Microbiological Research 166 (2011) 1—13

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Properties of different lactic acid bacteria isolatedfrom Apis mellifera L. bee-gut

M. Carina Audisioa,b,c,�, Marıa J. Torresa,b, Daniela C. Sabatea,b,Carolina Ibargurena,b, Marıa C. Apellab,d

aInstituto de Investigaciones para la Industria Quımica (INIQUI-CONICET), A4402FDC- Salta, Salta, ArgentinabConsejo Nacional de Investigaciones Cientıficas y Tecnicas (CONICET), ArgentinacFacultad de Ingenierıa, Universidad Nacional de Salta; Salta, ArgentinadCentro de Referencia para Lactobacilos (CERELA-CONICET); 4000-San Miguel de Tucuman, Tucuman, Argentina.

Received 11 November 2009; received in revised form 30 December 2009; accepted 3 January 2010

KEYWORDSLactobacillusjohnsonii;Apis mellifera L.;Lactic acid bacteria;Bee-gut microbiota

ee front matter & 2010micres.2010.01.003

ing autor at: INIQUI (Ina Quımica), Universidadogy, Av. Bolivia 5150, A487 4251006.ess: audisio@unsa.edu.

SummaryEight strains belonging to Lactobacillus spp. and five to Enterococcus spp. were isolatedfrom the gut of worker Apis mellifera L. bees. Studies based on 16S rRNA sequencingrevealed that AJ5, IG9, A15 and CRL1647 strains had a 99% identity with Lactobacillusjohnsonii, while SM21 showed a 99% similarity with Enterococcus faecium. L. johnsoniiCRL1647, AJ5 and IG9 were high lactic acid producers (values were between 177 and275 mM), and in vitro they inhibited different human food-borne pathogens andPaenibacillus larvae, the American foulbrood agent. This bacterium was the mostsensitive to the lactic acid effect being inhibited by 44 mM of this metabolite.L. johnsonii CRL1647, AJ5 and IG9 also presented important surface properties. Thesecells showed between 77% and 93% of auto-aggregation. The preliminary study of thechemical nature of the aggregating factors revealed that the molecules involved in thesurface of each L. johnsonii strain were quite complex; and something of a peptidicnature was mainly involved. E. faecium SM21 produced bacteriocin-like compounds withanti-Listeria effects. Furthermore, a band close to 6.0–7.5 kDA was detected by SDS-PAGE 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 beenisolated, identified, cultivated and some of their functional properties reported.& 2010 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights rese

stituto de InvestigacionesNacional de Salta (UNSa),402FDC-Salta, Argentina.

ar (M. Carina Audisio).

Introduction

The microbiota associated to the honeybee Apismellifera mellifera L. is complex and far frombeing fully understood, or even known. It has been

rved.

M. Carina Audisio et al.2

described as being mainly composed of yeasts, Gram-positive bacteria (such as Lactobacillus rigidus apis,L. constellatus, Bacillus spp., Streptococcus andClostridium), and Gram-negative or Gram variablebacteria (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 beendemic in the alimentary tract of adult bees andwere neither dependent on seasonal nor food factors.Glinski and Jarosz (1995), also suggests that this bees’normal microbiota comes from pollen consumption orthrough contact with older bees. The presence ofintestinal enterobacteria in hibernating honeybeeswas also extensively discussed by Lyapunov et al.(2008).

Interestingly, only five studies have been carriedout regarding the presence of lactic acid bacteriaassociated with honeybee microbiota, and theirisolation and identification have yielded inconclu-sive results (Rada et al. 1997; Gilliam, 1997;Machova et al., 1997; Kacaniova et al. 2004,Olofsson and Vasquez 2008). In some cases, bacteriaconsidered as probable members of Lactobacillusgroup, produced catalase and reduced nitrate(Gilliam, 1997). These properties are negative forthis genus (Hammes et al. 1992). Kacaniova et al.(2004) could not detect Lactobacillus spp. at all.Thus, only these scientific articles report thepresence of culturable bee-gut Lactobacillus,Bifidobacterium or Enterococcus strains associatedwith the intestinal tract of the honeybee.

Lactic acid bacteria are important inhabitantsof the intestinal tract of man and other mammalianand vertebrate animals. Lactobacillus andEnterococcus are members of this family and arealso present in food and fermentation processes.These microorganisms disclose interesting proper-ties not only for the food industry but also forhealth (Naidu et al., 1999). The antimicrobialpotential of these bacteria includes, among others,the synthesis of compounds such as lactic acid,short-chain-volatile-fatty acids, and bacteriocin-like molecules (Jack et al. 1995; Wilson et al.2005). Antagonistic studies are generally directedtowards food-spoilage and/or pathogenic microor-ganisms related to the host or product from whichthe lactic acid bacteria were isolated. Differentmicroorganisms affect the honeybee and the mostserious bacterial agent may be Paenibacilluslarvae, a sporulated Gram-positive bacillus thatcauses the American foulbrood disease in larvae(Williams, 2000). An indiscriminate antibiotic useagainst this bee pathogen has brought about not

only chemoresistant strains, but also honey con-tamination (Evans, 2003).

