemical studies of
molecules involve in the lactobacilli surface
interactions
In order to approach the chemical nature of the
molecules involved in the surface adsorption
phenomena, L. johnsonii cells were put in contact
with solutions of either 1 mg/mL of trypsin and
pepsin at 37 1C for 1 h, lipase and sodium mperiodate at 25 1C for 1 h, and heated to 100 1C for
15 min. All enzymes were from Sigma and were
prepared in their optimal buffer. Following this, the
aggregation assay was performed.
Statistical analyses
All analyses were carried out according to the
Tukey test and the results considered significant at
the Po0.05 level. Assays were carried out in
duplicate.
Properties of different lactic acid bacteria
5
Results
Antimicrobial spectrum
Screening and phenotypic characterization
of lactic acid bacteria associated to bee-gut
The agar-diffusion assay was used to ‘‘quick
screen’’ the antimicrobial spectra of the 13 lactic
bacteria strains isolated from the bee gut. It was
observed that only Lactobacillus AJ5, IG9, A15 and
CRL1647 were able to inhibit S. aureus ATCC29213,
B. cereus C1, E. coli O157:H7, L. monocytogenes
and P. larvae; but strain A15 failed to inhibit two of
the eight P. larvae strains tested. While, E. faecium
SM21 only exhibited anti-L. monocytogenes activity. The lactobacilli antagonistic effects disappeared in all cases after adjusting CFS pH to 6.0,
but the coccus kept its antilisteria activity under
this new condition (see Table 2).
Different types of colonies developed on the
surface of BHI agar plate after 1 day of incubation
at 37 1C, and after 48 or 72 h on the surface of MRS
agar. Thirteen microorganisms were purely isolated
and showed biochemical characteristic of lactic
acid bacteria: small, round, opaque and white
colonies, non-sporulated, cocci (5) or rods (8)
Gram-positive, non-motile cells, catalase negative
and nitrate negative. Classic biochemical tests
revealed that the eight bacilli were homofermentative and belonged to Lactobacillus genus. The
strains CRL1647 and IG9 were characterized as
acidophilus and crispatus, respectively; but the
species of the other strains could not be determined and they were designated as Lactobacillus
spp. The cocci were grouped into the Enterococcus
genus. Basic biochemical tests revealed that, at
least, SM21, CA12 and SM7 can be considered as
E. faecium (Table 1).
Phylogenetic identification and analyses of
selected isolates
Five strains were selected due to their antimicrobial
spectra and their species-specific identifications were
derived using a 16S-rRNA sequence analysis. Comparison of the near complete 16S rRNA gene sequence of
the bee-gut associated CRL1647, AJ5, IG9 and A15
Table 1. Characterization of the most representative lactic acid bacteria associated with summer adult worker bee
gut samples.
Isolates
Characters
Tentative
identification
IG9, CRL1647, AJ5, A15
C22, SM16, SM18, CA22
Gþ, long rods, non motile, non sporulated, nitrate (),
Catalase (), arginine (þ), esculin (þ), growth at 37 1C (þ),
grwoth at 45 1C (þ), glucose (þ) without gas production
Lactobacillus spp.
IG9, CRL1647
AJ5, A15
Gluconate (), esculin (þ), melezitose (), sacarose (þ),
Glycerol (), fructose (þ), mannose (þ), cellobiose (þ),
Rhamnose ()a, sorbitol (þ), manitol (þ), Darabinose (þ)
Salicin (wþ), trehalose (þ), xylitol (), L-fucose (þ),
Gentibiose (þ), maltose (þ), arbutine (þ), tagatose (þ),
Raffinose (þ), amygdaline (þ), melibiose ()a, lactose ()a
L. acidophilus CRL1647
L. crispatus IG9
Lactobacillus spp. A15
Lactobacillus spp. AJ5
CA12, SM21, SM7,
G(þ), cocci in short chains, immobile, non sporulated, nitrate
()
Catalase (), growth at 15, 37 and 451C (þ), growth at pH 9.6
(þ);
Growth at 6.5% NaCl (þ), Voges Proskauer (), arginine (þ),
Esculin (þ), glucose (þ)
Enterococcus spp.