The aim of this work was to isolate, characterize,cultivate, preserve and pheno and genotypicallyidentify lactic acid bacteria associated to bee gutin order to improve the understanding of thismicrobial environment. Different properties werealso analyzed such as their surface characteristicsand in vitro antimicrobial spectra against thebacterial 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 beesfrom different hives of El Galpon (Salta, Argentina)were used to obtain homogenates in physiologicalsolution (0.85% w/w NaCl). The whole intestinaltracts (esophagus to rectum) were analyzed andaseptically handled to avoid contamination withthe external surface of the bee body.

Culture media and growth conditions forlactic acid bacteria isolation

Known aliquots of the different homogenateswere 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%; sodiumsulphite 0.022%; L-cysteine 0.022%; pH 6.5; Audisioet al., 2005), on MRS agar (Britania, Argentina) andon brain-heart infusion agar (BHI, Britania, Argen-tina). The plates were incubated at 37 1C for 24–48 hexcept for the MRS plates which were incubated at37 1C for 48–72 h under microaerophilic conditions. Inall cases, the incubation period was extended untilvisible colonies appeared. White, small, roundcolonies from the MSS and BHI agar were recoveredand placed into BHI broth and incubated at 37 1C.Colonies with similar characteristics from MRS agarwere transferred to MRS broth.

Lactic acid bacteria phenotypiccharacterization

Morphological observations were carried outusing Gram staining. Carbohydrate fermentationpatterns (APICH50, Biomerieux) and phenotypicalidentification according to basic biochemical testswere carried out for Lactobacillus (Hammes et al.,1992). Enterococcus strains were also characterized

Properties of different lactic acid bacteria 3

according to typical biochemical tests for this genus(Devriese et al., 1992).

Antimicrobial spectrum analyses

Antimicrobial substance synthesis by the isolatedlactic acid bacteria was analyzed in the cell-freesupernatants (CFS), obtained from cultures of thelactobacilli (strains AJ5, A15, IG9 and CRL1647) andenterococci (strains SM21 and CA12) in MRS and BHIbroths, respectively. After 24 h of incubation at37 1C and under the appropiate atmosphere foreach 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 monocytogenes99/287 was provided by Instituto ‘‘Dr. CarlosMalbran’’ (Buenos Aires, Argentina). Bacillus cereusC1 was supplied by Dr. Morea (ISPA, Bari, Italy).Escherichia coli 0157:H7 was supplied by Dr. Raya(CERELA, Argentina). Staphylococcus aureus 29213from ATCC (American Type Culture Collection). Sixdifferent 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 werefrom Unidad de Bacteriologıa of CIDEFI (La Plata,Buenos Aires – Argentina). P. larvae strains wereactivated in MPYGP (Dingman and Stahly, 1983)agar at 37 1C for 72 h in a microaerophilic atmo-sphere. The other indicator strains were grown inBHI 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 wasadjusted to pH 6.0 with NaOH 2.0 N; both aliquotswere filter-sterilized (0.22 mm pore size celluloseacetate filter) and kept at 4 1C until used. Differentlawns of the human pathogen strains were obtainedon BHI agar (1.5%) for the agar diffusion assay.P. larvae cell suspensions were prepared byrecovering the colonies grown on MPYGP agar, witha sterile cotton swab soaked in buffered-peptonewater. They were resuspended in 2 mL of MPYGPbroth (ca. 1� 107 CFU/mL) and 300 mL were inocu-lated in 10 mL of molten MPYGP agar. Twenty fivemicroliters of each CFS were placed in wells (5 mmdiameter) cut in agar plates previously sown withthe indicator strains. The plates were incubated at30 1C for 12–24 h and examined for inhibition halos.For the microplaque technique, 100 mL of each CFSfractions were placed into 96 wells microplaqueand 10 mL of the different indicator strain cellsuspensions (final concentration varies from 106 to108 CFU/mL) were added. Then, the microtitula-tion plaques were incubated at 37 1C for 2, 4 and24 h under the optimal conditions for the tested

bacteria. Lack of turbidity in the wells sown withthe pathogen indicated its inhibition by the sampleunder analysis. Peptone–water (0.1%) dilutionswere made and viable indicator cell number wasdetermined by plate counting. In the particularcase of P. larvae, aliquots of 5 ml of those suspen-sions that presented slight turbidity were sown onMPYGP agar and the plates were incubated in orderto determine P. larvae residual viability. All assayswere carried out in triplicate.