Cellobiose (þ), melibiose (þ), melezitose (), sacarose (),
Starch (þ), glycerol (þ), L-arabinose (þ), ribose (þ), fructose
(þ),
Galactose (þ), manitol (þ), D-mannose (þ), rhamnose (þ),
Sorbitol (þ), lactose (þ), salicin (þ), trehalose (þ), xylitol (),
D-arabinose (þ), gentibiose (þ), maltose (þ), arbutine(þ),
Amygdaline (þ), gluconate (þ)
Enterococcus faecium
CA10, CA11
CA12, SM21, SM7
þ, positive; wþ, weak positive; , negative.
(þ) for AJ5 and A15 strains.
a
6
Table 2.
M. Carina Audisio et al.
Property profile of the selected lactic acid bacteria isolated from the bee-gut of Apis mellifera L.
Phylogenetic identification
Genbank accession code
Lactic acid synthesis (mM)
Bacteriocin production
Surface property
Inhibitory spectrum:
P. larvae
L. monocytogenes
S. aureus ATCC29213
B. cereus C1
E. coli O157:H7
CRL1647
AJ5
IG9
A15
SM21
L. johnsonii
EU428007
17777
–
þ
L. johnsonii
EU428008
27578
–
þ
L. johnsonii
EU780913
18575
–
þ
L. johnsonii
EU428009
10678
–
–
E. faecium
EU428012
3475
þ
–
8/8
3/3
þ
þ
þ
8/8
3/3
þ
þ
þ
8/8
3/3
þ
þ
þ
6/8
3/3
þ
þ
þ
0/8
3/3
–
–
–
Figure 1. Phylogenetic tree, based in the 16S-rRNA gen sequences (1339 bp) of the four L. johnsonii strains culturable
lactic acid bacteria from bee-gut, was constructed using the Tree Builder program of the Ribosomal Database Project II
(http://rdp.cme.msu.edu) (Cole et al., 2009).
strains with the corresponding Lactobacillus sequences
from the GenBank database showed all strains lay in
the evolutionary clade of Lactobacillus. The four
strains had a 99% identity with known Lactobacillus
johnsonii, and their sequences were introduced into
the GenBank database (Table 2). A phylogenetic tree
of these lactobacilli was built with the neighbourjoining algorithms, using different sequences from the
L. acidophilus group with, at least, 95% identity as
shown in Figure 1. L. johnsonii NCC533, a type strain
with its complete genome known, was also included in
this analysis. It can be seen that the bee-gut
associated strains are closely related to L. johnsonii.
A similar analysis was done with SM21 strain
confirmed as an E. faecium with a 99% identity with
E. faecium strain DSM20477 (=ATCC19434), a type
strain, and other known and reported sequences
(see Table 2 and Figure 2).
Lactic acid synthesis and antagonistic effect
analyses
The lactic acid production by L. johnsonii
CRL1647, IG9, AJ5, A15 and E. faecium SM21 was
determined by HPLC. Interestingly, even though
the pH values of the lactobacilli cultures were
similar with an average value of 3.870.5; the
lactic acid concentration profile was characteristic
of each strain. L. johnsonii AJ5 synthesized
the largest amount of this metabolite followed
by IG9, CRL1647 and A15 (Table 2). While,
E. faecium SM21, also a homofermentative lactic
acid bacteria, acidified BHI broth at pH 4.5
and produced the lowest concentration of
lactic acid. BHI broth was selected to culture
Enterococcus spp. because it was determined in
the laboratory that MRS broth was not as efficient
for cocci growth.
E. coli O157:H7, S. aureus ATCC29213, B. cereus
C1 and the three L. monocytogenes assayed showed
the same behaviour against lactic acid; all of them
were inhibited by 177 mM of this acid (Table 2).
But, 8875 mM had no impact on cell viability,
independently of the detection method, i.e. welldiffusion agar or direct contact in microplaques. In
particular, Figure 3A demonstrates the effect of
177 mM lactic acid synthesized by L. johnsonii
CRL1647 on L. monocytogenes 99/287 nonproliferative cells viability.