Phylogenetic analyses of selected bacteria

Lactobacilli CRL1647, A15, AJ5 and IG9 and theEnterococcus faecium SM21 were selected forgenotypical characterization and further analyses.Their total genomic DNA were extracted fromovernight MRS or BHI cultures by alkaline lysis(Pospiech and Neumann 1995). Two oligonucleo-tides, based on the report of Daffonchio et al.(1998), were used to determine 16S rDNA of theisolate: S-D-BACT-1494-A-S-20 (GTCGTAACAAGG-TAGCCGTA) and L-D-BACT-0035-A-A-15 (CAAGG-CATCCACCGT). PCR amplification was performedin a 25 mL reaction mix containing 2.5 mL 10X STRreaction buffer (Promega), 20 ng total DNA, 0.5 mMof each primer and 1 U of Taq DNA polymerase(Promega). Amplification consisted of an initialdenaturation step at 94 1C for 5 min, followed by 35cycles at 94 1C for 1 min, 50 1C for 2 min and 72 1Cfor 2 min. A final extension step consisting of 7 minat 72 1C was included (Sabate et al., 2009). ThePCR amplification fragments were resolved byagarose 0.8% gel electrophoresis at 75 V for 1 h30 min. The gel was stained with ethidium bromide,and the bands were visualized under UV illumina-tion at 254 nm. The 16S rDNA amplified with PCRwas purified and the DNA sequencing carried out byMacrogen Services. Sequences were compared andaligned with sequences from the GenBank databaseby using the BLAST program of the National Centerfor Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) network server. Phylogenetic trees,based in the 16S-rRNA gen sequences (1,339 bp) of theisolate and culturable lactic acid bacteria wereconstructed using the Tree Builder program of theRibosomal Database Project II (http://rdp.cme.msu.edu) (Cole et al. 2009). The neighbour-joining tree wasbootstrapped 1000 times. Lactobacillus acidophilus(acc. Number 780913) was used as an outgroup.

Physicochemical characterization of theantagonistic compounds

The concentration of all organic acids wasdetermined by HPLC. Prior to the assay, CFS from

M. Carina Audisio et al.4

MRS (lactobacilli) and BHI (enterococci) cultureswere deproteinized. In brief, 1 mL of sample wasadded to 2 mL of a solution in 1.8% Ba(OH)2, adding2 mL of a solution of 2% ZnSO4. The mixture wasvortex shaken, allowed to settle for 10 min andcentrifuged at 3500g for 5 min. Each supernatantwas filter sterilized (0.45 mm) before the HPLCanalyses. The sample amount injected was 20 mL.The column temperature was 55 1C and the flow rateof the H2SO4 10 mM mobile phase was 0.6 mL/min.Detection was carried out by determining therefraction index using a 2142 LKB DifferentialRefractometer. The chromatography column (RezecOrganic Acid, Phenomenex) had a diameter of7.8 mm and a length of 300 mm. Peak profiles,integration, and quantification were obtained with aCR601 Shimadzu chromatopac integrator (ShimadzuCorporation, Analytical Instrument Division, Kyoto,Japan). All tests were carried out in duplicate.

Bacteriocin-like substances production

Bacteriocin-like substances production was de-termined by the well-diffusion assay withL. monocytogenes 99/287 as the indicator strain.Trypsin, proteinase K, pronase E, a-chymotrypsinand pepsin (final concentration of 1 mg/L inphosphate buffer pH 7.00, 0.05 M), were assayedto test the proteinaceous nature of the inhibitors.Catalase, a-amylase and lipase effects (final con-centration of 1 mg/L in phosphate buffer pH 7.00,0.05 M), were also analyzed. Heat resistance wasstudied after CFSs were heated to 70 1C for 30 min,100 1C for 15 min and 121 1C for 15 min (in anautoclave) while another aliquot without thermaltreatment was used as control. Then, they werecooled and tested for antimicrobial activity.

To determine bacteriocin molecular size, thepeptides present in the CFS of E. faecium SM21were separated by Tricine-SDS-PAGE as describedby Schager (2006). Polyacrylamide concentrationon the separating gel was 16% (w/v). Electrophor-esis was conducted at a constant voltage of 50 V for1 h, 75 V for 2 h and 150 V for 1 h. A low rainbowmolecular weight marker with sizes ranging from3.5 to 38 kDa (Amersham Pharmacia, Germany)was used. The gel was fixed with an isopropanol(20% v/v) – acetic acid (10% v/v) solution, exhaus-tively washed with distilled water, and overlaid onBHI agar inoculated with L. monocytogenes 99/287.