Properties of different lactic acid bacteria
7
7.5
9.5
7
9
6.5
8.5
log CFU/mL
log CFU/mL
Figure 2. Phylogenetic tree, based in the 16S-rRNA gen sequences (1339 bp) of the E. faecium SM21 culturable lactic
acid bacterium from bee-gut, was constructed using the Tree Builder program of the Ribosomal Database Project II
(http://rdp.cme.msu.edu) (Cole et al., 2009).
6
5.5
8
7.5
5
7
4.5
6.5
6
4
0
1
Time (h)
2
0
1
2
Time (h)
Figure 3. Listeria monocytogenes 99/287 inhibition by effect of: (A) lactic acid produced by L. johnsonii CRL1647
(~ control; m 177 mM lactic acid at pH 6.0; ’ 177 mM lactic acid at pH 3.9) and (B) bacteriocin-like compounds
produced by E. faecium SM21 (m control; ’ crude CFS at pH 6.0; ~ CFS at pH 6.0 and trypsin treated).
The vegetative cells of P. larvae were the most
sensitive to lactic acid action; no colonies were
detected on MPYGP agar after a 2 h contact period
with 44 mM of lactic acid. These results were
confirmed by the microplaque technique when
different aliquots of cultures, from wells of the
microplaque with slight turbidity, showed a viable
plate count number three orders less in the log of
CFU/mL than the control (Figure 4).
Bacteriocin-like compounds synthesis
E. faecium SM7, SM21 and CA12 produced
compounds with strong anti-L. monocytogenes
activity. The antagonistic effect had no significant
difference when the filtered cell-free supernatants
were adjusted to pH 6.0 with NaOH. Also lipase,
catalase and a-amylase did not affect the antiListeria activity, but disappeared after a treatment
8
M. Carina Audisio et al.
8
1
3
log CFU/mL
7
2
6
5
4
0
2
Time (h)
4
Figure 4. Paenibacillus larvae IV response in the presence of different CFS from L. johnsonii CRL1647. (A) By the welldifussion assay (1: pH 3.9 and 177 mM lactic acid; 2: pH 5 and 177 mM lactic acid, and 3: pH 5 and 44 mM lactic acid) and
(B) by the microplaque technique ((~) control of P. larvae IV cells, (m) P. larvae IV cells in contact with 177 mM lactic
acid; and (&) in contact with 44 mM lactic acid).
with pepsin, a-chymotrypsin, trypsin, pronase E
and proteinase K, confirming their peptidic nature.
The compounds were also highly thermostable
keeping their anti-listeria effect after autoclave
sterilization (121 1C for 15 min).
A bactericidal effect of E. faecium SM21 cellfree supernatant on L. monocytogenes 99/287
non-proliferative cells viability was observed
(Figure 3B). Also, it was confirmed that the
antagonistic effect was mainly due to bacteriocinlike compounds because when the CFS was treated
with trypsin, its antilisteria effect disappeared
(Figure 3B).
In particular, the analysis of E. faecium SM21
antimicrobial peptides by Tricine-SDS-PAGE, and
further antimicrobial assays against L. monocytogenes 99/287, showed a broad band with a
molecular weight of approximately 6.0–7.5 kDa
(Figure 5A). The analyses of different structural
enterocin genes in the DNA of E. faecium SM21 by
PCR reactions was also done and revealed that the
entA, B and P genes were amplified (Figure 4B). The
size of the fragments of entA (126 bp) and entB
(168 bp) were identical to those obtained for
E. faecium CRL1385, the strain used as the
positive control. The specific PCR fragment of
120 bp, corresponding to the entP gene, was also
amplified. Moreover, no specific PCR fragments
were detected with the set of primers for
bacteriocins L50A/B and 31 (Figure 5B).
L. johnsonii surface properties
The presence of cells adhered to the tube wall were
observed only in three of the fourteen lactobacilli
isolated from bee-gut, as shown in Figure 6A for L.
johnsonii CRL1647. A similar situation was observed
for L. johnsonii AJ5 and IG9 cultures. After 3 h,
without agitation, a reduction in the OD (600 nm)
values were determined from 24-hour-old cells of IG9
and AJ5 (93.2% and 83.4%, respectively) resuspended
in PBS. While, L. johnsonii CRL1647 showed 77.3% of
autoaggregation. By scanning microscopy analyses,
lactobacilli cells were aligned like bricks in a wall and
some random coils were also observed (Figure 6B).
Flocculation was not suspected as this phenomena is
more common in yeast than in bacteria cells (Table 3).
The preliminary study of the chemical nature of
the autoaggregating factors, revealed the molecules involved on the surface of each L. johnsonii
strain were quite complex. All being sensitive to
trypsin, sodium m-periodate and highly thermostable. But, only those of AJ5 were affected by
pepsin. Also lipase action was more pronounced on
IG9 cells autoaggregation (Table 4).
Discussion
The scientific works where the presence of lactic
acid bacteria associated to honeybee-gut is re-
Properties of different lactic acid bacteria
9
38 KDa
31 KDa
24 KDa
17 KDa
12 KDa
8.5 KDa
3.5 KDa
1
2
1
2
3
4
5
6
Figure 5. (A) Tricine-SDS-PAGE gel showing the zone of growth inhibition of L. monocytogenes 99/287 by the band of
the CFS of E. faecium SM21. Lane 1: molecular mass marker; lane 2: CFS of E. faecium SM21; (B) Amplification of
structural enterocin genes from DNA of E. faecium SM21. Lane 1: 100 bp Ladder (invitrogen), lane 2: ent A (126 bp),
lane 3: ent B (162 bp), lane 4: ent P (120 bp), lane 5: ent bac 31 and lane 6: ent L50.
Figure 6. Auto-aggregating phenomena of L. johnsonii CRL1647 cells. (A) macroscopic observation, (B) scanning
electron microscopy.
Table 3. Autoaggregation percentage of the different
L. johnsonii cultures.
Strain
CRL1647
AJ5 4
IG9
Time (h)
0
1
2
3
33.170.5
36.670.6
31.671.1
36.871.2
49.670.8
81.771.1
59.170.9
71.470.4
92.170.8
77.370.5
83.470.7
93.271.1
ported are limited. They were carried out with
different purposes. For example, Mohr and Tebbe
(2006) revealed the presence of Lactobacillus in
bee-gut only by a genetic analysis, but no isolations
were carried out and the strains detected were
uncultured. Mrázek et al. (2008) also analyzed the
diversity of insect intestinal microbiota, without
any strain isolation, and only reported the possible
presence of Bifidabacterium sp. in honeyebees.
Rada et al. (1997) were able to isolate Lactobacillus sp., Enterococcus sp. and some Bifidobacterium sp. when they were studying the microbiota in
the honeybee digestive tract. However, they only
characterized a Bifidobacterium asteroides by
biochemical tests and analyzed the sensitivity of
these bacteria to veterinary drugs.
In this work, Lactobacillus and Enterococcus
strains were isolated from the gut of the summer
adult worker Apis mellifera L. bees of the
Argentinean Northwest region. The Streptococcus
10
Table 4.
M. Carina Audisio et al.
Autoaggregation percentagea of the different L. johnsonii strains after different treatments.
Treatment
Control
Trypsin
Pepsin
Lipase
Sodium m-periodate
Boiling temperature for 15 min
a
Strains
CRL1647
IG9
AJ5
77.370.5
30.871.2
75.170.5
61.670.8
59.370.4
75.671.1
93.270.7
59.470.3
90.371.1
67.371.3
70.570.7
92.470.6
83.471.1
45.370.5
65.370.8
83.170.4
67.771.2
82.870.7
Each point corresponds to an average of at least four readings.
selective medium (MSS), modified by Audisio et al.