The presence of enterocin-encoding genes wasstudied by PCR amplification with primers for thefollowing well-known enterococcal bacteriocins:enterocin A; enterocin B; enterocin P; enterocin31 and enterocin L50A/B (du Toit et al., 2000).

Total genomic DNA from strains was used. PCRproducts were analyzed by electrophoresis (85 V for1 h 5 min) on 2% (w/v) agarose gels in 1�TAEbuffer. Gels were stained GelRed (Invitrogen) andobserved under UV light. The sizes of the amplifiedfragments were determined using 100-bp DNALadder (Invitrogen) as a molecular weight marker.As a positive control, the entA, B and P bacteriocin-gene sequenced of E. faecium CRL1385 were used(accession numbers to the GeneBank databaseGQ369790, GQ369791 and FJ57726, respectively).

Lactobacillus cell surface properties

When AJ5, IG9 and CRL1647 cells were grown inan MRS (Britania, Argentina) medium, discerniblecell grouping appeared to the naked-eye, whichadhered to the tube walls or sedimented to thebottom of the tube, due to this, their aggregationproperties were studied. These properties wereanalyzed according to Handley et al. (1987) withsome modifications. Lactobacilli cells were washedtwice with PBS (NaCl 0.8; KH2PO4, 0.034; K2HPO4,0.121, pH 7.4), resuspended in the same buffer andadjusted to OD600=0.6070.05. Cell suspensions(2 mL) were centrifuged at 2000g for 15 min andoptic density (OD) at 600 nm was measured, with-out agitation, at room temperature every hour for4 h. The percentage of autoaggregation was ex-pressed as: A%=(A0�At)/A0� 100, where A0 repre-sents the absorbance (A600) at 0 h and At representsthe absorbance (A600) taken at 1, 2 and 3 hours.

Preliminary physic-chemical studies ofmolecules involve in the lactobacilli surfaceinteractions

In order to approach the chemical nature of themolecules involved in the surface adsorptionphenomena, L. johnsonii cells were put in contactwith solutions of either 1 mg/mL of trypsin andpepsin at 37 1C for 1 h, lipase and sodium m-periodate at 25 1C for 1 h, and heated to 100 1C for15 min. All enzymes were from Sigma and wereprepared in their optimal buffer. Following this, theaggregation assay was performed.

Statistical analyses

All analyses were carried out according to theTukey test and the results considered significant atthe Po0.05 level. Assays were carried out induplicate.

Properties of different lactic acid bacteria 5

Results

Screening and phenotypic characterizationof lactic acid bacteria associated to bee-gut

Different types of colonies developed on thesurface of BHI agar plate after 1 day of incubationat 37 1C, and after 48 or 72 h on the surface of MRSagar. Thirteen microorganisms were purely isolatedand showed biochemical characteristic of lacticacid bacteria: small, round, opaque and whitecolonies, non-sporulated, cocci (5) or rods (8)Gram-positive, non-motile cells, catalase negativeand nitrate negative. Classic biochemical testsrevealed that the eight bacilli were homofermen-tative and belonged to Lactobacillus genus. Thestrains CRL1647 and IG9 were characterized asacidophilus and crispatus, respectively; but thespecies of the other strains could not be deter-mined and they were designated as Lactobacillusspp. The cocci were grouped into the Enterococcusgenus. Basic biochemical tests revealed that, atleast, SM21, CA12 and SM7 can be considered asE. faecium (Table 1).

Table 1. Characterization of the most representative lactigut samples.

Isolates Characters

IG9, CRL1647, AJ5, A15 Gþ, long rods, non motile, non sC22, SM16, SM18, CA22 Catalase (�), arginine (þ), escul

grwoth at 45 1C (þ), glucose (þ)

IG9, CRL1647 Gluconate (�), esculin (þ), meleAJ5, A15 Glycerol (�), fructose (þ), mann

Rhamnose (�)a, sorbitol (þ), manSalicin (wþ), trehalose (þ), xylitGentibiose (þ), maltose (þ), arbRaffinose (þ), amygdaline (þ), m

CA12, SM21, SM7, G(þ), cocci in short chains, immo(�)

CA10, CA11 Catalase (�), growth at 15, 37 an(þ);Growth at 6.5% NaCl (þ), Voges PEsculin (þ), glucose (þ)

CA12, SM21, SM7 Cellobiose (þ), melibiose (þ), meStarch (þ), glycerol (þ), L-arabin(þ),Galactose (þ), manitol (þ), D-maSorbitol (þ), lactose (þ), salicin (D-arabinose (þ), gentibiose (þ),Amygdaline (þ), gluconate (þ)

þ, positive; wþ, weak positive; �, negative.a(þ) for AJ5 and A15 strains.