(2005), was selective only for Enterococcus isolation. However Lactobacillus isolation was somewhat difficult. If MRS agar was prepared adjusting
the pH close to 5.0 with acetic acid, as indicated by
the manufacturer, scant colony development was
observed and sometimes, there was no colony at
all. On the other hand, if MRS agar was prepared
without an acidic final pH, i.e. equal to 6.5, a
higher number of typical lactic acid bacteria
colonies were detected, including many others.
Thus, to isolate lactobacilli from the bee-gut
environment, the samples were seeded both on
MRS at pH 6.5 and on MRS at pH 5.0, and small,
round, opaque and white typical lactic acid
bacteria colonies were picked up from both media.
The response of classic biochemical tests performed for Enterococcus (Devriese et al., 1992)
allowed us to correctly identify some of the
enterococci strains, confirmed by a phylogenetic
analysis for the SM21 strain. The isolation of these
microorganisms from bee-gut was an important
step as there are only four scientific papers
reporting their presence in the honeybee intestinal
environment (Rousseau et al., 1969; Snowdon and
Cliver, 1996; Rada et al., 1997; Kačániova et al.,
2004) and with this information a clearer understanding of the bee-gut microbiota composition can
be achieved.
Although there were differences between the
phenotypical characterization of strains CRL 1647,
IG9, AJ5, A15 and their genotypical profile, they
were not surprising, as authors had already
reported similar situations (Berger et al., 2007).
Other articles have shown that there are six
Lactobacillus strains of distinct homology groups
(A1, A2, A3, A4, B1 and B2) that respond phenotypically to the characteristics of the acidophilus
species, but a study with molecular biology tools
revealed that the species may be crispatus, gasseri,
johnsonii, gallinarum, amylovorus or acidophilus
(Johnson et al., 1980; Pot et al. 1993). All the
lactobacilli preselected in this work were homo-
fermentative, i.e., they do not produce gas from
glucose. These results agree with those reported by
Olofsson and Vásquez (2008) who isolated and
characterized many homofermentative lactobacilli
from the honeybee stomach. They observed
that lactobacilli and bifidobacteria were predominant within the lactic microbiota associated to
the honeybee stomach, but failed to report
the presence of L. johnsonii or Streptococcus or
Enterococcus in this bee environment. However, in
this work it was determined, by phylogenetic
analyses, that the homofermentative lactobacilli
associated to bee-gut were closely related to
L. johnsonii. In particular, the IG9 strain showed a
97% (ca. 400 bp) identity with the 50 end of L.
johnsonii NCC533, a type strain with its complete
genome known (Boekhorst et al., 2004).
A high in vitro susceptibility of P. larvae
vegetative cells to a low pH, unreported so far for
this bacterium, was observed in this work. The
three L. johnsonii strains selected were able to
inhibit the vegetative cells of the American
foulbrood agent by lactic acid synthesis and
44 mM of this compound were necessary.
E. faecium SM21, cultured in BHI broth, reached a
pH close to 5.0 and produced 34 mM of lactic acid,
but this concentration was not enough to affect P.
larvae viability. Among the pre-selected lactobacilli and enterococci, the final pH values for the CFS
used in the antagonistic tests were close to 4.0 or
4.5 in most cases, but organic acid concentration
was variable and not all lactic acid bacteria
selected, particularly enterococci, inhibited E. coli
O157:H7, S. aureus ATCC29213, B. cereus C1 or
P. larvae, by acidity. Although lactic acid production is an essential characteristic of the bacteria
studied here, the amount of this metabolite that is
synthesized depends on each particular strain.
Susceptibility to the antagonistic activity of lactic
acid also varies among the pathogenic microorganisms (Ogawa et al., 2001; Wilson et al., 2005). So,
the present in vitro study revealed that certain
Lactobacillus species, such as L. johnsonii exert
Properties of different lactic acid bacteria
growth inhibition on the indicator microorganisms
assayed. To the authors’ knowledge, this would be
the first report on P. larvae growth inhibition due to
acidity generated by lactobacilli isolated from bee
gut. In vitro inhibition of P. larvae by Bacillus sp.
bacteria has been previously reported (Evans and
Armstrong, 2005; Alippi and Reynaldi, 2006; Sabaté
et al., 2009). However, Bacillus sp. is a genus
normally related to the honeybee environment
(Gilliam, 1987, 1997; Snowdon and Cliver, 1996).
The selected enterococci were remarkably active
against L. monocytogenes. This inhibition was not
due to lactic acidity but to a synthesis of
bacteriocin-like compounds by four E. faecium of
the nine isolated. These compounds were thermostable and showed both a proteinaceous nature
and a bactericidal effect against L. monocytogenes
99/287; all these characteristics agree with those
found for different enterocins produced by
E. faecium isolated from other sources (FoulquiéMoreno et al., 2006). It has been informed that the
structural gene of enterocin A is widely distributed
among E. faecium strains, whereas that of enterocin B always occurs in the presence of enterocin A;
besides, the high-frequency and variability of
enterocin structural genes among enterococci of
different origin by means of simple PCR reactions,
without purification or sequentiation, was also
reported (Poeta et al., 2007). This trend was
confirmed in this paper and this would be the first
case where these genes have been detected in
enterococci associated with the bee digestive
tract.
Bacterial aggregation/surface property at bacterial cell envelope level can be determined and
quantified by the reduction of optic density values
at different times and should not be confused/
misunderstood with the flocculation phenomena
(Handley et al., 1987; Kos et al., 2003). Flocculation is a phenomena more common/current for
yeasts than bacteria. Furthermore, yeast flocculation is a non-sexual and reversible cell aggregation
in which cells adhere to each other to form flocs
(Zhao and Bai 2009). In this work, an important
auto-aggregation was observed in three different
L. johnsonii strains isolated from bee-gut. The
assays carried out to determine the chemical
nature of the factor involved in the lactobacilli
auto-aggregation revealed in the cell envelope,
different compounds for each strain; a lipopetide
for IG9 and a glicopeptide for CRL1647 and AJ5. The
involvement of proteinaceous bacterial surface
compounds in lactobacilli adhesion has been analyzed and characterized for strains from human,
fish and dairy product origins (Ventura et al., 2002;
Ying et al., 2007). However, no reports exist on the
11
study of this property in Lactobacillus with regards
to insects, furthermore no analysis has been
performed on honeybee-derived Lactobacillus until
now. Further studies are needed to determine
which molecules are present in the autoaggregation
phenomena.
Even though this work analyzes human foodborne
pathogens, it should be pointed out that the
honeybee or its products are not E. coli O157:H7
or L. monocytogenes hosts or vectors. The origin of
a strain was not considered to be an obstacle to
ascertain its full antimicrobial potential; thus, the
findings may open the way for different applications other than apiculture practices.
Conclusions
Lactobacillus and Enterococcus strains associated
with adult worker bees were isolated and characterized to enhance the knowledge of microorganisms found in the intestinal environment of this
insect. The presence of L. johnsonii has now been
reported for the first time in the bee gut.
Furthermore, this is the first report on P. larvae
growth inhibition due to acidity generated by
lactobacilli isolated from the bee gut ecosystem.
Acknowledgements
This paper was supported by Agencia Nacional de
Promoción Cientı́fica y Técnica (ANPCyT, Grant
PICTR890/06), CIUNSa (PI No 1725) and Consejo
Nacional de Investigaciones Cientı́ficas y Técnicas
(CONICET). M.C. Audisio and M.C. Apella are
members of the Research Career of CONICET. The
authors thank Dr. S.A. Cuozzo and Dr. R.R. Raya for
the excellent assistance with the phylogenetic
analyses of the isolated strains.
References
Alippi AM, Reynaldi FJ. Inhibition of the growth of
Paenibacillus larvae the causal agent of American
Foulbrood of honey bees by selected strains of aerobic
spore forming bacteria isolated from apiarian sources.
J Invertebr Pathol 2006;91:141–6.
Audisio MC, Terzolo HR, Apella MC. Bacteriocin from
honeybee beebread Enterococcus avium, active
against Listeria monocytogenes. Appl Environ Microbiol 2005;71:3373–5.