Antimicrobial spectrum

The agar-diffusion assay was used to ‘‘quickscreen’’ the antimicrobial spectra of the 13 lacticbacteria strains isolated from the bee gut. It wasobserved that only Lactobacillus AJ5, IG9, A15 andCRL1647 were able to inhibit S. aureus ATCC29213,B. cereus C1, E. coli O157:H7, L. monocytogenesand P. larvae; but strain A15 failed to inhibit two ofthe eight P. larvae strains tested. While, E. faeciumSM21 only exhibited anti-L. monocytogenes activ-ity. The lactobacilli antagonistic effects disap-peared in all cases after adjusting CFS pH to 6.0,but the coccus kept its antilisteria activity underthis new condition (see Table 2).

Phylogenetic identification and analyses ofselected isolates

Five strains were selected due to their antimicrobialspectra and their species-specific identifications werederived using a 16S-rRNA sequence analysis. Compar-ison of the near complete 16S rRNA gene sequence ofthe bee-gut associated CRL1647, AJ5, IG9 and A15

c acid bacteria associated with summer adult worker bee

Tentativeidentification

porulated, nitrate (�), Lactobacillus spp.in (þ), growth at 37 1C (þ),without gas production

zitose (�), sacarose (þ), L. acidophilus CRL1647ose (þ), cellobiose (þ), L. crispatus IG9itol (þ), D�arabinose (þ) Lactobacillus spp. A15ol (�), L-fucose (þ), Lactobacillus spp. AJ5utine (þ), tagatose (þ),elibiose (�)a, lactose (�)a

bile, non sporulated, nitrate Enterococcus spp.

d 451C (þ), growth at pH 9.6

roskauer (�), arginine (þ),

lezitose (�), sacarose (�), Enterococcus faeciumose (þ), ribose (þ), fructose

nnose (þ), rhamnose (þ),þ), trehalose (þ), xylitol (�),maltose (þ), arbutine(þ),

Figure 1. Phylogenetic tree, based in the 16S-rRNA gen sequences (1339 bp) of the four L. johnsonii strains culturablelactic 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).

Table 2. Property profile of the selected lactic acid bacteria isolated from the bee-gut of Apis mellifera L.

CRL1647 AJ5 IG9 A15 SM21

Phylogenetic identification L. johnsonii L. johnsonii L. johnsonii L. johnsonii E. faeciumGenbank accession code EU428007 EU428008 EU780913 EU428009 EU428012Lactic acid synthesis (mM) 17777 27578 18575 10678 3475Bacteriocin production – – – – þ

Surface property þ þ þ – –Inhibitory spectrum:P. larvae 8/8 8/8 8/8 6/8 0/8L. monocytogenes 3/3 3/3 3/3 3/3 3/3S. aureus ATCC29213 þ þ þ þ –B. cereus C1 þ þ þ þ –E. coli O157:H7 þ þ þ þ –

M. Carina Audisio et al.6

strains with the corresponding Lactobacillus sequencesfrom the GenBank database showed all strains lay inthe evolutionary clade of Lactobacillus. The fourstrains had a 99% identity with known Lactobacillusjohnsonii, and their sequences were introduced intothe GenBank database (Table 2). A phylogenetic treeof these lactobacilli was built with the neighbour-joining algorithms, using different sequences from theL. acidophilus group with, at least, 95% identity asshown in Figure 1. L. johnsonii NCC533, a type strainwith its complete genome known, was also included inthis analysis. It can be seen that the bee-gutassociated strains are closely related to L. johnsonii.

A similar analysis was done with SM21 strainconfirmed as an E. faecium with a 99% identity withE. faecium strain DSM20477 (=ATCC19434), a typestrain, and other known and reported sequences(see Table 2 and Figure 2).

Lactic acid synthesis and antagonistic effectanalyses

The lactic acid production by L. johnsoniiCRL1647, IG9, AJ5, A15 and E. faecium SM21 was

determined by HPLC. Interestingly, even thoughthe pH values of the lactobacilli cultures weresimilar with an average value of 3.870.5; thelactic acid concentration profile was characteristicof each strain. L. johnsonii AJ5 synthesizedthe largest amount of this metabolite followedby IG9, CRL1647 and A15 (Table 2). While,E. faecium SM21, also a homofermentative lacticacid bacteria, acidified BHI broth at pH 4.5and produced the lowest concentration oflactic acid. BHI broth was selected to cultureEnterococcus spp. because it was determined inthe laboratory that MRS broth was not as efficientfor cocci growth.

E. coli O157:H7, S. aureus ATCC29213, B. cereusC1 and the three L. monocytogenes assayed showedthe same behaviour against lactic acid; all of themwere 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. well-diffusion agar or direct contact in microplaques. Inparticular, Figure 3A demonstrates the effect of177 mM lactic acid synthesized by L. johnsoniiCRL1647 on L. monocytogenes 99/287 non-proliferative cells viability.