Berger BR, Pridmore D, Barretto C, Delmas-Julien F,
Schreiber K, Arigoni F, et al. Similarity and differences in the Lactobacillus acidophilus group identified
12
by polyphasic analysis and comparative genomics. J
Bacteriol 2007;189:1311–21.
Boekhorst J, Siezen RJ, Zwahlen MC, Vilanova D,
Pridmore RD, Mercenier A, et al. The complete
genomes of Lactobacillus plantarum and Lactobacillus
johnsonii reveal extensive differences in chromosome
organization and gene content. Microbiol 2004;150:
3601–11.
Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, et al.
The ribosomal database project: improved alignments
and new tools for rRNA analysis. Nucl Acids Res
2009;37:D141–5.
Daffonchio D, Borin S, Frova G, Manachini P, Sorlini C. PCR
fingerprinting of whole genomes: the spacers between
the 16S and 23S rRNA genes and of intergenic tRNA
gene regions reveals a different intraespecific genomic variability of Bacillus cereus and Bacillus licheniformis. Int J Syst Bacteriol 1998;48:107–16.
Devriese LA, Collins MD, Wirth R. The genus Enterococcus. In: Balows A, Trüper HG, Dworkin M, Harder W,
Heinz Schleifer K, editors. The Prokaryotes, vol. II.
New York: Springer-Verlag; 1992. p. 1465–81.
Dingman DW, Stahly DP. Medium promoting sporulation of
Bacillus larvae and metabolism of medium components. Appl Environ Microbiol 1983;46:860–9.
du Toit M, Franz CM, Dicks LM, Holzapfel WH. Preliminary
characterization of bacteriocins produced by
Enterococcus faecium and Enterococcus faecalis
isolated from pig faeces. J Appl Microbiol 2000;88:
482–94.
Evans JD. Diverse origins of tetracycline resistance in the
honey bee bacterial pathogen Paenibacillus larvae. J
Invertebr Pathol 2003;83:46–50.
Evans JD, Armstrong TM. Inhibition of the American
foulbrood bacterium, Paenibacillus larvae larvae, by
bacteria isolated from honey bees. J Apicult Res
2005;44:168–71.
Foulquié-Moreno MR, Sarantinopoulos P, Tsakalidou E, De
Vuyst L. The role and application of enterococci in
food and health. Int J Food Microbiol 2006;1:1–24.
Gilliam M, Morton HL. Bacteria belonging to the genus
Bacillus isolated from honey bees, Apis mellifera, fed
2,4-D and antibiotics. Apidologie 1978;9:213–22.
Gilliam M, Prest DB. Microbiology of feces of the larval
honey bee, Apis mellifera. J Invert Pathol 1987;49:
70–5.
Gilliam M. Identification and roles of non-pathogenic
microflora associated with honey bees. FEMS Microbiol
Lett 1997;155:1–10.
Glinski Z, Jarosz J. Mechanical and biochemical defences
of honey bees. Bee World 1995;76:110–8.
Hammes WP, Weiss N, Holzapfel W. The genera Lactobacillus and Carnobacterium. In: Balows A, Trüper HG,
Dworkin M, Harder W, Heinz Schleifer K, editors. The
Prokaryotes, vol. II. New York: Springer-Verlag; 1992.
p. 1535–94.
Handley PS, Harty DWS, Wyatt JE, Brown CR, Doran JP,
Gibbs ACC. A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of
fibrilar and fimbriate strains of Streptococcus salivarius. J Gen Microbiol 1987;133:3207–17.
M. Carina Audisio et al.
Jack R, Tagg H, Ray B. Bacteriocins of Gram-positive
bacteria. Microbiol Rev 1995;59:171–200.
Johnson JL, Phelps CF, Cummins CS, London J, Gasser F.
Taxonomy of Lactobacillus acidophilus group. Int J
Syst Bacteriol 1980;30:53–68.
Kačániova M, Chlebo R, Kopernichý M, Trakocvická A.
Microflora of the honeybee gastrointestinal tract.
Folia Microbiol 2004;49:169–71.
Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic
S. Adhesion and aggregation ability of probiotic strain
Lactobacillus acidophilus M92. J Appl Microbiol 2003;
94:981–7.
Lyapunov Y, Kuzyaev RZ, Khismatullin RG, Bezgodova OA.
Intestinal enterobacteria of the hibernating Apis
mellifera mellifera L. bees. Microbiology 2008;77:
373–9.
Máchová M, Rada V, Huk J, Smékal Fr. Development of
probiotics for bees. Apiacta 1997;4:99–111.
Mohr KI, Tebbe C. Diversity and phylotype consistency
of bacteria in the guts of three bee species (Apoidea)
at an oilseed rape field. Environ Microbiol 2006;8:
258–72.
Mrázek J, Štrosová L, Fliegerova K, Kott T, Kopečný J.
Diversity of insect intestinal microflora. Folia Microbiol 2008;53:229–33.
Naidu AS, Bidlack WR, Clemens RA. Probiotic spectra of
lactic acid bacteria (LAB). Crit Rev Food Sci Nutr
1999;39:13–26.
Ogawa M, Shimizu K, Nomoto K, Tanaka R, Hamabata T,
Yamasaki S, et al. Inhibition of in vitro growth of Shiga
toxin-producing Escherichia coli O157:H7 by probiotic
Lactobacillus strains due to production of lactic acid.
Int J Food Microbiol 2001;68:135–40.
Olofsson TC, Vásquez A. Detection and identification of a
novel lactic acid bacterial flora within the honey
stomach of the honeybee Apis mellifera. Curr Microbiol 2008;57:356–63.
Poeta P, Costa D, Rojo-Bezares B, Zarazaga M, Klibi N,
Rodrigues C, et al.
Detection of antimicrobial
activities and bacteriocin structural genes in faecal
enterococci of wild animals. Microbiol Res 2007;162:
257–63.
Pospiech A, Neumann B. A versatile quick-prep of
genomic DNA from Gram-positive bacteria. Trends
Genet 1995;11:217–8.
Pot B, Hertel C, Ludwig W, Descheemaeker P, Kersters K,
Schleifer KH. Identification and classification of
Lactobacillus acidophilus, L. gasseri and L. johnsonii
strains by SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization. J Gen Microbiol 1993;139:
513–7.
Rada V, Máchová M, Huk J, Marounek M, Duskova B.
Microflora in the honeybee digestive tract: count,
characteristics and sensitivity to veterinary drugs.
Apidologie 1997;28:357–65.
Rousseau M, Tysset C, Durand C. Presence of streptococci of
the Lancefield D group in healthy working bees (Alpis
mellifica L.). Interpretation of their presence in alimentary bacteriology. Bull Acad Vet Fr 1969;42:173–86.
Sabaté DC, Carrillo L, Audisio MC. Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis
Properties of different lactic acid bacteria
isolated from honeybee gut and honey samples. Res
Microbiol 2009;160:193–9.
Schäger H. Tricine-SDS-PAGE. Nature protocols 2006;1:16–22.
Snowdon JA, Cliver DO. Microorganisms in honey. Int J
Food Microbiol 1996;31:1–26.
Ventura M, Jankovic I, Walker DC, Pridmore RC, Zink R.
Identification and characterization of novel surface
proteins in Lactobacillus johnsonii and Lactobacillus
gasseri. Appl Environ Microbiol 2002;68:6172–81.
Ying C, Lei W, Jiazhong L, Zhantao S, Liguo A. Identification and purification of a novel adhesion-associated
protein in a new strain of Lactobacillus, L15, from
13
flounder (Paralichthys olivaceus). Vet Microbiol 2007;
122:116–22.
Williams DL. Review. A veterinary approach to the
European honey bee (Apis mellifera). Vet J 2000;160:
61–73.
Wilson AR, Sigee D, Epton HA. Anti-bacterial activity of
Lactobacillus plantarum strain SK1 against Listeria
monocytogenes is due to lactic acid production. J Appl
Microbiol 2005;99:1516–22.
Zhao XQ, Bai FW. Yeast flocculation: new story in
fuel ethanol production. Biotechnol Adv 2009;27:
849–56.