Figure 2. Phylogenetic tree, based in the 16S-rRNA gen sequences (1339 bp) of the E. faecium SM21 culturable lacticacid 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).

4

4.5

5

5.5

6

6.5

7

7.5

Time (h)

log

CFU

/mL

6

6.5

7

7.5

8

8.5

9

9.5

0Time (h)

log

CFU

/mL

1 20 1 2

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 compoundsproduced by E. faecium SM21 (m control; ’ crude CFS at pH 6.0; ~ CFS at pH 6.0 and trypsin treated).

Properties of different lactic acid bacteria 7

The vegetative cells of P. larvae were the mostsensitive to lactic acid action; no colonies weredetected on MPYGP agar after a 2 h contact periodwith 44 mM of lactic acid. These results wereconfirmed by the microplaque technique whendifferent aliquots of cultures, from wells of themicroplaque with slight turbidity, showed a viableplate count number three orders less in the log ofCFU/mL than the control (Figure 4).

Bacteriocin-like compounds synthesis

E. faecium SM7, SM21 and CA12 producedcompounds with strong anti-L. monocytogenesactivity. The antagonistic effect had no significantdifference when the filtered cell-free supernatantswere adjusted to pH 6.0 with NaOH. Also lipase,catalase and a-amylase did not affect the anti-Listeria activity, but disappeared after a treatment

1

3

2

4

5

6

7

8

0Time (h)

log

CFU

/mL

2 4

Figure 4. Paenibacillus larvae IV response in the presence of different CFS from L. johnsonii CRL1647. (A) By the well-difussion 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 lacticacid; and (&) in contact with 44 mM lactic acid).

M. Carina Audisio et al.8

with pepsin, a-chymotrypsin, trypsin, pronase Eand proteinase K, confirming their peptidic nature.The compounds were also highly thermostablekeeping their anti-listeria effect after autoclavesterilization (121 1C for 15 min).

A bactericidal effect of E. faecium SM21 cell-free supernatant on L. monocytogenes 99/287non-proliferative cells viability was observed(Figure 3B). Also, it was confirmed that theantagonistic effect was mainly due to bacteriocin-like compounds because when the CFS was treatedwith trypsin, its antilisteria effect disappeared(Figure 3B).

In particular, the analysis of E. faecium SM21antimicrobial peptides by Tricine-SDS-PAGE, andfurther antimicrobial assays against L. monocyto-genes 99/287, showed a broad band with amolecular weight of approximately 6.0–7.5 kDa(Figure 5A). The analyses of different structuralenterocin genes in the DNA of E. faecium SM21 byPCR reactions was also done and revealed that theentA, B and P genes were amplified (Figure 4B). Thesize of the fragments of entA (126 bp) and entB(168 bp) were identical to those obtained forE. faecium CRL1385, the strain used as thepositive control. The specific PCR fragment of120 bp, corresponding to the entP gene, was alsoamplified. Moreover, no specific PCR fragmentswere detected with the set of primers forbacteriocins L50A/B and 31 (Figure 5B).

L. johnsonii surface properties

The presence of cells adhered to the tube wall wereobserved only in three of the fourteen lactobacilliisolated from bee-gut, as shown in Figure 6A for L.johnsonii CRL1647. A similar situation was observedfor 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 IG9and AJ5 (93.2% and 83.4%, respectively) resuspendedin PBS. While, L. johnsonii CRL1647 showed 77.3% ofautoaggregation. By scanning microscopy analyses,lactobacilli cells were aligned like bricks in a wall andsome random coils were also observed (Figure 6B).Flocculation was not suspected as this phenomena ismore common in yeast than in bacteria cells (Table 3).

The preliminary study of the chemical nature ofthe autoaggregating factors, revealed the mole-cules involved on the surface of each L. johnsoniistrain were quite complex. All being sensitive totrypsin, sodium m-periodate and highly thermo-stable. But, only those of AJ5 were affected bypepsin. Also lipase action was more pronounced onIG9 cells autoaggregation (Table 4).

Discussion

The scientific works where the presence of lacticacid bacteria associated to honeybee-gut is re-

38 KDa

31 KDa

24 KDa

17 KDa

12 KDa

8.5 KDa

3.5 KDa

1 2 3 4 5 6 1 2

Figure 5. (A) Tricine-SDS-PAGE gel showing the zone of growth inhibition of L. monocytogenes 99/287 by the band ofthe CFS of E. faecium SM21. Lane 1: molecular mass marker; lane 2: CFS of E. faecium SM21; (B) Amplification ofstructural 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) scanningelectron microscopy.

Table 3. Autoaggregation percentage of the differentL. johnsonii cultures.

Strain Time (h)

0 1 2 3

CRL1647 33.170.5 36.871.2 59.170.9 77.370.5AJ5 4 36.670.6 49.670.8 71.470.4 83.470.7IG9 31.671.1 81.771.1 92.170.8 93.271.1

Properties of different lactic acid bacteria 9

ported are limited. They were carried out withdifferent purposes. For example, Mohr and Tebbe(2006) revealed the presence of Lactobacillus inbee-gut only by a genetic analysis, but no isolationswere carried out and the strains detected were

uncultured. Mrazek et al. (2008) also analyzed thediversity of insect intestinal microbiota, withoutany strain isolation, and only reported the possiblepresence of Bifidabacterium sp. in honeyebees.Rada et al. (1997) were able to isolate Lactoba-cillus sp., Enterococcus sp. and some Bifidobacter-ium sp. when they were studying the microbiota inthe honeybee digestive tract. However, they onlycharacterized a Bifidobacterium asteroides bybiochemical tests and analyzed the sensitivity ofthese bacteria to veterinary drugs.

In this work, Lactobacillus and Enterococcusstrains were isolated from the gut of the summeradult worker Apis mellifera L. bees of theArgentinean Northwest region. The Streptococcus

Table 4. Autoaggregation percentagea of the different L. johnsonii strains after different treatments.

Treatment Strains

CRL1647 IG9 AJ5

Control 77.370.5 93.270.7 83.471.1Trypsin 30.871.2 59.470.3 45.370.5Pepsin 75.170.5 90.371.1 65.370.8Lipase 61.670.8 67.371.3 83.170.4Sodium m-periodate 59.370.4 70.570.7 67.771.2Boiling temperature for 15 min 75.671.1 92.470.6 82.870.7

aEach point corresponds to an average of at least four readings.

M. Carina Audisio et al.10

selective medium (MSS), modified by Audisio et al.(2005), was selective only for Enterococcus isola-tion. However Lactobacillus isolation was some-what difficult. If MRS agar was prepared adjustingthe pH close to 5.0 with acetic acid, as indicated bythe manufacturer, scant colony development wasobserved and sometimes, there was no colony atall. On the other hand, if MRS agar was preparedwithout an acidic final pH, i.e. equal to 6.5, ahigher number of typical lactic acid bacteriacolonies were detected, including many others.Thus, to isolate lactobacilli from the bee-gutenvironment, the samples were seeded both onMRS at pH 6.5 and on MRS at pH 5.0, and small,round, opaque and white typical lactic acidbacteria colonies were picked up from both media.

The response of classic biochemical tests per-formed for Enterococcus (Devriese et al., 1992)allowed us to correctly identify some of theenterococci strains, confirmed by a phylogeneticanalysis for the SM21 strain. The isolation of thesemicroorganisms from bee-gut was an importantstep as there are only four scientific papersreporting their presence in the honeybee intestinalenvironment (Rousseau et al., 1969; Snowdon andCliver, 1996; Rada et al., 1997; Kacaniova et al.,2004) and with this information a clearer under-standing of the bee-gut microbiota composition canbe achieved.

Although there were differences between thephenotypical characterization of strains CRL 1647,IG9, AJ5, A15 and their genotypical profile, theywere not surprising, as authors had alreadyreported similar situations (Berger et al., 2007).Other articles have shown that there are sixLactobacillus strains of distinct homology groups(A1, A2, A3, A4, B1 and B2) that respond phenoty-pically to the characteristics of the acidophilusspecies, but a study with molecular biology toolsrevealed that the species may be crispatus, gasseri,johnsonii, gallinarum, amylovorus or acidophilus(Johnson et al., 1980; Pot et al. 1993). All thelactobacilli preselected in this work were homo-

fermentative, i.e., they do not produce gas fromglucose. These results agree with those reported byOlofsson and Vasquez (2008) who isolated andcharacterized many homofermentative lactobacillifrom the honeybee stomach. They observedthat lactobacilli and bifidobacteria were predomi-nant within the lactic microbiota associated tothe honeybee stomach, but failed to reportthe presence of L. johnsonii or Streptococcus orEnterococcus in this bee environment. However, inthis work it was determined, by phylogeneticanalyses, that the homofermentative lactobacilliassociated to bee-gut were closely related toL. johnsonii. In particular, the IG9 strain showed a97% (ca. 400 bp) identity with the 50 end of L.johnsonii NCC533, a type strain with its completegenome known (Boekhorst et al., 2004).

A high in vitro susceptibility of P. larvaevegetative cells to a low pH, unreported so far forthis bacterium, was observed in this work. Thethree L. johnsonii strains selected were able toinhibit the vegetative cells of the Americanfoulbrood agent by lactic acid synthesis and44 mM of this compound were necessary.E. faecium SM21, cultured in BHI broth, reached apH 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 lactoba-cilli and enterococci, the final pH values for the CFSused in the antagonistic tests were close to 4.0 or4.5 in most cases, but organic acid concentrationwas variable and not all lactic acid bacteriaselected, particularly enterococci, inhibited E. coliO157:H7, S. aureus ATCC29213, B. cereus C1 orP. larvae, by acidity. Although lactic acid produc-tion is an essential characteristic of the bacteriastudied here, the amount of this metabolite that issynthesized depends on each particular strain.Susceptibility to the antagonistic activity of lacticacid also varies among the pathogenic microorgan-isms (Ogawa et al., 2001; Wilson et al., 2005). So,the present in vitro study revealed that certainLactobacillus species, such as L. johnsonii exert

Properties of different lactic acid bacteria 11

growth inhibition on the indicator microorganismsassayed. To the authors’ knowledge, this would bethe first report on P. larvae growth inhibition due toacidity generated by lactobacilli isolated from beegut. In vitro inhibition of P. larvae by Bacillus sp.bacteria has been previously reported (Evans andArmstrong, 2005; Alippi and Reynaldi, 2006; Sabateet al., 2009). However, Bacillus sp. is a genusnormally related to the honeybee environment(Gilliam, 1987, 1997; Snowdon and Cliver, 1996).

The selected enterococci were remarkably activeagainst L. monocytogenes. This inhibition was notdue to lactic acidity but to a synthesis ofbacteriocin-like compounds by four E. faecium ofthe nine isolated. These compounds were thermo-stable and showed both a proteinaceous natureand a bactericidal effect against L. monocytogenes99/287; all these characteristics agree with thosefound for different enterocins produced byE. faecium isolated from other sources (Foulquie-Moreno et al., 2006). It has been informed that thestructural gene of enterocin A is widely distributedamong E. faecium strains, whereas that of enter-ocin B always occurs in the presence of enterocin A;besides, the high-frequency and variability ofenterocin structural genes among enterococci ofdifferent origin by means of simple PCR reactions,without purification or sequentiation, was alsoreported (Poeta et al., 2007). This trend wasconfirmed in this paper and this would be the firstcase where these genes have been detected inenterococci associated with the bee digestivetract.

Bacterial aggregation/surface property at bac-terial cell envelope level can be determined andquantified by the reduction of optic density valuesat different times and should not be confused/misunderstood with the flocculation phenomena(Handley et al., 1987; Kos et al., 2003). Floccula-tion is a phenomena more common/current foryeasts than bacteria. Furthermore, yeast floccula-tion is a non-sexual and reversible cell aggregationin which cells adhere to each other to form flocs(Zhao and Bai 2009). In this work, an importantauto-aggregation was observed in three differentL. johnsonii strains isolated from bee-gut. Theassays carried out to determine the chemicalnature of the factor involved in the lactobacilliauto-aggregation revealed in the cell envelope,different compounds for each strain; a lipopetidefor IG9 and a glicopeptide for CRL1647 and AJ5. Theinvolvement of proteinaceous bacterial surfacecompounds in lactobacilli adhesion has been ana-lyzed 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

study of this property in Lactobacillus with regardsto insects, furthermore no analysis has beenperformed on honeybee-derived Lactobacillus untilnow. Further studies are needed to determinewhich molecules are present in the autoaggregationphenomena.

Even though this work analyzes human foodbornepathogens, it should be pointed out that thehoneybee or its products are not E. coli O157:H7or L. monocytogenes hosts or vectors. The origin ofa strain was not considered to be an obstacle toascertain its full antimicrobial potential; thus, thefindings may open the way for different applica-tions other than apiculture practices.

Conclusions

Lactobacillus and Enterococcus strains associatedwith adult worker bees were isolated and char-acterized to enhance the knowledge of microorgan-isms found in the intestinal environment of thisinsect. The presence of L. johnsonii has now beenreported for the first time in the bee gut.Furthermore, this is the first report on P. larvaegrowth inhibition due to acidity generated bylactobacilli isolated from the bee gut ecosystem.

Acknowledgements

This paper was supported by Agencia Nacional dePromocion Cientıfica y Tecnica (ANPCyT, GrantPICTR890/06), CIUNSa (PI No 1725) and ConsejoNacional de Investigaciones Cientıficas y Tecnicas(CONICET). M.C. Audisio and M.C. Apella aremembers of the Research Career of CONICET. Theauthors thank Dr. S.A. Cuozzo and Dr. R.R. Raya forthe excellent assistance with the phylogeneticanalyses of the isolated strains.

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